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Navy Experimental Diving Unit
321 Bullfinch Rd
Panama City, FL 32407-7015
LT08-02
NEDU TR 13-06
JANUARY 2015
DEVELOPMENT OF A HYPERBARIC UPRIGHT EPIFLUORESCENCE HOFFMAN

CONTRAST MICROSCOPE FOR WHOLE-CELL ELECTROPHYSIOLOGY
RECORDINGS FOLLOWING EXPOSURE TO VARYING GAS MIXTURES
Authors
LCDR Denis G. Colomb Ph.D. (MSC, USN)
HM1 Kris G. Barber
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1. REPORT DATE (DD-MM-YYYY)

30-08-2013
4. TITLE AND SUBTITLE
2. REPORT TYPE
Technical Report
The Development of a Hyperbaric Upright Epifluorescence Hoffman Contrast Microscope for

Whole-Cell Electrophysiology Recordings Following Exposure to Varying Gas Mixtures
MIXTURES
6. AUTHOR(S)
LCDR Colomb, Denis G. Jr. Ph.D.(MSC, USN) and HM1 Barber, Kris G.
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)

Navy Experimental Diving Unit/ NAVSEA
321 Bullfinch Road
Panama City, Florida 32407
9. SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)
Office of Naval Research
Undersea Medicine Code 342
875 North Randolph Street, Suite 1425
Arlington, VA 22203-1995
3. DATES COVERED (From - To)

October 2007 April 2014
5a. CONTRACT NUMBER
N0001412WX20784
5b. GRANT NUMBER
ONR N0001412WX20784
5c. PROGRAM ELEMENT NUMBER
0602236N
5d. PROJECT NUMBER
N/A
5e. TASK NUMBER
LT 08-02
5f. WORK UNIT NUMBER
N/A
8. PERFORMING ORGANIZATION
REPORT NUMBER
TR 13-06
10. SPONSOR/MONITORS
ACRONYM(S)
N/A
11. SPONSOR/MONITORS REPORT
NUMBER(S)
12. DISTRIBUTION / AVAILABILITY STATEMENT

A: Approved for public release; distribution is unlimited.
13. SUPPLEMENTARY NOTES
None
14. ABSTRACT
Two of the long studied, but poorly understood stimuli that adversely affect CNS function, are pressure per se (barosensitivity) and gas narcosis. Likewise, the
mechanisms underlying their interesting interactionsanesthetic/narcotic gas-reversal of pressure effects and vice versais poorly understood at the cellular level. To address this
issue we have designed and fabricated a hyperbaric (3.457 MPa) patch-clamp system to address said effects on voltage- and ligand-gated ionic conductances. While presynaptic
mechanisms are implicated in the neurogenesis of barosensitivity (i.e., modulation of neurotransmitter release); evidence also exists for postsynaptic mechanisms in the formation of
barosensitivity (i.e. likely changes in receptor affinity and ion channel modulation). Therefore, an in vitro analysis of barosensitivity/gas narcosis is warranted. We have created a
HYPERBARIC (3.457 MPa) UPRIGHT EPIFLUORESCENCE HOFFMAN CONTRAST MICROSCOPE FOR WHOLE-CELL ELECTROPHYSIOLOGY RECORDINGS
FOLLOWING EXPOSURE TO VARYING PARTIAL PRESSURES OF He, N 2, AND A He/N2 MIX AT A CONSTANT 21 KPa O2 and 5 KPa CO2 as defined in the title. The system is
designed as an enabling technology in the nascent field of hyperbaric pharmacology.
15. SUBJECT TERMS

Cellular, Chamber Diving, Electrophysiology, Epifluorescence Excitable, Fluorescence, Gas, Helium, Giga Ohm Seal, Hyperbaric, Inert, Ion
Channel, Microscopy, Narcosis, Nitrogen, Oxygen, Pascal Partial Pressure, Patch Clamp, Perfusion, Recordings, Voltage-Clamp, WholeCell
16. SECURITY CLASSIFICATION OF:
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PERSON NEDU Librarian
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Ms. Nancy
Hicks NUMBER (include
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c. THIS PAGE
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area code)
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Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std. Z39.18
ACKNOWLEDGMENTS
The work defined in this document was funded in whole by the Office of Naval Research
(ONR), Under Sea Medicine Program (Warfighter Protection and Application; Division
342, Code 34) under the award numbers N0001412WX20784 [1] and
N0001412WX20610 [2]. The authors thank the various program managers and support
staff of ONR over the life of the project
The authors wish to specifically thank Master Model Maker Mr. James Arehart
(Contracts N0463A-08-M-0038, N0463A-10-P-0009, N0463A-10-P-0057, N0463A-12-P0016, & N0463A-13-P-0004; Arehart Model Maker, LLC) for his mentoring in design
considerations and practical aspects of equipment design. Mr. Areharts mentorship was
critical for the success of this project. His professionalism, knowledge (60+ years), and
commitment to the project and our individual growth is highly noteworthy.
Additionally, we would like to thank Dr. Jay Dean Ph.D. (University of South
Florida, Ta MPa, FL) for his guidance in the early phases of the project under Contract
N0463A-08-M-007, Mr. Bob Miller (Wright State University; Dayton, OH) for his
expertise in electrical/electronic design, and Ms. Ann Crawford (NEDU) for editing and
formatting.
The authors also wish to thank Mr. Richard Steckel (NEDU) and Mr. Rob Gray
(NEDU) for their role in chamber certification and installation, CAPT Richard Mahon
M.D.(MC, USN; Naval Medical Research Command, Bethesda; NMRC) for the transfer
of the surplus chamber and electrophysiology equipment, Mr. Raymond Glasgow
(NEDU) for his expertise in development of the gas supply system, Mr. Vincent Villani
(Nikon Instruments; Contracts N0463A-08-F-0004, N0463A-12-F-0003, N0463A-12-F0004, N0463A-12-F-0007, & N0463A-12-F-0037) for his numerous hours of support and
input related to microscopy, and Mr. Greg Grismore and the staff of Swagelok
Alabama Fluid Systems Technologies (Pelham, AL) for their input on pressure fittings
and pressure system design. We would like to thank CDRs Patrick T. Hennessey M.D.
Ph.D. (MC, USN), Rene Hernandez Ph.D. (MSC, USN), CAPT Thomas Herzig Ph.D.
(MSC, USN), and Dr. John Clarke Ph.D. for their independent review of the manuscript.
While not named specifically, this work could not have been accomplished without the
unnamed civilian, contactor, and military men and women of NEDU.
ii
NOTICES
The opinions and assertions contained herein are the sole views of the authors and are
not to be construed as the official views of the Department of Defense (DOD), United
States Navy (USN), or the Navy Experimental Diving Unit (NEDU) at large.
While the USN holds patent rights on information directly provided by active duty
service members and government employees, the system design is the intellectual
property of the authors. Patent related issues related to non-government employees
shall be handled via their respective legal representation and appropriate DOD offices.
This document has U.S. Government created or contracted drawings,
specifications, and related data. When U.S. Government drawings or contracted
drawings, specifications, or other data are used for any purpose other than a definitely
related Government procurement operation, the Government thereby incurs no
responsibility nor any obligation whatsoever, and the fact that the Government may
have formulated, furnished or in any way supplied the said drawings , specifications, or
other data is not to be regarded by implication or otherwise, as in any manner licensing
the holder or any other person or corporation, or conveying any rights or permission to
manufacture, use, or sell any patented invention that may in any way be related thereto.
This technical report has been reviewed by the NEDU Scientific Director for
scientific integrity, and has been approved for release by the NEDU Commanding
Officer as per the defined distribution statement. Correspondences related to technical
details, scientific matters, and/or manuscript reprint requests should be sent to the
NEDU Public Affairs Officer and/or Scientific Director for consideration. The NEDU
quarterdeck number is 850-230-3100. DOD agencies registered with the Defense
Technical Information Center should direct requests for copies of this report to: Defense
Technical Information Center, Cameron Station; Alexandria, VA.
iii
CONTENTS
REPORT DOCUMENTATION PAGE.............................................................................................................................. i
PREFACE ...................................................................................................................................................................... ii
Acknowledgments ......................................................................................................................................................... iii
NOTICES ...................................................................................................................................................................... iii
CONTENTS ................................................................................................................................................................... v
FIGURES ..................................................................................................................................................................... vii
INTRODUCTION............................................................................................................................................................ 1
System Design and Build ............................................................................................................................................... 4
General ...................................................................................................................................................................... 6
Microscope Design and Construction....................................................................................................................... 25
Transmitted Light Source ......................................................................................................................................... 75
Imaging .................................................................................................................................................................... 88
Epifluorescence Microscopy .................................................................................................................................... 98
Sled Assembly: ...................................................................................................................................................... 107
High Pressure Cellular Perfusion System .............................................................................................................. 112
High Pressure Cellular Perfusion System 750 mL Cylinder Characterization ........................................................ 137
Negative-Pressure System (NPS): ......................................................................................................................... 143
Electrophysiology Methods and Equipment Standard Patch Clamp. ..................................................................... 151
High Pressure Cellular Perfusion System Standard bath Solution Characterization.......157
Chamber Ancillary and Electrical Systems ............................................................................................................. 161
Discussion .................................................................................................................................................................. 173
References ................................................................................................................................................................. 175
APPENDIX A MOBILE TEST CHAMBER 4 (MTC-4) CONTROLLED ASSEMBLY DOCUMENTATION NOTEBOOK
....................................................................................................................................................................................... 1
APPENDIX B
MOBILE TEST CHAMBER 4 (MTC-4) FIBER OPTIC (VS18-SS-150-1-FO-96-48) AND USB (PT-16SS-150-1-USBAFEMALE-USBAMODE) ........................................................................................................................ 1
APPENDIX C MOBILE TEST CHAMBER 4 (MTC-4) SYSTEM PRESSURE RELIEF VALVES ................................ 1
APPENDIX D MOBILE TEST CHAMBER 4 (MTC-4) MODIFIED REAR FLANGE INSTRUMENT GROUND, AND
REAR FLANGE ELECTRICAL ....................................................................................................................................... 1
APPENDIX E MOBILE TEST CHAMBER 4 (MTC-4) VIEWPORT AND UNIVERSAL SERIAL BUS (USB) .............. 1
APPENDIX F MOBILE TEST CHAMBER 4 (MTC-4) PRESSURIZATION MODIFICATION AND IEEE 1394 HIGH
SPEED SERIAL BUS (FIREWIRE) ................................................................................................................................ 1
APPENDIX G MOBILE TEST CHAMBER 4 (MTC-4) ELECTRICAL ......................................................................... 1
APPENDIX H MOBILE TEST CHAMBER 4 (MTC-4) PQS ........................................................................................ 1
APPENDIX I MOBILE TEST CHAMBER 4 (MTC4) PROCEDURES FOR WORKING WITH EXCEPTIONALLY
DANGEROUS CHEMICAL OR BIOLOGICAL AGENTS ................................................................................................ 1
APPENDIX J MOBILE TEST CHAMBER 4 (MTC-4) OPERATIONAL AND EMERGENCY PROCEDURES ............ 1
APPENDIX K MOBILE TEST CHAMBER 4 (MTC-4) PRESSURE BASED PERFUSION DIAGRAM........................ 1
APPENDIX L MOBILE TEST CHAMBER 4 (MTC-4) LOWER OPTICAL UNIT AND TRANSMITTED LIGHT
SOURCE ........................................................................................................................................................................ 1
APPENDIX M MOBILE TEST CHAMBER 4 (MTC-4) FLUORSCENCE .................................................................... 1
APPENDIX N MOBILE TEST CHAMBER 4 (MTC-4) PERFUSION ........................................................................... 1
APPENDIX O MOBILE TEST CHAMBER 4 (MTC-4) O 2 QUANTIFICATION ............................................................ 1
APPENDIX P MOBILE TEST CHAMBER 4 (MTC-4) ELECTROPHYSIOLOGY........................................................ 1
APPENDIX Q MOBILE TEST CHAMBER 4 (MTC-4) ELECTRICAL AND ANCILLARY COMPONENTS ................. 1
APPENDIX R MOBILE TEST CHAMBER 4 (MTC-4) ANCILLARY ............................................................................ 1
APPENDIX S MOBILE TEST CHAMBER 4 (MTC-4) CCD FAILURE ........................................................................ 1
iv
APPENDIX T
APPENDIX U
MOBILE TEST CHAMBER 4 (MTC-4) GAS PANEL FOR 1 L CYLINDER CHARACTERIZATION ..... 1
MOBILE TEST CHAMBER 4 (MTC-4) PRESSURE RELIEF CHECK .................................................. 1
FIGURES
Figure 1: MTC-4 System Overview. ............................................................................................................................ 5
Figure 2 : MTC-4 Internal View (Fore). ...................................................................................................................... 12
Figure 3: MTC-4 Chamber Banks 1 and 2. ............................................................................................................... 13
Figure 4: Gas System MTC-4..................................................................................................................................... 14
Figure 5: MTC-4 Chamber Bank One. ....................................................................................................................... 15
Figure 6: MTC-4 Main Chamber Gas Bank (Bank 2). ............................................................................................... 16
Figure 7: MTC-4 Chamber Control Panel. ................................................................................................................ 17
Figure 8: MTC-4 Port View. ........................................................................................................................................ 18
Figure 9: MTC-4 Starboard View. ............................................................................................................................. 19
Figure 10: MTC-4 Aft View Rear Flange. .................................................................................................................. 20
Figure 11: MTC-4 External Electrical Panel. ............................................................................................................ 21
Figure 12: MTC-4 Internal Fore Sled Electrical Panel. ............................................................................................ 22
Figure 13: MTC-4 Internal Aft Sled Electrical Panel. ............................................................................................... 23
Figure 14: MTC-4 Port Microscope Components. ................................................................................................... 32
Figure 15: MTC-4 Starboard Main Components. ..................................................................................................... 33
Figure 16: MTC-4 Upper Optical Pathways. ............................................................................................................. 34
Figure 17: MTC-4 Objectives and Head Stage Main Components. ........................................................................ 35
Figure 18: MTC-4 Objectives. .................................................................................................................................... 36
Figure 19: MTC-4 Tube Len Assemblies. ................................................................................................................. 37
Figure 20: MTC-4 Upper Horizontal Optical Assemblies. ....................................................................................... 38
Figure 21: MTC-4 Upper Horizontal Optical Assemblies View Two. ...................................................................... 39
Figure 22: MTC-4 Upper Horizontal Optical Assemblies View Three. ................................................................... 40
Figure 23: MTC-4 Upper Horizontal Optical Assemblies View Four. ..................................................................... 41
Figure 24: MTC-4 Upper Horizontal and Vertical Optical Assemblies and Fluorescence Adaptor. .................... 42
Figure 25: MTC-4 Upper Vertical Optical Train and Fluorescence Adaptor. ......................................................... 43
Figure 26: MTC-4 Upper Vertical Optical Cube Placement of Filter Cube Mount View One. ............................... 44
Figure 27: MTC-4 Upper Vertical Optical Cube Placement of Fluorescence Adaptor View Two......................... 45
Figure 28: MTC-4 Upper Vertical Optical Cube Placement of Filter Cube Mount View Three. ............................ 46
Figure 29: MTC-4 Upper Vertical Optical Assembly Fore View. ............................................................................. 47
Figure 30: MTC-4 Upper Vertical and Horizontal Assembly. .................................................................................. 48
Figure 31: MTC-4 Objective Manipulator Assemblies. ............................................................................................ 49
Figure 32: MTC-4 Objective Manipulator View One................................................................................................. 50
Figure 33: MTC-4 Course Objective Manipulator View Two. .................................................................................. 51
Figure 34: MTC-4 NAI MM-4M-F-50-HT Z- Axis Manipulator Unit. .......................................................................... 52
Figure 35: MTC-4 NAI MM-4 M-F-50-HT Z- Axis Unit Manipulator Mounting Adaptors. ....................................... 53
Figure 36: MTC-4 NAI MM-4M-F-50-HT Z- Axis Unit Manipulator Mounting. ......................................................... 54
Figure 37: MTC-4 Objective Mount (Upper View). ................................................................................................... 55
Figure 38: MTC-4 Lens Mount (Lower View). ........................................................................................................... 56
Figure 39: MTC-4 Objective Units (Lower View). ..................................................................................................... 57
Figure 40: MTC-4 Objective Unit (Lower Unit) View Two. ....................................................................................... 58
Figure 41: MTC-4 Objective Unit View Three (Bottom). .......................................................................................... 59
Figure 42: MTC-4 Objectives Unit and Adaptor Plate View One. ........................................................................... 60
Figure 43: MTC-4 Objective Units and Adaptor Plate View Two. ........................................................................... 61
Figure 44: MTC-4 Objective Manipulator Primary Mounting Arm. ......................................................................... 62
Figure 45: MTC-4 Objective Manipulator Arm and Objectives Control System View One. .................................. 63
Figure 46: MTC-4 Objective Manipulator Arm and Objective Control System View Two. ................................... 64
Figure 47: MTC-4 Objective Manipulator Arm, Objective Control System, and Objective Unit. .......................... 65
Figure 48: MTC-4 Objective Mounting Arm Mounting. ............................................................................................ 66
Figure 49: MTC-4 Initial Fitting and Placement of Objective Manipulator Assembly. .......................................... 67
Figure 50: Mobile Test Chamber 4 (MTC-4) Upper Optical Train Mounting Systems. .......................................... 68
Figure 51: MTC-4 Final Assemblies Microscope Objective System Aft View. ...................................................... 69
Figure 52: MTC-4 Lower Optical Train Assemblies. ................................................................................................ 77
Figure 53: MTC-4 Lower Optical Train Assemblies and Manipulator. ................................................................... 78
Figure 54: MTC-4 Lower Optical Train Assemblies. ................................................................................................ 79
Figure 55: MTC-4 Hoffman Modulation Contrast Condenser............................................................................... 80
Figure 56: MTC-4 Condenser Manipulator Arm. ...................................................................................................... 81
Figure 57: MTC-4 Condenser Manipulator Arm and Condenser In Fore Position. ............................................... 82
Figure 58: MTC-4 HMC Condenser Slit Apertures. ............................................................................................... 83
Figure 59: MTC-4 HMC Polarizer Assembly View One. ........................................................................................... 84
Figure 60: MTC-4 HMC Polarizer Assembly View Two. ........................................................................................ 85
vi
Figure 61: MTC-4 HMC Polarizer Assembly View Three. ..................................................................................... 86

Figure 62: MTC-4 HMC Condenser and Polarizer Assemblies View Four. ......................................................... 87
Figure 63: MTC-4 Mounting of Camera View One. .................................................................................................. 90
Figure 64: Mobile Test Chamber 4 (MTC-4) Mounting of Camera View Two (Aft). ............................................... 91
Figure 65: MTC-4 Mounting Adaptor for the Modified QIClick............................................................................ 92
Figure 66: MTC-4 Filter Wheel Adaptor Second Version. ....................................................................................... 93
Figure 67: MTC-4 Nikon 0.7x DXM to Modified QIClick. ...................................................................................... 94
Figure 68: MTC-4 Modified QIClick With Alternative Mounting. ......................................................................... 95
Figure 69: MTC-4 PixeLINK PL-B741E CMOS. ...................................................................................................... 96
Figure 70: MTC-4 PixeLINK PL-B741E CMOS Aft Front Sled Mount. .................................................................. 97
Figure 71: MTC-4 Epifluorescence Excitation and Emission Pathways. ............................................................. 100
Figure 72: MTC-4 Upper Vertical Train Fluorescence Adaptors. ......................................................................... 101
Figure 73: MTC-4 Upper Vertical Fluorescence Adaptors Installation. ............................................................... 102
Figure 74: MTC-4 Upper Vertical Optical Quartz Multi-Modal Fiber Optic Cable Installation. ........................... 103
Figure 75: MTC-4 Emissions Pathway Initial Alignment. ...................................................................................... 104
Figure 76: MTC-4 Emission Pathway Initial Alignment View Two........................................................................ 105
Figure 77: MTC-4 Fluorescence Adaptor Installations. ........................................................................................ 106
Figure 78: MTC-4 Aft Sled Assemblies. .................................................................................................................. 108
Figure 79: MTC-4 Sled Clamps................................................................................................................................ 109
Figure 80: MTC-4 Front Sled Solution Heater and Stage Assemblies Mounts. .................................................. 110
Figure 81: MTC-4 Stage Mounting. ......................................................................................................................... 111
Figure 82: MTC-4 Perfusion Gas Compression Systems. .................................................................................... 117
Figure 83: MTC-4 HPLC Pump Based Perfusion Systems Overall Design. ........................................................ 118
Figure 84: MTC-4 Perfusion Pressurization System External. ............................................................................. 119
Figure 85: MTC-4 Perfusion Pressurization Systems External Primary Control Panel. ..................................... 120
Figure 86: MTC-4 HPLC Pump Based Perfusion Systems View One. ................................................................. 121
Figure 87: MTC-4 HPLC Pump Based Perfusion Systems View Two. ................................................................. 122
Figure 88: MTC-4 HPLC Pump Based Perfusion Systems View Three. ............................................................... 123
Figure 89: MTC-4 HPLC Pump Based Perfusion Systems View Four. ................................................................ 124
Figure 90: MTC-4 HPLC Based Perfusion Systems View Five. ............................................................................ 125
Figure 91: MTC-4 HPLC Pump Based Perfusion Systems View Six. ................................................................... 126
Figure 92: Mobile Test Chamber 4 (MTC-4) Perfusion Bank Internal Supply Panel. .......................................... 127
Figure 93: Mobile Test Chamber 4 (MTC-4) Perfusion Bank Gas Fluid Integration Control Panel. .................. 128
Figure 94: MTC-4 Perfusion System Integration Panel Gas and HPLC. .............................................................. 129
Figure 95: Experimental Set-Up Extrel Questor QGP Process Mass Spectrometer (QCPMS) 750 mL (1 L)
Sample Cylinder De-gassing and On-gassing. ...................................................................................................... 139
Figure 96: 750 mL (1 L) Swagelok CT8079 Perfusion Reserve Characterization................................................ 140
Figure 97: MTC-4 A211SS-120-A 4 Way 2 Position Solenoid Valve and Local Equipment. ............................... 147
Figure 98: MTC-4 Negative Pressure System (NOS) Schematic Representation. .............................................. 148
Figure 99: MTC-4 Negative Pressure System (NPS) A211SS-120-A 4 Way 2 Position Solenoid Valve. ........... 149
Figure 100: MTC-4 Negative Pressure System (NPS) External Controls. ............................................................ 150
Figure 101: MTC-4 Pipette Placement. ................................................................................................................... 152
Figure 102: MTC-4 Manipulator Placement. ........................................................................................................... 153
Figure 103: Mobile Test Chamber 4 (MTC-4) Bath Reference. ............................................................................. 154
Figure 104: MTC-4 RC-27LD Geometry. ................................................................................................................. 155
Figure 105: Mobile Test Chamber 4 (MTC-4).......................................................................................................... 156
Figure 106: MTC-4 RC-27LD 4mm Red Eye Probes Configuration. .................................................................. 157
Figure 107: Mobile Test Chamber 4 (MTC-4) RC-27D 4mm Red Eye Probes Experimental Set-up. ............... 158
Figure 108: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Mounted. ...................... 160
Figure 109: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Components. ............... 163
Figure 110: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Mounting Upper View. . 164
Figure 111: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Affluent and Effluent Line
Connections. ............................................................................................................................................................ 165
Figure 112: MTC-4 Prior H29/30 Stage Wiring Pin Out. ......................................................................................... 166
Figure 113: MTC-4 Prior H29/30 X- and Y-Axis L120/K120 Stage Wiring Pin Out. .............................................. 167
Figure 114: MTC-4 Prior MM-3M-F-1.5-HT Stage Wiring Pin Out.......................................................................... 168
Figure 115: MTC-4 HF110 10 Position Filter Wheel Pin Out. ................................................................................ 169
Figure 116: MTC-4 SH-27B Inline Heater. ............................................................................................................... 170
Figure 117: MTC-4 Inline-Solution heater Cable wiring (Solder side view). ........................................................ 171
Figure 118: MTC-4 CC-28 Cable Assembly Heater Plate Cell Bath. ..................................................................... 172
vii
TABLES
Table 1: MTC-4 Electrical Penetrations and Drawings 24
Table 2: MTC-4 Scope Assemblies Technical Drawings.. 70
Table 3: MTC-4 Pressure Head and HPLC Operating Procedures.. 131
Table 4: MTC-4 Mass Spec Gas Control Panel 141
Table 5: MTC-4 Negative Pressure System (NPS) Procedures 145
viii
INTRODUCTION
The specific aim of this project was the development of a hyperbaric electrophysiology
system for whole-cell voltage-/current-clamp and epifluorescence recordings to a
maximal pressure of 3.457 MPa over ambient pressure in excitable cell types. The
system allows analysis of macroscopic cellular excitability (i.e., ion channel function)
within the USNs published operational limit of 304.8 meters sea water (msw) [3]. Both
ligand- and voltage-gated ion channels (LGIC and VGIC; respectively), are the basic
functional subunits of cellular excitability. Their respective open probability (P O)
following a defined stimulus dictates the influx/efflux of ionic constituents; i.e.,
1
membrane resistance (RM) and conductance (G; G= ). This manuscript is intended to
provide a blueprint of the development and construction of a cellular system for

utilization under hyperbaric conditions (3.457 MPa 335 msw 1,100 feet of sea water;
fsw).
VGIC [4] are stimulated (activate and/or deactivate/inactivate) due to changes in
membrane potential (VM). This VM induces the voltage-sensing mechanisms [5-7] of
the VGIC (i.e., S4 segment) to undergo a conformational shift [8, 9], thereby allowing
the respective channels to enter a conductive state or non-conductive state depending
on the polarity of the stimulus and the channel superfamily. VGIC can be grouped into
four major families based upon their respective selectivity filters, as defined via
mutational analysis and related bacterial crystal structure [4, 10, 11]; Na+ (DEKA) [1219], K+ (GYG) [10, 20-27], Ca2+(EEEE) [28-31], & Cl- (GSGIP(106-110), G(K/R)EGP(146-150),
GXFXP(355-359) & Tyr445) [32-36].
In contrast LGIC are directly modulated via binding of specific ligands. The
extracellular LGIC consist of three superfamilies [37]: 1) Cys-Loop [nicotinic
acetylcholine receptors (AChR) [38], 2) serotonin receptors (5-HT3R) [39], glycine
receptors (GLyR), and -aminobutyric acid alpha receptors (GABAAR) [40], adenosine
triphosphate receptors (P2X; [41] ) and 3) ionotropic glutamate receptors (iGluR) [42,
43]. In the case of VGIC and LGIC it is this increase in G, which allows for the net
influx/efflux of charged particles; VM. Due to the relatively high input resistance (RInput)
of the cell body and/or axon terminal, even minimal shifts in ionic conductance (current;
I) are capable of large changes in voltage; based upon Ohms Law (V=IR; G=1/R).
While the system is designed for the elucidation of He and N2, effects of other
chemically inert gases may be tested. This equipment allows for analysis of the
modulation of macroscopic ionic current/s as based upon the effects of narcosis and/or
pressure per se. The system allows for initial analysis of the antagonistic relationship
between gas narcosis and hyperbaric pressure. However, the system pressure range is
insufficient to fully address the latter. It should be noted that our laboratory follows the
assumption that hydrostatic and gas compression methodologies differ in effect
although no detailed cellular analysis of the difference between hydrostatic and

pneumatic pressure has been completed to date.
The role of the chemically inert gases [44] (specifically N2) [45, 46] and pressure
per se in cognitive [47-53] and muscular performance [50, 54-56] have been well
defined in the literature since the 1930s [57-60]. However, the physiological
significance of the inert gas N2 and noble gases He, Ar, Ne, Kr, and Xe in single cell
neuronal excitability under hyperbaric conditions are areas of research that have had
minimal consideration. While CO2 is narcotic [61-63] and O2 would be predicted to be as
per their respective lipid solubilitys, these gases are clamped to standard cell culture
values (101.353 KPa; O2 20.270 KPa and CO2 5.068 KPa). The lack of basic cellular
physiology is interesting since saturation diving relies on the antagonistic
pharmacological relationship [64-68] between pressure per se and narcosis to reach
depths in excess of 182msw (1.935 MPa). This approach is based on the classic
finding of the pressure reversal of anesthetic gases in animal models and the quenching
of luciferase activity by hydrostatic pressure.
There are numerous approaches to define neuronal excitability under normobaric
conditions. However, the development of next generation pharmacological approaches
to mitigating gas-based diving issues requires elucidation of the molecular mechanisms
responsible for shifts in cellular physiology under hyperbaric conditions.
Whole-cell voltage- and current-clamp (WCVC/WCCC) recordings (WCR) are
quantitative approaches included under the suite of techniques referred to as patchclamping. In the academic and pharmaceutical arena standard and automated [69-75]
WCR are considered the gold-standard in the analysis of VGIC and/or LGIC
modulation following exposure to an agonist or antagonist [76, 77]. While planar based
approaches are mechanistically superior to pipette based WCR approaches and offer
increased throughput, a significant issue is the increased capacitance [78], increased
series resistance (RSeries), and inability to consistently form giga-ohm (G) seals (i.e.,
lower seal resistance; RSeal); i.e., recordings tend to have issues when analysing
kinetics and small current profiles. The ability to form a G seal is critical since the
membrane is electrically in parallel with RSeal and therefore a decreased RSeal will
significantly affect the characteristic of the membrane RC circuit. This issue currently
limits the resolution of the planar systems when defining channel kinetics. However, the
seal formation by classic pipettes and planar chips has received significant attention
and their respective properties have improved [79-81]. For an outstanding review of
planar electrophysiology we recommend the reviews of Py [82], Bebarova [76], and
Farre [73].
The utilization of standard WCVC and WCCC under hyperbaric (i.e., in excess of
ambient pressure; 101.32 KPa) conditions to address the significance of increasing
inert gas concentration and/or pressure per se on macroscopic ionic conductance is an
area of research that has received little attention due to methodological and technical
difficulties. To date we can find no researchers using whole-cell recordings (WCVC or
WCCC) in mammalian cell types under sustained hyperbaric conditions. Prior research
has focused on the utilization of sharp microelectrodes [83-87] (high series resistance),
field potential recordings [88], and the loose macro-patch technique [89].
In summary, we completed the primary aim of this project; the development of a
hyperbaric electrophysiology system for WCR (both WCVC and WCCC) and
epifluorescence recordings to a maximal pressure of 3.457 MPa over ambient pressure
in excitable cell types. We provide the first detailed starting point and road map to
develop further enabling technology for the analysis of modulations in cellular
physiology under hyperbaric conditions of relevance to the USN and DOD.
SYSTEM DESIGN AND BUILD

OVERVIEW
Figure 1 shows an overview of the Hyperbaric Upright Epifluorescence Hoffman
Contrast Microscope system in relationship to MTC-4. Cells are exposed to varying
partial pressure [90-92] of gases in solution. Solution and chamber gas make-up are the
same. Solutions may be varied depending on the experimental question. Using an
upright scope, a borosilicate pipette (2-3 M) is maneuvered into close geometry with
the cell. The distance is verified via variations in R following a square V pulse. A light
suction is applied to form a G seal. Following stabilization, a negative pressure is
applied to the seal allowing for the formation of the whole-cell configuration.
Cells are compressed to the target partial pressure of gases while maintaining a
constant O2 and CO2 partial pressure. The utilization of varying ionic backgrounds, and
defined antagonists/agonists, allow for the quantification and identification of the various
ion channels and the development of a list of VGICs affected by increasing gas
concentration.
The system also allows concomitant study of second messenger pathways using
epifluorescence based methodologies. While the epifluorescence capability of this
scope is qualitative [93-95], when combined with whole-cell recording, the fluorescence
based recordings provide a detailed picture of cellular modifications in excitability.
Manipulators for pipette placement and microscope focus are externally
controlled. The mounting of the system in a hyperbaric chamber allows for analysis of
various gas configurations. This nascent system provided the USN with the first wholecell recording system for the elucidation of variations in VGICs and LGICs.
5
Figure 1: MTC-4 System Overview.
The figure shows a schematic representation of the hyperbaric electrophysiology and epifluorescence system. Calibration gases are equilibrated in 1 L cylinders
(750 mL working volume). Following selection (
) the equilibrated solution is maintained at a 2 mL min -1 rate by the Mass Flow Controller (MFC). Alternatively,
equilibrated solution may be inserted distal to the MFC by the High Pressure Liquid Chromatography (HPLC) pump. Both systems utilize a pressure head to allow
for transfer of solution across the primary boundary (MFC utilizes a gas pressure head with the HPLC utilizing a mechanical pressure head). Affluent solutions
equilibrated with gas bathes the cells ( 2 mL min-1). The solution is removed by a micro-handling pump (Pump). Effluent solution is captured in a reserve
container (T-175; culture flask); pump is set to a slightly greater flow rate then the affluent input. In the event the reserve is full, a chamber pressure driven
emergency dump (ED) can be opened to remove the media. Variations in ion channel activity are measured using the Axon Instruments head-stage (Head) and
amplifier (AXON). A LED light source is used for Brightfield and Hoffman illumination. An epifluorescence source (Fluor Source) illuminated samples via the
objective with a defined excitation (EX) frequency. The fluorophore releases photons in the emission (EM) frequency. The EM, BF, and Hoffman signals are
collected at the CCD. The EX frequency pathway is inhibited by band-pass filters. The EM frequency is filtered by the band-pass and filter wheel assemblies.
Chamber compression (COMP) and decompression (DECO) are controlled by external mechanical controls. The numerous motorized controllers that cross the
primary pressure boundary for optical focusing and manipulator control are not defined (Various Equipment). Final configuration is dependent upon experimental
requirements.
GENERAL
Appendixes are defined as letters with respective pages; i.e., Appendix A page 20
would be defined as A-20 in the text.
A surplus (NMRC) military hyperbaric chamber was designated as MTC-4. MTC4 was refurbished and internally certified to a maximal working pressure of 3.457 MPa
at the USN NEDU (Panama City, Florida) according to the standards set forth in the
USN Diving and Manned Hyperbaric Systems Safety Certification Manual [96]. The
chamber (US Vessel # H-176-2 BFM) was manufactured by the Bethlehem Corporation
(Bethlehem, Pennsylvania) in 1968. MTC-4 was non-destructive tested
[97-99] to MIL-STD-278 (Military Standard Welding and Casting Standard) by the
Tecnico Corporation (Job # 202432; Appendix A), Chesapeake, VA. Non-destructive
testing was completed off-site. MTC-4 was down-graded from its original maximal
working pressure of 6.899 MPa to a maximal working pressure of 3.457 MPa. The
chamber was tested to 10.342 MPa. Following temperature stabilization, the system
was stable (<2% P) for 24hrs. The down-grading of MTC-4 was due to lack of
maintenance documentation, overall chamber status, and hull modifications post
manufacturing by previous facilities.
MTC-4 was mounted on a NEDU designed table and was vibration-isolated via
varying density rubber padding at the footings. The system was mounted to the floor
utilizing 12.70mm diameter stainless steel (SS) bolts. The chamber was mounted to the
NEDU constructed table in a similar fashion. It is highly recommend that future systems
be mounted on vibration isolation tables of sufficient lifting capability. Initially, the
chamber was mounted on air bladders (N2) at the table foot regions. However this
configuration was not stable as based on the swing-arm clam-shell design. The
chamber clam-shell seal was mounted on a SS swing-arm. The arm was covered with
high density foam to limit injuries when the chamber was in the non-pressurized or
pressurized states. Failure to place the protective foam on the SS arm has the potential
to result in significant injury.
System maximal pressure was defined to address biophysical modifications in
ionic conductance following exposure to operational and experimental breathing gases
within the 3.457 MPa published limitations of USN Diving [3]. MTC-4 certification was
completed internally at NEDU to meet the intent of the Naval Facilities Engineering
Command (NAVFAC) certification standards. However, it should be noted MTC-4 was
not a NAVFAC certified chamber system. Chamber certification (NEDU) and nondestructive testing documentation are defined in Appendix A (A-2 to A-247 and A-248 to
A-264; respectively). Standard engineering symbols are utilized in all schematic
diagrams (A-265 and A-266). Entry control documents are provided in Appendixes as
follows:
1) Appendix B: MTC-4 Fiber Optic and Universal Serial Bus (USB),
2) Appendix C: MTC-4 Fiber Optic and USB,
3) Appendix D: MTC-4 Modified Rear Flange, Instrumentation Ground, and Rear
Flange Electrical,
6
4) Appendix E: MTC-4 Viewport and USB),

5) Appendix F: MTC-4 Pressurization Modification and IEEE 1394 High Speed
Serial Bus (FireWire),
6) Appendix G: Electrical,
7) Appendix U: System Pressure Relief Valves
Laboratory personel were responsible for completion of entry control
documentation, associated notes, and quality assurance. Additionally, all military
laboratory personnel were qualified as chamber operators and craftsmen with the
laboratory principle investigator serving as the chamber supervisor. All personnel
operating MTC-4 were certified as chamber operators as per NEDU MTC-4 Personal
Qualification Standards (Appendix H). Chamber operation required two chamber
qualified personnel for safety. Failure to operate the chamber using a two person
approach is a significant safety risk that is not warranted and may result in injury or
death in the event of system failure.
Chamber certification and entry control documentation are provided to set forth
the standard of safety. Chamber preventative maintenance scheduling and tasking was
completed as per NAVSEA Standard Specification for Ship Repair and Alteration
Program [100]. MTC-4 was housed in a room with multiple Analox O2 Buddy oxygen
analyzers (Analox Sensor Technology Ltd.; UK) to ensure audible alarm in case O2
dropped below 20%. MTC-4 gas supplies are a significant asphyxiation hazard. The O 2
sensors were checked weekly in a <20% 02 background. Sensors were replaced when
they no longer were within manufacture specification or system battery was exhausted.
The system and sensors are housed at 101.352 KPa ambient (+/- barometric
pressure).
MTC- 4 has an internal volume of 0.67 m3 (166.52 L floodable); 1.02 m length
by 0.46 m diameter. An overall internal view of the chamber can be seen in Figure 3. A
technical drawing of the MTC-4 geometry is provided in A-242 to A-244.
Chamber maximum descent rate is 224.148 KPa min-1 (22.24 msw min-1) with a
maximum ascent rate of 179.332 KPa min-1 (17.79 msw min-1). Chamber is limited to
21% O2 at 101.325 KPa. Inlet and outlet supplies where 12.7mm SS tubing (o.d.) with a
0.889 mm wall thickness. Chamber gas was supplied by K-bottles (0.05 m3), with a total
of 10 bottles aligned in two parallel gas fields (5 bottles per field; total volume at 24.132
MPa 118.66 m3). Bottle banks may be operated individually or in parallel as required
by experimentation. The chamber is He certified. Compression profiles utilizing nonhelium gas mixes required switching of K-bottles to ensure He-bottle certification was
maintained. One-way check valves were installed post installation to minimize
contamination issues following gas/bank change.
Procedures for utilization of exceptionally dangerous chemicals under hyperbaric
conditions are defined in Appendix I (NAVXDIVINGU 6240.4B [101]); the overall
approach is utilize a minimal volume in the event of a compromise of the primary
pressure boundary and/or perfusion system integrity (i.e., aerosol development during
7
rapid decompression). It should be noted that in many cases the effects of aerosols of
standard ion channel agonist and antagonists, to include biological agents and
molecular biology reagents, are undefined in the literature and therefore safety
precautions are required but speculative. Precautions were defined to allow for
functional experimentation and to minimize hazards; i.e., it is not realistic to place a
hyperbaric system in a Biosafety Cabinet. Perfusion solution volumes are held to a
minimum volume (released aerosol in relationship to area and know LD50s) in case of
system failure. In addition to NAVXDIVINU 6240.4B, laboratory and hyperbaric
experiments meet the requirements of the following NEDU programs; (1) Chemical
Hygiene Program (CHP) [102], (2) Biological Safety Cabinet Program [103], (3)
Molecular Biology Lab Safety Program [104], and as relevant the (4) Hazardous
Communication (HAZCOM) Program and (5) Hazardous Materials (HAZMAT) Program
[105]. During the technical report period there were no negative reports or chemical
exposures; outside reviews of the programs resulted in zero discrepancies. Additionally,
there were no issues related to chamber pressurization/depressurization. EnviroMed
Services (Panama City) was the vendor for disposal of biological and chemical agents.
MTC-4 Operating and Emergency Procedures (EP/OP) are defined in Appendix
J. Technical drawings of the MTC-4 bottle bank and pressurization controls are provided
in Figures 3 and 4. Figures 5 to 7 show chamber control and the overall bottle field setup to include charging station integration point. The chamber gas (He) was charged
from the main NEDU He banks via a SS charging whip. Chamber documentation
ensured personnel were adequately trained in all aspects of OP and EP of the defined
high energy system. Chamber personnel were Planned Maintenance System (PMS)
301 and 303 qualified. PMS is a subsystem of the USNs Maintenance and Material
Management System (3-M). Briefly, the system allows standardization, efficiency,
documentation, and analysis of preventative maintenance. All relevant PMS
requirements were completed and internally documented.
Installation of Swagelok components were completed as per manufacture
recommendation; LCDR Colomb (Cert # A2242) and HM1 Barber (Cert # A2243)
completed the Swagelok Alabama Tube Fitting Safety and Installation Course. All
penetrators were bi-directionally (atmosphere/vacuum) pressure tested to 51.710 MPa
(100% He). Helium testing (4.002602 +/-0.000002 gmol-1; molecular weight) was
defined as the standard. Figures 8 to 11 show the pressure hull penetration layout and
defining relative joint identification number (J.I.D.s; Note: J.I.D. numbers are specific for
tasking and will vary on re-entry control documentation).
General electrical, fluorescence light source, universal serial bus (USB), and
IEEE 1394 High Performance Serial Bus (FireWire) chamber penetrators utilized for
crossing the primary pressure boundary where purchased from PAVE Technology
Company (Dayton, Ohio [106]) or Swagelok Alabama (Birmingham, AL [107]). Custom
penetrators were ordered from PAVE. These two companies were utilized for all nonpressure hull modifications to MTC-4; Hazardous Category (HAZCAT) 1 -3. HAZCAT 1
was defined as the primary pressure boundary (MTC-4) to which the staff is exposed
and the first isolation valves connected to the pressure boundary. HAZCAT 2 was
8
defined as systems, equipment, and components sufficiently isolated from the primary
pressure boundary but that may cause injury unless pressure, gas flow, fluid flow,
and/or electrical power are secured. HAZCAT 3 was defined as items that during failure
would compromise the research/testing but would not result in direct injury due to items
listed under HAZCAT 1 and 2.
The penetrators installed are as follows (J.I.D.#/ manufacture designation/PN# if
applicable):
(1) J.I.D.#001; Chamber Reference (Appendix A),
(2) J.I.D.#002; Swagelok (Chamber Bias to Aschroft Volumetric Controller
Model 3000),
(3) J.I.D.#003 Swagelok (Volumetric Control from Aschroft Volumetric
Controller Model 3000),
(4) J.I.D.#004 PAVE PT16-SS-200-30-TE20-X-X (G-16) ,
(5) J.I.D.#005 PAVE PT-16-SS-150-1-USBAFEMALE TO USBAMALE (B-19),
(6) J.I.D.#006 PAVE PT-16-SS-150-1-USBAFEMALE TO USBAMALE (B-19)
(7) J.I.D.#007 PAVE PT16-B-150-1-AXOPATCH200B-70-48 (PN#3618),
(8) J.I.D.#008 Swagelok Perfusion In ,
(9) J.I.D.#009 Swagelok Perfusion Out,
(10) J.I.D.#010 Vent (Appendix A),
(11) J.I.D.#011 Pressurization Line from Gas Field (Appendix A),
(12) J.I.D.#012 Swagelok B-8-P (Appendix A),
(13) J.I.D.#013 PAVE VS15L-SS-150-1-USBAmale-USBAfemale PN#3529 (E18),
(14) J.I.D.#014 acrylic window (Appendix A),
(15) J.I.D.#015 PAVE PT8-SS-150-1-.25CU-3-3 (D-23 to D-24),
(16) J.I.D. # 016 Swagelok B-4-P (Appendix A),
(17) J.I.D. # 017 JM Canty, Inc. (Buffalo, NY) LB6535-11313,
(18) J.I.D.#018 PAVE VS15L-SS-150-6-28-IEEE1394 FIREWIRE (PN#3321),
(19) J.I.D.#019 B-4-P (Appendix A),
(22) J.I.D.#022 PAVE PT16-SS-200-30-TE20-X-X (G-16),
(23) J.I.D.#023 Swagelok B-16-P (Appendix A),
(26) J.I.D.#026 PAVE VS18-SS-150-1-FO-96-48-TEE20-48-48 Quartz Fiber (B18),
(27) J.I.D.#027 PAVE VS18-SS-150 Plug (D-20)
(28) J.I.D.#028 PAVE VS18-SS-150-1RG-178-36-36 (D-41), and
(29) J.I.D.#029 PAVE VS18-SS-150 Plug (D-20).
Penetrators not defined were capped with National Pipe Thread (NPT) Brass
Pipe Plugs of the Swagelok B-XX-P series or as defined in Appendix A to T. Brass
was utilized to not damage hull penetrator threading in the event of cross-threading.
Penetrators and location on chamber are referenced to the Split-Locking Ring entrance
(i.e., Fore). All penetrations and defined locations are shown in Figures 7 to 9.
Power was provided to all systems via Spectrum Power Management Systems
72RA120BFLBA-10 metered power strips connected to facility power (UPS) as defined
in Appendix Q (Q-18). The power management systems were adapted to standard
National Electrical Manufacture Association (NEMA) 5-15R utilizing NEMA 5-15P to
NEMA L5-20R plug adaptors. MTC-4 electrical components were integrated into the
facility uninterrupted power supply (UPS) to ensure the integrity of experimentation
during a power failure and for system safety. All pressure control systems are
independent (mechanical) of facility power. The automation of diving controls is
recommended in future systems. Components were purchased for automation of the
chamber but were not installed due to operational commitments. Emergency lighting is
located by chamber compression/decompression controls.
MTC-4 internal lighting was provided using an externally mounted JM Canty Inc.
LB6535-11313 [108] Chamber Light (50 W, 12 V, wide lens pipe). Chamber lighting was
also provided utilizing magnet mounted custom LED Lighting Tubes (24 V; rheostat).
The Chamber LED lighting system is not shown in the figures as it was not required for
current experimentation.
MTC-4 had independent chamber and system grounds, allowing troubleshooting
of electrical interference and ground loops [109]. Electrical noise was a significant
problem in the early design phases. Instrumentation ground points are defined on the
sled and can be modified as required.
Electrical numbering and associated cables are defined in Table 1. System and
the electrical drawings are provided in Appendix Q. An overall view of the electrical
approach can be seen on Q-2. Electrical connections utilized a junction box approach
from the primary external panel to the rear and front sled internal electrical junctions.
Internal and external electrical panel layout and construction are defined as; A) Inside
front junction panel 293511-1 (Q-3), B) Inside rear junction panel 293511-2 (Q-4), and
C) Outside junction panel 293511-3 (Q-5). Jumper cables to test cable integrity were
built for ease of troubleshooting during set-up and modifications. Figure 10 shows the
installed external panel. Figures 12 and 13 defining the relationship of the internal rear
and front panels (Note: System removed to allow for ease of viewing. Rear panel
mounts to back of rear sled and front panel mounts to undercarriage of front sled).
System overall wiring breakout is defined in Q-7.
Wiring related to microscope systems was run along the sides of the upper and
lower portions of the sleds in a configuration to minimize clutter; cabling is not shown to
allow for ease of viewing and due to varying placement depending on experimental
requirements. Final placement of electrical lines and utilization of mounting systems for
wiring are at discretion of the user. Bending radius of wires and associated armoring are
critical to minimize wire compromise. Electrical shielding is required to minimize system
noise.
10
Various clamps and electrical risers are defined in Q-21 to Q-26 and were utilized
to secure electrical connections, specifically system ribbon cables. It is critical to ensure
ribbon cables are secured in a fashion not to compromise integrity of the conductive
pathway (Figure 13). Unfortunately, ribbon cables are highly susceptible to internal
compromise and were replaced often. Various electrical components, adaptors, and
information considered in installation and/or utilized in the electrical system are provided
in Q-27 to Q-36. The specific mounting approaches for connectors are not discussed as
they will vary from system to system. Mounting options will vary depending on user
preference.
The microscope and sled in figures external to the chamber are on an internal
geometry mock-up. This allows for placement and verification of fit prior to final
fabrication.
The electrical system is designed for a 21% O2 (101.325 KPa 1 ATA) with a
chemically inert gas background. System utilization at >21% O2 may result in system
compromise and possible injury. MTC-4 components are not designed for environments
in which O2 concentrations place the system in the fire zone; THIS IS NOT A HIGH O2
SYSTEM (>21%). Pressurized system components defined as HAZCAT 2 and 3 were
tested at >34.373 MPa (He or N2), with the respective ports sealed with Swagelok caps
or plugs (1.587 and 3.175 mm tubing). Caps or plugs are utilized when ports are not
functional. System components were isolated and maintained at >34.473 KPa during
storage (He or N2).
11
17
12
Chamber
Railing System
Figure 2 : MTC-4 Internal View (Fore).

The figure shows the MTC-4 front (Fore) view. MTC-4 has a split-locking ring with an internal volume of 0.67 m3 (166.52 L floodable volume); 1.02 m length by
0.46 m diameter.
Figure 3: MTC-4 Chamber Banks 1 and 2.
13
1914
Figure 4:
Gas System MTC-4
The figure shows a schematic representation of MTC-4 pressure banks (1 and 2) from Figure 3 in parallel configuration with chamber controls. See Appendix J for
chamber EP and OP.
Gas Flow Chamber
20
15
He Bottles 6-10: Bank 2

Perfusion
System Bottles
Figure 5: MTC-4 Chamber Bank One.

The figure shows one of the two main gas banks for chamber compression. MTC-4 Bank 2 is shown for reference. Figure shows overall design of pressurization
Bank 1. Standard procedures utilize He, He/N2, or N2 for bank operations. For utilization of mixes other than He, bottles are switched out due to difficulty in recertifying the bottles to 100% He following contamination of storage bottles. When gases other than He were utilized, a 10 min flush was completed with an
independent He source to re-certify system to 100% He prior to charging on the main NEDU He Bank. The flush procedure was completed 3 times prior to recertification. Bottles to left of He are for the perfusion system; Bank 2 was designed in a similar approach. All bottles are labeled using hang tags with specific gas
mixes, charging date, and certification. Arrows show direction of gas flow.
21
16

Charging Whip Storage
and System Tie In:
System Charged with He
from NEDU Main Gas
Bank
Figure 6: MTC-4 Main Chamber Gas Bank (Bank 2).
The figure shows one of the two main gas banks for chamber compression. Figure shows overall design of pressurization Bank 2. Standard procedures utilize He,
He/N2, or N2 for Bank operation. For utilization of mixes other than He, bottles are switched out due to the difficulty of re-certifying the bottles to 100% He following
contamination. When gases other than He were utilized, a 10 min flush was completed with an independent He source to re-certify system to 100% He. Bank recertification required 3 flushes. Bottles to left of He are for perfusion system. Charging of Bank 1 and/or Bank 2 He was completed using a manufactured (NEDU)
30.48m charging whip. Bottles were charged from the main NEDU He gas bank as defined in Appendix J.
Figure 7: MTC-4 Chamber Control Panel.

The figure shows the chamber mechanical controls for compression and decompression. Procedures for Chamber
Operations are defined in Appendix J. Chamber controls are mechanical. Note: A secondary stop watch (hanging) is
utilized to verify ascent and descent rates. Chamber personnel are required to dive/ ascend MTC-4 rates within the
system limitations. Rate of descent/ascent is defined by stop watch and pressure gauge. Chamber certification for
operators required five successful controlled descents/ascents prior to certification.
17
2318
K L
O*
*
Figure 8: MTC-4 Port View.

The figure shows the port penetrators. View defined from front (chamber door) of chamber. Penetrator labeling port view (Joint Identification Number; J.I.D.#XXX),
viewports were replaced with SS blanks machined to defined standards (mm); (A) 6.35 NPT (J.I.D.#001), (B) 6.35 NPT (J.I.D.#002), (C) 6.35 NPT (J.I.D.#003, (D)
25.4 NPT (J.I.D.#004), (E) 25.4 NPT (J.I.D.#005), (F) 25.4 NPT (J.I.D.#006), (G) 25.4 NPT (J.I.D.#007), (H) 6.35 NPT (J.I.D.#008), (I) 6.35 NPT (J.I.D.#009), (J)
19.05 NPT (J.I.D.#010), (K) 19.05 NPT (J.I.D.#011), (L) 12.7 NPT (J.I.D.#012), (M) 25.4 NPT (J.I.D.#013), (N) acrylic viewport (J.I.D.#014), and (O) 12.7 NPT
(J.I.D.#015).
24
19
Figure 9: MTC-4 Starboard View.

The figure shows the starboard penetrators. Views defined from front of chamber. Penetrator labeling starboard view (Joint Identification Number; J.I.D.#XXX),
viewports labeled were replaced with SS blanks machined to defined standards (mm); (P) 6.35 NPT (J.I.D.#016), (Q) 25.4 NPT (J.I.D.#017), (R) 25.4 NPT
(J.I.D.#018), (S) 3.175 NPT (J.I.D.#019), (T) 3.175 NPT (J.I.D.#020), (U) 3.175 NPT (J.I.D. #021), (V) 25.4 NPT (J.I.D.#022), (W) 25.4 NPT (J.I.D.#023), (X) 25.4
NPT (J.I.D.#024), and (Y) 25.4 NPT (J.I.D.#025).
2520
AA
CC
BB
NOTE: OVERPRESSURIZATION
RELIEF
Figure 10: MTC-4 Aft View Rear Flange.

The figure shows the aft flange. The aft flange was machined to required specification (Appendix A) at NEDU. Aft SS flange; Z, AA, BB, and CC (mm) 31.75 NPTs
(J.I.D.#s 26 to 29; respectively). Figure 2 shows rear flange following installation of 31.75 NPT. An independent chassis ground is connected to the aft MTC-4
mounting leg. A 3.792 MPa over-pressurization relief is installed on the compression line. Penetrators are mounted on the vacuum side for clearance. Penetrators
were He checked bi-directional for integrity. Seals were tested using Snoop Liquid Leak Detector (Swagelok; PN# MS-SNOOP-8OZ).
Figure 11: MTC-4 External Electrical Panel.

The figure shows the external electrical panel MTC-4 (Q5). Front panel connections are designed to be modified as
experimental requirements define. Spare connections allow upgrading and expansion of electrical capabilities. Panel
is shown in an early configuration.
21
Fore Chamber Electrical Panel
Figure 12: MTC-4 Internal Fore Sled Electrical Panel.

The figure shows the fore electrical panel MTC-4 (Q5). Front panel connections are designed to be modified as
experimental requirements define. Spare (blanks) connections allow for upgrading and expansion of electrical
capabilities.
22
Aft
Fore
23
28
Aft Chamber Electrical Panel
Figure 13: MTC-4 Internal Aft Sled Electrical Panel.

The figure shows the aft internal electrical panel MTC-4 (Q5). Rear panel connections are designed to be modified as experimental requirements define. Spare
connections allow for upgrading and expansion of electrical capabilities. Arrow shows direction of system mounting in MTC-4. System is mounted on chamber
mock-up.
Table One: MTC-4 Electrical Penetrations and Drawings

MATERIALS:
N/A
Exterior Panel (293511-3; Q-5)
Function
1
2
3
4
5
6
7
8
9
10
11
12
13
14
20
Microscope Stage Info

X Axis Microscope Stage
Y Axis Microscope Stage
Micro Manipulator
Micro Manipulator
Micro Drive Lens
Micro Drive Lens
Inline Heater
Bath Heater
Camera Power
LED Light
Filter Wheel
Spare
Spare
Intensity Polarizer
21
Intensity Polarizer
22
23
24
Intensity Polarizer
Intensity Polarizer
Power Module (Multiple
Drawings)
25
Spare
END TABLE ONE
24
Appendix Q Drawing #
(Page)
293511-8 (Q-9)
293511-11 (Q-12)
293511-11 (Q-12)
293511-9 (Q-10)
293511-9 (Q-10)
293511-10 (Q-11)
293511-10 (Q-11)
293511-9 (Q-10)
293511-9 (Q-10)
293511-11 (Q-12)
293511-11 (Q-12)
293511-8 (Q-9)
293511-10 (Q-11)
293511-10 (Q-11)
293511-21 (Q-13),
293511-23 (Q-15),
295311-24 (Q-16)
293511-23 (Q-15),
295311-24 (Q-16)
293511-23 (Q-15)
293511-23 (Q-15)
293511-21 (Q-13),
293511-22 (Q-14),
293511-23 (Q-15),
293511-24 (Q-16)
293511-21 (Q-13)
MICROSCOPE DESIGN AND CONSTRUCTION

A fixed-stage upright Hoffman Modulation Contrast (HMC) microscope [110] with
video microscopy capability was constructed by Mr. James Arehart (Arehart Model
Maker, Dayton, OH; retired) for utilization in MTC-4. The design of the scope was based
upon the work of Jay Dean at the University of South Florida [84, 111] and Mr. James
Arehart. The system was significantly modified to meet ONR tasking. Scope assemblies
were manufactured from 6061 aluminum (UNS A96061). This aluminum alloy was
chosen due to it corrosion resistance. All components were black anodized unless
denoted in the respective Appendixes/ Tables. Technical drawings for the system are
provided in Appendix K and L. For a detailed review of optical microscopy terminology
and principles the authors recommend the review of Davidson and Abramowitz [112].
A master list of manufactured components (scope and main optical pathway) is
provided in Table 2. The system is built on a platform of three pre-tapped metric
Edmunds Optical Bread Boards (Edmund Optics; PN 56-935) modified for chamber
geometry. Machined sleds are fitted with mounting brackets and slide adaptors for final
installation on the chamber railing. The fore board includes assembly components
independent of the microscope design (e.g., micro-positioners, main stage, front sled
electrical connections, ancillary connectors, overview imaging system..), with the aft
board housing the microscope assembly, micro-dispensing pump (effluent solution),
electrical board/ground assembly, filter wheel, and system imaging. Secondary
equipment, such as non-microscope imaging systems, can be mounted on the fore or
aft assembly as required by experimental requirements. An overall view of the system
and its core components is shown in Figure 14.
The optical path of the microscope system for transmitted light, HMC, and
epifluorescence microscopy can be seen in Figure 15, K-69, and M-9. An infinity
corrected optical train was utilized for the design [113]. For Brightfield microscopy, the
light source is focused on the specimen by the condenser following being converted into
parallel waves by the focusing mirror distal to the light source. The light source
configuration is shown in K-62. Non-deviated and diffracted light (180 out of phase),
reach the front focal plane of the objective. The converging light waves summate and
result in destructive interference; i.e., an optical image. The infinity corrected Nikon
Instruments Inc. (Nikon) objectives projects the light source in parallel rays to the 35.5
by 36 mm Nikon Silver Mirror (21010 SIL MIR C106756; mounted in Nikon MXA22030
Blank Filter Cube ) mounted in the optical housing. Objectives utilized in the system
include; 1) 2X Plan Apo (NA 0.1; /-, WD 8.5), 20X HMC (P/N 200455) Fluor (NA
0.50; /0, WD 2.0), 3) 20X HMC (P/N 29635) ELWD Plan Fluor (NA 0.45; /0-2, WD
7.4), and 4) 40X HMC (P/N 200446) Fluor (NA 0.80; /0, WD 2.0). For a guide on the
principle of lens selection the authors recommend the publications of Drent [114],
Matsumoto [115], Abramowitz, M [116-118].
The silver mirror is highly sensitive to condensation during decompression and
requires weekly replacement during times of significant experimentation. Parallel light
waves are projected onto the front focal plane of a variable placement CF160 infinity
25
objective compatible tube lens unit (Nikon; MXA20696/70 g, tube focal length f/200 m),
distance to front focal plane of the reduction lens is set at 151.2 mm. Distance from
objective shoulder to CF160 front focal plane is variable from 100 to 200 mm. A 0.7X
DXM Reducing lens (17.5 mm) was utilized to match the image to the modified ChargeCouple Device (CCD) Camera. The placement was based upon the manufacture
recommended distance from the reducing lens rear face to the camera chip set. While
discussed latter, it is critical to note that the camera CCD has no protective covering.
The modification allows for compression and decompression without compromise of the
CCD.
Epifluorescence experiments utilized a similar light train with the exception of the
transmitted light source and condenser. The excitation frequency enters the microscope
as show in Figure 15, and is reflected downward towards the objective via a band-pass
filter. The objective acts as the condenser unit, focusing the excitation signal on the
sample based upon the focal plane. It should be noted that the signal is not limited to
the defined focal plane. The emission signal travels through the objective in a fashion
similar to a bright field image and is band-passed at the fluorescence filter. Filters are
defined based upon the excitation and emission frequency of the fluorophore. For a
detailed review the authors recommend the reviews of Drummen [119, 120] and Fischer
[121].
Hoffman Contrast optics allow visualization of neurons in pseudo-3-dimensional
(pseudo 3-D) relief. However, in retrospect we recommend future systems utilize a
phase-contrast inverted scope as the 3-D capabilities are not needed for electrode
placement. Hoffman modified Nikon [122] water immersion objectives were positioned
on a cantilever system. The configuration allowed a maximal of two objectives per
experiment. Objectives must be chosen to ensure stage clearance.
Objective positioning was accomplished using a National Aperture Industries
(NAI [123]) MM-3M-F-1.5-HT micro-mini motorized stage (S/N 61988; K-64 and K-65).
The motorized stage allows alignment of objectives with respect to the 45 silver coated
relay mirror. Course focusing (Z-axis) is accomplished using the stage. The high torque
MM-3M-F-1.5 HT has a travel range of 38 mm.
Fine positioning of objective lens in the Z-axis was accomplished utilizing a NAI
MM-4M-F-50-HT micro-mini motorized stage (S/N 15599; K-66 and K-67). The micromini stages where operated in conjunction with MC-5B controllers (S/N 1111 & 1112;
respectively) using NAI Software Version 900-0001_104.
A Prior Scientific [124] H101AF1 stage (S/N 68997), with manufactured stage
inserts, was utilized for specimen placement. The stage was utilized for pipette based
electrophysiology and epifluorescence experiments. Positioning of the H101AF1 stage
was accomplished using a Prior Scientific ProScan II controller (S/N 08832). While we
utilized the controller in the joy stick configuration, the system can be upgraded for
computer based control.
26
For a general review of alignment procedures and theory related to brightfield

and HMC the authors recommend the reviews of Friedman and Davidson [112, 125], in
addition to the works of Hoffman [126-128]. Alignment of the HMC system was
completed as per manufacture recommendation [129].
Centering of the field aperture in the optical pathway required removing the rear
camera adaptor and 0.7X DXM reducing lens. The system must be in mock-up
configuration to align as shown in Figure 14. Gross alignment cannot be accomplished
with the scope in the chamber. Following centering the field aperture is opened. The
condenser turret is moved to the appropriate split aperture for the HMC objectives.
Prior to installation of the objectives it is critical to mark the dark side of the
objective. This allows alignment with the slit aperture. The slit apertures are in spring
mounted housing in the turret, allowing for easy rotation and lateral movement. Do not
remove the spring housing. Extreme care should be used as oils and contaminates will
compromise the slit apertures and require reordering [130]. While observing through the
rear of the scope (i.e., the Nikon CF160 tube lens), reach under the condenser unit and
align the non-variable slit component with the modulators gray region (parallel
configuration) by turning the spring loaded slit aperture. Once parallel, move the slit
holder so that the non-variable portion of the slit image is registered within the gray
region of the modulator. At this point, rotate the polarizer assembly and the P2 section
of the slit will change from minimal to maximal contrast as you transition. Adjust the
polarizer for maximal contrast. Align the system for the secondary objective as required.
Replace rear 0.7X DXM reducing lens and camera adaptor. The system is aligned for
the HMC configuration.
Prior to alignment verify the condenser working distance (WD). The condenser
unit defined above has a 2.0 mm WD. Verify lens with a variable objective correction
collar are set to the appropriate thickness for the selected sample holder. Visualize
samples and modify slit aperture as needed to optimize.
Carefully move system to the chamber and utilize the CCD camera to fine adjust
slit aperture to compensate for movement during placement in chamber. This task is
very challenging; great care and experience is required to successfully transfer the
aligned system to the chamber without modifying the objective placement and the slit
aperture in the condenser turret. Maximal resolution requires significant attention to
alignment. The majority of electrophysiology experiments do not require this level of
resolution.
To define assembly, the system is separated into the upper optical and lower
optical trains (i.e., the optics (upper) and the light source/condenser turret (lower)).
Figures 14 and 15 show the horizontal upper optical unit (Item A) and the vertical upper
optical unit (Item B) in relationship to the overall microscope design. While assembly
can begin with either unit, discussion will begin with the horizontal upper optical unit.
27
Figure 15 (Item C) shows the placement of the objective in relationship to the

two optical units as previously defined. The objective focus (Z-axis) is driven by the NAI
MM-4M-F-50-HT micro-mini motorized stage. High torque (HT) motors were utilized. An
overview of the upper optical train is provided in K-62. Figure 16 shows the MM-3M-F1.5-HT micro-mini motorized stage in relationship to the Figure 15 Item C. The
horizontal upper optical unit houses the 45 silver coated relay mirror, CF160 tube lens,
and 0.7X DXM reducing lens. The relay mirror may be blown clean with dry compressed
gases such as air, N2, or He. Care must be taken to not allow condensation from the
compressed air cans to interact with the silver mirror. Condensation will damage the
silver coated relay mirror. Hyperbaric conditions required frequent mirror changes.
Figure 18 shows the tube lens holder with the CF160 tube lens removed. The
lens must be configured towards the distal end of the optical path within 151.2 mm of
the reducing lens for optimal optical performance. The rack (K-18) is mounted to the
tube lens holder (K-28). The lens is stabilized with manufactured 3.5 mm padded
locking screws.
The assembly is placed in the horizontal upper optical unit (K-51) as shown in
Figure 19 and 20. Ensure the rack is facing the bottom of the scope. Mounted on the
horizontal upper optical unit is the pinion shaft housing for integration into the CF160
rack mounting. The unit consists of the housing (K-17), gear (K-19), and shaft (K-20).
Items are lubricated with MZ-2 Extra Fine Graphite Dry Lubricant (AGS). The rack and
pinion interface was lubricated with 111 Silicone Grease (Dow Corning).
The 45 silver coated relay mirror holder (K-27) is mounted at the distal end of
the 45 silver coated relay mirror with SS 3 mm screws. The mirror allows the collimated
image to be transferred to the CF160 tube lens. Relay mirror, tube lens assembly, and
horizontal upper optical unit geometry can be seen in Figures 19 to 22.
Figure 22 shows installation of the upper (K-34) and lower (K-35) mounting
angles (3.97 mm hex head; 6.35 mm). This allows integration with the vertical upper
optical unit. Leave sufficient slack to allow for alignment prior to tightening. Figure 23
shows the final installation of the vertical upper unit with the fluorescence system
adaptor installed.
The complete vertical upper optical unit is shown in Figures 23 to 29. Figure 24
shows the fluorescence optical cube in alignment with florescence side port. Using 4/40
SS screws, the optical cube holder (K-25) is installed in the vertical upper optical unit
(K-50). The cube set screw must be placed in alignment with the optical cover plate (K23) and secured with 2.78 mm hex keys. The fluorescence cube is secured in alignment
with 3.5 mm screws. Note that upper and lower mounting angles may be installed on
either unit prior to assembly as seen in Figures 25 to 29. The completed assembly can
be seen in Figure 30. During fluorescence-based experiments external lighting must be
minimized.
28
The objective manipulator system is utilized to place the microscope objectives in

alignment with the completed upper optical train (Figure 31). Note that the optical train
is installed on the rear mounting platform. For clarity, we will discuss its assembly prior
to discussing the respective front and rear sleds.
Objective placement in the X-, Y-, and Z-axis are controlled by the manipulators
(Figure 32). Manipulation in the Z-axis is for focusing and sample placement, while
manipulation in the X- and Y-axis is for optical alignment. All manipulators were verified
to work under hyperbaric conditions.
As seen in Figure 33, the adaptor plate was required for the integration of the
NAI MM-4M-F-50-HT micro-mini motorized stage 50 mm carrier back face (K-67; Figure
34) to the course Z-axis manipulator. The adaptor (K-7) is shown installed in Figure 35
using multiple 2.78 mm hex keys. Figure 36 shows the integration of the NAI MM-4M-F50-HT micro-mini motorized stage 50 mm back face to the manipulator adaptor unit;
2.78 mm hex keys.
As shown in Figure 31, the lens selection unit is integrated into the motorized Zaxis control. In order to allow for integration of the objectives, a mounting bracket was
designed to allow for mounting with the NAI MM-4M-F-50-HT micro-mini motorized
stage 50 mm carrier stage to the objective main mount (K-9) as seen in Figure 37. The
objective mount (K-29) slides into the beveled adaptor unit as shown in Figure 38. This
allows for motorized transition. Resistance between manufactured units is minimal.
Resistance between the objective mount holder and the objective main mount was
controlled using manufactured 3.505 mm Teflon tip set screws. For experiments using
a single objective, Teflon tipped set screws can be used to lock the lens holder mount
in proper alignment. Alignment of the units is shown in Figures 39 to 42.
The MM-3M-F-1.5-HT (back face) is mounted (1.19 mm) to the underside
(beveled face of the adaptor unit) of the adaptor unit using an auxiliary mounting plate
(K-30). Screws (2.5 mm) secure the unit to the adaptor unit.
The manipulator arm (K31) is attached (1.57 mm) to the MM-3M-F-1.5-HT slider
mounting surface (K-65) via the manipulator spacer (K-31). Once assembled, the
manipulator adaptor (K-21) is installed to the angled end of the beveled adaptor as
shown in Figure 42 and 43 with 4 mm (12.7 mm) hex keys. The unit is attached to the Zaxis of the course manipulator (2.52 mm).
The objective manipulator system is attached to manipulator mounting plate (K-8)
using 9.525 mm hex keys (12.7 mm). Figure 44 shows the manipulator mounting prior
to installation. A wire mounting system is utilized to minimize stress on the ribbon cables
for control of the MM-3M-F-1.5-HT and NAI MM-4M-F-50-HT motorized stages. The
manufactured ribbon cable clamp and electrical wire bundle mount clamp post can be
found in Q-22 and Q-26; respectively.
29
The completed manipulator mounting plate, manipulator system, and wiring

management system is shown in Figures 45 and 46. Figure 47 shows installation of a
Nikon objective. Ensure objectives are compatible with the threads and diameter of the
objective mount. The system was designed on Nikon threading. The MM-3M-F-1.5-HT
ribbon cable is secured to the upper face of the beveled adaptor (K-9) with a flat clamp
(Q-22; 4.76 mm). This completes the assembly of the objective mounting components
of the optical train.
System objective control and assembled upper vertical/horizontal optical
assembly are integrated into the aft sled (Edmund Optics; PN 56-935). As previously
addressed, the sleds are broken into two units (fore and aft). The fore sled allows for
placement of the sample and experimental manipulation. Figure 15 shows the final
configuration of the sled assemblies. Note that the aft sled is made of two sleds.
In Figure 48, the aft sleds are in isolation. The rear aft sled has not been cut to
final length. Placement of the manipulator mounting plate is shown. As seen in the
figure, the manipulator mounting plate is mounted on a riser pad (K-43) allowing for
proper height. This configuration also optimizes travel in the motorized and manual
manipulators. The design of the fore sled, aft front sled, and aft rear sled are shown in
K-2 to K-4; respectively.
Integration of the aft front and rear sleds required manufactured connector links
(K-6) secured with 6.35 mm hex keys. The aft sleds are mounted to the chamber railing
(A-242 to A-244) as shown in Figure 15. Ultra-high-molecular-weight polyethylene
(UHMWPE or UHMW) manufactured under-carriage risers (K-26) were utilized. This
material has a low coefficient of friction, is pressure insensitive ( 3.457 MPa), is
relatively moisture insensitive, and acts to provide vibration dampening. A similar
approach was utilized for the fore sled. Some figures do not include the risers for ease
of placement during documentation. All experimental procedures required the
installation of the risers.
Figure 49 shows the relationship between the objective manipulator system and
the upper optical train mounting block (K-45; 6.35 mm). The upper optical train
mounting assembly consists of a series of brackets as seen in Figure 50. This allows
the upper optical train to be mounted on the aft sled assembly.
In order of assembly, the unit consists of the; (A) upper optical train mounting
block (K-45), (B) vertical microscope support arm (K-39), (C) horizontal microscope
support arm (K-40), and (D) upper optical train integration support arm (K-41). All are
mounted with 6.35 mm hex keys. The upper optical train is attached to the assembly
using hex keys (4.851 mm). It is critical to note the position of the mounted optical train
and the manipulator assembly.
The assembly should be centered on the fore sled opening for the condenser/
transmitted light assembly. Figure 51 shows a non-optimum alignment. The objectives
and the upper optical train are slightly out of plane. Image quality and field of view are
30
the defining criteria. Significant time and effort must be invested in understanding the
system for a proper alignment.
In the next section we will discuss the assembly of the condenser/ transmitted
light assembly. This will be defined as the lower optical train assembly.
31
Fore
B
36
32
Aft
C
Figure 14: MTC-4 Port Microscope Components.

The figure shows the MTC-4 Hyperbaric Epifluorescence Upright Hoffman Contrast Microscope Assembly optical and stage components. System is shown in the
mock chamber geometry configuration. The A) Upper Horizontal Optic Train, B) Upper Vertical Optical Train, C) Fore Sled Assembly, D) Aft Front Sled Assembly,
and E) Aft Rear Sled Assembly are defined. Fore manipulator stands are installed; manipulators are not shown. Stand placement varies on experimental
requirements. Mounting system consists of SS 50.8 mm posts (Edmund Optics; EO) PN 58-952 with EO PN 53-357 right angle post clamps. The mock chamber
geometry configuration is the recommended storage and travel configuration.
D
3733
Figure 15: MTC-4 Starboard Main Components.

The figure shows the MTC-4 Hyperbaric Epifluorescence Upright Hoffman Contrast Microscope assembly optical and stage components. System is shown in the
mock chamber geometry for developmental and equipment placement purposes. The A) Upper Horizontal Optical Train, B) Upper Vertical Optical Train, C)
Condenser Manipulator Assembly, and D) Rear Electrical Panel (Q-4) placement are defined. Electrical considerations are discussed in relevant sections. The
connector wiring is defined in Appendix Q. Model MS314 Marzhauser Wetzlar [131] bi-directional (X/Y/Z- axes) controllers (WPI; SN 95533 O3J & 91009 O6H) are
shown installed to Edmund Optics mounting accessories. Motorized micromanipulators are shown.
B
C
3438
Figure 16: MTC-4 Upper Optical Pathways.

The figure shows the upper optical train in final configuration. Vertical and horizontal units assemblies are shown. The A) excitation pathway for fluorescence
source (90) to sample, B) emission pathway from sample via band-pass filter to rear filter (See K-62 for basic optical path; fluorescence filter distal to objective
and proximal to 45 silver mirror), and C) transmitted light pathway (LED) are defined.
Set Screw Head

Stage
Set Screw to Define

Fine Control
Placement
3935
Figure 17: MTC-4 Objectives and Head Stage Main Components.
The figure shows the MTC-4 objectives mounting installation in relationship to completed upper optical assembly. The pipette holder and Axon 200B head-stage
are shown. The head-stage is secured using Teflon tipped set screws.
4036
Figure 18: MTC-4 Objectives.
The figure shows the mounted objectives in relationship to objective manipulator system/upper optical assembly. The configuration allows for clearance following
sample placement. All objectives are vented to allow for pressurization/depressurization. System is shown in non-anodized state to allow for fitting of assembly
prior to finalization of design. Note that the distal objective face is open to contaminants. Attention must be given to daily maintenance.
Optical Pathway Horizontal
41
37
Fore
A
Aft (Camera
Side
Figure 19: MTC-4 Tube Len Assemblies.

The figure shows tube lens assemblies. The tube lens assemblies include; A) Drive rack, B) Relay lens mount, and C) CF160 tube lens. CF160 tube lens must be
mounted towards the Nikon 0.7x DXM reducing lens.
Bottom
42
38
Fore
Aft (CCD Side)

Optical Pathway Horizontal
Top
Figure 20: MTC-4 Upper Horizontal Optical Assemblies.

The figure shows the completed upper horizontal (K-51) optical assembly for MTC-4. The 45 silver mirror and mounted CF160 tube lens are shown installed in the
respective sub-assemblies. Rear reducing lens/camera mount assembly has been removed for ease of viewing. Arrow defines optical pathway to CCD. Top and
bottom are in reference to installation.
Fore
Aft (CCD Side)
4239
Optical Pathway
Horizontal
Figure 21: MTC-4 Upper Horizontal Optical Assemblies View Two.

respective sub-assemblies. The rear reducing lens and the camera mount assemblies have been removed for ease of viewing. Arrow defines optical path to CCD.
4240
Optical Pathway
Horizontal
Figure 22: MTC-4 Upper Horizontal Optical Assemblies View Three.

The figure shows the completed upper horizontal (K-51) optical assemblies for MTC-4. The 45 silver mirror and mounted CF160 tube lens are shown installed in
the respective sub-assemblies. Arrow defines optical path to CCD.
42
41
Optical Pathway
Horizontal
Figure 23: MTC-4 Upper Horizontal Optical Assemblies View Four.

respective sub-assemblies. Rear reducing lens and the camera mount assemblies are removed for ease of viewing. Arrow defines optical path to CCD.
Figure 24: MTC-4 Upper Horizontal and Vertical Optical Assemblies and Fluorescence Adaptor.
The figure shows completed optical assemblies (K-50 and K-51) to include the fluorescence mount adaptor. Upper
optical mounting hex keys are left loose for alignment. Arrow defines optical pathway for transmitter light and
fluorescence excitation frequency to CCD.
42
To Camera
Emission
Path
Excitation Path
To Sample
Figure 25: MTC-4 Upper Vertical Optical Train and fluorescence Adaptor.
The figure shows the adaptor (A) that allows for integration of the SPECTRA X light engine quartz multimodal fiber
optic cable (Lumencor P/N 10-10618 [132]) to the upper vertical housing (K-50). Emission and excitation pathways
are shown in reference to sample and camera locations (90); dashed line = emission and solid line = excitation. The
fluorescence cube acts as a band-pass filter. Filters were purchased from Chroma Technology [133] and mounted in
the Nikon filter cube.
43
4844
Beveled
(Filter Cube
Adaptor)
Upper Angle
Connector
Figure 26: MTC-4 Upper Vertical Optical Cube Placement of Filter Cube Mount View One.
The figure shows the installed filter cube adaptor. The filter cube adaptor (inside upper vertical housing; K-50) is beveled to allow for placement of cube in the
proper alignment. A Teflon tipped mounting screw is utilized to secure the filter cube in final alignment. The upper angle connector is shown installed .
Upper Angle
Connector
Fluorescence Adaptor
45
49
Figure 27: MTC-4 Upper Vertical Optical Cube Placement of fluorescence Adaptor View Two.
The figure shows the upper vertical optical assembly with the fluorescence adaptor mount installed. Upper angle connector is shown from port view.
50
46
Figure 28: MTC-4 Upper Vertical Optical Cube Placement of Filter Cube Mount View Three.
The figure shows the installed filter cube beveled mount (inside upper vertical housing; K50). A Teflon tipped mounting screw is utilized to secure the filter cube
in the final alignment. The fluorescence cube mounting adjustment viewport (K-23) is removed (cover plate not shown).
Figure 29: MTC-4 Upper Vertical Optical Assembly Fore View.

The assembly shows the final configuration with fluorescence cube adjustment port removed. The Teflon tipped
adjustment screw for the filter cube is shown installed.
47
5248
CCD Side (Aft)
Figure 30: MTC-4 Upper Vertical and Horizontal Assembly.

The figure shows the completed assembly with fluorescence adaptor removed. The CCD camera adaptor mount for the 0.7x DXM reducing lens is installed on the
aft side.
Optical
Z-Axis Movement
53
49
Condenser Movement
X-, Y-, and Z-Axis
Movement
Figure 31: MTC-4 Objective Manipulator Assemblies.

The figure shows the motorized Z-axis manipulator (NAI MM-4M-F-50-HT) assembly in relationship to the condenser gross manual manipulator (X-, Y-, and Z-axis;
EO PN 03-607; non-motorized manipulator). The system is shown in a non-anodized state. Ribbon cable is in the storage configuration.
Z-Axis
Y-Axis
X-Axis
Figure 32: MTC-4 Objective Manipulator View One.

The figure shows the 50 mm Diameter, X-Y-Z Positioning Movement (EO PN 03-607) with adaptor plate accessory
(EO PN 54-491).
50
Adaptor
Plate
55
5551
Figure 1: MTC-4 Course Objective Manipulator View Two.

The figure shows the course objective manipulator. The 50 mm Diameter X-Y-Z Positioning Movement (EO; PN 03-607) is shown with the adaptor plate installed
(EO; PN 54-491). Manipulators are centered prior to installation to maximize travel.
56
5252
Carrier Unit
Figure 2: MTC-4 NAI MM-4M-F-50-HT Z- Axis Manipulator Unit.

The figure shows the carrier unit installed on the NAI MM-4M-F-50-HT for mounting on the X-Y-Z Positioning Movement (EO; PN 03-607) with the adaptor plate
(EO; PN 54-491); K-66 and K-67.
Figure 3: MTC-4 NAI MM-4 M-F-50-HT Z- Axis Unit Manipulator Mounting Adaptors.
The figure shows the X-Y-Z Positioning Movement (EO PN 03-607) adaptor plate (EO PN 54-491) installed on the
NAI MM-4M-F-50-HT. Final placement of the adaptor plate is dependent on alignment requirements.
53
Figure 4: MTC-4 NAI MM-4M-F-50-HT Z- Axis Unit Manipulator Mounting.

The figure shows the NAI MM-4M-F-50-HT mounted to X-, Y-, and Z-axis manipulator.
54
55
59
55
Figure 5: MTC-4 Objective Mount (Upper View).

The figure shows the mount (K-9) adaptor for installation of the objectives holder (K-29).
Objective Lock Screws/ Balance Adjustments

(Teflon Tipped)
Drag/Balance Adjustments
Figure 6: MTC-4 Lens Mount (Lower View).

The figure shows the mount (K-9) adaptor for installation of the objectives holder (K-29). The objective and imaging
port must align for optimal performance. Units are shown in a non-aligned configuration. Set screws for varying drag
and balance (Z-axis) on the MM-3M-F-1.5-HT motor. Teflon tipped lock screws are used to secure the objectives
when in single lens configuration or may be used for adjusting balance or drag (X-axis).
56
Screw Link
Figure 7: MTC-4 Objective Units (Lower View).

The figure shows the objective mounting components in the proper alignment. The manipulator arm (K-31) is
attached to the mounted (adaptor; K-30) MM-3M-F-1.5-HT motor. A 3.5718 mm screw links the lens adaptor to the
manipulator arm. Optimal length for the screw link is 25.4 mm. Washers allow for final gross alignment.
57
Figure 8: MTC-4 Objective Unit (Lower Unit) View Two.

The figure shows the objectives mounting components in the proper alignment.
58
Figure 9: MTC-4 Objective Unit View Three (Bottom).

The figure shows the objectives mounting components in the proper alignment.
59
Adaptor Plate
64
60
60
Figure 10: MTC-4 Objectives Unit and Adaptor Plate View One.
The figure shows the installed adaptor plate for mounting of the NAI MM-4M-F-50-HT Z- axis carrier unit to angled end of the lens mount adaptor. Ribbon cables
are extremely sensitive to compromise. Placement of ribbon cables is critical to avoid damage. Standard ribbon cable placement is shown.
Adaptor Plate
65
6161
Figure 11: MTC-4 Objective Units and Adaptor Plate View Two.
The figure shows the adaptor plate for mounting the NAI MM-4M-F-50-HT Z- axis unit carrier to the angled end of the lens mount adaptor.
62
62
66
Figure 12: MTC-4 Objective Manipulator Primary Mounting Arm.
The figure shows the primary mounting arm (K-8) for the objective manipulator and cable management systems. Cable placement varies depending on
experimental requirements. Risers and mounts are dependent on experimental requirements.
Figure 13: MTC-4 Objective Manipulator Arm and Objectives Control System View One.
The figure shows the final configuration of the objective control manipulators (manual and motorized) in relationship
to the mounting arm. Significant caution should be taken as the assembly is unbalanced. Ribbon cables are not
mounted for ease of viewing. The mounting arm installation points are slotted for variable placement.
63
Figure 14: MTC-4 Objective Manipulator Arm and Objective Control System View Two.
to the mounting arm. Ribbon cables are not in mounting configuration for ease of viewing. Ribbon mounting clip is
visible on objective mount. Objective mount ribbon cable mounting is standard.
64
Marking Made for

HMC Alignment
Figure 15: MTC-4 Objective Manipulator Arm, Objective Control System, and Objective Unit.
to the mounting arm. A 20X HMC Modified Nikon Lens is shown installed. Marking the position of the slit aperture
on the outside of the lens is for the alignment of the HMC slit aperture. Extreme caution should be taken, lens are
susceptible to damage. Lens should only be cleaned with vendor approved solutions. Objective mount ribbon cable is
shown secure with manufactured holder.
65
66
66
70
B
C
Figure 16: MTC-4 Objective Mounting Arm Mounting.

The figure shows the objective mounting arm mounted to the mounting plate. This allows proper alignment with the upper optical train and appropriate distance to
the CF160 Tube Lens. Components include (plate); A) Aft rear, B) Aft front, C) Fore (K-2), D) Mounting (K-43), and E) Objective mounting (K-8). Bread board
assemblies were not anodized during design phase. The railing riser unit is not manufactured to final configuration. Aft units are shown connected with sled
connector links (K-6).
Placement is in Relationship to the Fore

Sled Transmitted Light Source Opening
67
71
67
Figure 17: MTC-4 Initial Fitting and Placement of Objective Manipulator Assembly.
The figure shows the placement of objective manipulator system. Marking line on fore sled denotes area to be removed.
System Prior to Being

Anodized
68
68
72
B
A
E
Figure 18: Mobile Test Chamber 4 (MTC-4) Upper Optical Train Mounting Systems.
The figure shows the installation of upper optical train in reference to objective manipulator system. Components shown are; A) Microscope mounting block (K-45),
B) Microscope support arm long (K-39), C) Microscope support arm short (K-40), and D) Microscope support arm (K-41). The condenser manipulator mounting
plate has variable placement. Final version aft mounting screw slots are machined for maximal movement. The placement of the microscope mounting block is
defined in K-10.
69
73
69
Figure 19: MTC-4 Final Assemblies Microscope Objective System Aft View.
Table Two: MTC-4 Scope Assemblies Technical Drawings

- Appendices K, L and P.
MATERIALS:
Edmond Optics Metric Bread Bench Plates Machined to Front and Aft Sled
Requirements (www.edmondoptics.com)
6061 (6061 Aluminum (Alum)/ Black Anodized after Final Design (Black
Anodized; Anod)
Stainless Steel (SS)
Ultra-High-Molecular Weight Polyethylene (UHMW)
Brass
Component
Front (Fore) Sled
Technical Drawing #
283505-1
Appendix Page
K-2
Rear Sled (Aft) Fore
283505-2
K-3
Rear Sled (Aft) Aft
283505-3
K-4
Positioner Standoffs
283505-4
K-5
Sled Connector Link 283505-5
K-6
283505-6
K-7
283505-7
K-8
283505-8
K-9
283505-9
K-10
283505-10
K-11
283505-11
K-12
283505-12
K-13
283505-13
K-14
283505-14
K-15
283505-15
K-16
NA MM-4M-EF-50
Mount
Mounting Arm Z
Axis Manipulator
Optics
Lens Primary Mount
Arm
Locator Microscope
Mount Sled
(283505-2)
Filter Wheel/Scope
Adaptor
Prior Stage Rear
Supports
Locating Spacer for
283505-1/283505-2
Stage Locating
Blank for 283505-1
Sled Edge Mount
Secure
Sled Clamp
CONTINUED:
70
Info
12.7 mm Bench
Plate
12.7 mm Bench
Plate
12.7 mm Bench
Plate
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
UHMW
Table Two: MTC-4 Scope Assemblies Technical Drawings Continued.

MATERIALS LEGEND:
Requirements
6061 Aluminum (Alum)/ Black Anodized after Final Design (Black Anodized;
Anod)
Brass
Component
Drive Rack GRB
Technical Drawing #
283505-17
Appendix Page
K-18
Pinion Gear
Pinion Shaft
Holder Lens Arm
283505-18
283505-19
283505-20
K-19
K-20
K-21
Rear Mounting
Plate CCD Camera
Cover Plate Lower
Optical Path
Main Scope
Manipulator Pad
Cube Lens Holder
Lower Optical Path
(Flour)
Cube Lens Holder
Upper Optical Path
(Silver Mirror)
Riser Block Front
and Rear Sleds
Lens Mount MXA
20696
Lens Mount
Objective Lens
Mounting Plate MM3M-F-65HT
Manipulator Arm
283505-21
K-22
283505-22
K-23
283505-23
K-24
283505-24
K-25
283505-26
K-27
6.35 mm 6061
Alum/ Black Anod
283505-25A
K-26
283505-27
K-28
283505-28
K-29
283505-29
K-30
283505-30
K-31
Rail Clamps Rear

Sleds
Riser Pad Scope
283505-31
K-32
6.35 mm 6061
Alum/ Black Anod
6061 Alum/ Black
Anod
6.35 mm 6061
Alum/ Black Anod
3.175 mm 6061
Alum/ Black Anod
6061 Alum/ Black
Anod
UHMW
283505-32
K-33
CONTINUED:
71
Info
GRB-32/20-12
Small Parts Inc.
Brass
SS
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
6.35 mm 6061
Alum/ Black Anod
7.93 mm 6061
Alum/ Black Anod

MATERIALS LEGEND:
Requirements
Anod)
Brass
Component
Magnetic Mounting
Plate (Optional)
Microscope Main
Support Arm
Microscope Main
Support Arm 2
Microscope Support
Arm
Filler Bar
Microscope Support
Arm
Extension Arm
Riser
Manipulator
Condenser Arm
Mounting Plate
Microscope Main
Mounting Block
Condenser Lens
Adjusting Arm
Condenser Lens
Mount Main Mount
Condenser Lens
Mount
Condenser Lens
Mount
Vertical (Lower)
Scope Section Main
Horizontal (Upper)
Scope Section Main
Lens Holder Side
Mounting Block
Technical Drawing #
283505-37
Appendix Page
K-38
Info
9.525 mm 1018 Steel
283505-38
K-39
283505-39
K-40
283505-40
K-41
283505-41
K-42
12.7 mm 6061 Alum/

Black Anod
12.7 mm 6061 Alum/
Black Anod
12.7 mm 6061 Alum/
Black Anod
6.35 mm 6061 Alum/
Black Anod
283505-42
K-43
283505-43
K-44
283505-44
K-45
283505-45
K-46
283505-46
K-47
283505-47
K-48
283505-48
K-49
283505-49
K-50
19.05 mm 6061
Alum/ Black Anod
9.525 mm 6061
Alum/ Black Anod
9.525 mm 6061
Alum/ Black Anod
9.525 mm 6061
Alum/ Black Anod
6.35 mm 6061 Alum/
Black Anod
Multiple; drawings
283505-50
K-51
Multiple; drawings
123550-2
L-3
25.4 mm 6061 Alum/

Black Anod
CONTINUED:
72
6.35 mm Bench
Plate/ Black Anod
6.35 mm 6061 Alum/
Black Anod

MATERIALS LEGEND:
Requirements
Anod)
Brass
Component
Inner Slide Block
Technical Drawing #
123550-4
Appendix Page
L-5
Outer Slide Block
123550-5
L-6
Mounting Base
Plate Right/Left
Motor Holder
123550-6
L-7
123550-7
L-8
Motor Stop Sleeve
123550-8
L-9
Gear Collar
Polarizing Gear
Lens Holder
45 mm Lens Holder
Notes:
123550-9
123550-10
L-10
L-11
Info
6.35 mm 6061 Alum/
Black Anod
6.35 mm 6061 Alum/
Black Anod
6.35 mm 6061 Alum/
Black Anod
6061 Alum/ Black
Anod
6061 Alum/ Black
Anod
Brass
UHMW
123550-11
L-12
6061 Alum
CONTINUED:
73

Sample Specific Components.
- Materials are Clear Acrylic and 6061 Aluminum as required
Component
Technical Drawing Appendix Page
#
283505-52
K-52
Microscope
Specimen Holder
Standard Square
283505-53
K-53
Sample Slide
Clamps
283505-54
K-54
Sample Slide
Heater Clamps
123565-01
K-55
Microscope
Specimen Holder
Standard Circle
283505-51
P-21
Microscope
Specimen Holder
Non Heated
Notes:
END TABLE TWO.

74
Info
6.35 mm Acrylic
3.175 mm Acrylic
3.175 mm Alum
6.35 mm Acrylic
6.35 mm Acrylic
TRANSMITTED LIGHT SOURCE

Transmitted light was provided with a Light Emitting Device (LED; Switronix Torch LED
TL-50 [134]). Upgrading of the LED light source is recommended with future systems.
The condenser unit was mounted to a manual manipulator arm on the aft front sled. The
LED assembly was mounted on the fore sled assembly in alignment with the condenser
unit. This unit is in alignment with the upper optical train as shown in Figure 52. The unit
shall be defined as the lower optical train.
Final placement of the transmitted light source is adjustable to bring the source in
alignment with the upper optical train. The 45 mm polarization lens was housed in a
motorized assembly. A multi-directional externally mounted control unit (Q-14 to Q-18)
was utilized for control.
Gear reduction was accomplished using post-production machined Sterling
Instruments (Division of Designatronics Inc., NY [135]) hubless gears; S1268Z-032S019
(motor) and S1268Z-032S072 (45 mm polarizing lens).Set screws are utilized to
minimize slippage/drag during gear movement. The S1268Z-032SO19/S1268Z032S072 interface was lubricated with MZ-2 Extra Fine Graphite Dry Lubricant (AGS
[136]). Dry lubrication was utilized to minimize compromise of the optical components.
Technical drawings for the transmitted light/polarization assembly are included in
Appendix L (L-2 to L-11). A system quote is provided as a reference for sources (H-13).
It should be noted that it is critical to properly align the lower light source with the upper
optical train. Failure will result in uneven illumination of the sample and limited image
quality.
Assembly of the lower optical train begins with the condenser manipulator
assembly and installation of the condenser unit. Figure 53 defines the placement of the
condenser manipulator assembly with respect to the upper optical train and objective
mounts. The manipulator is mounted to the manipulator mounting plate (K-44) using
6.35mm hex keys. Components of the assembly include the; A) Manual manipulator, B)
Condenser main manipulator arm assembly (condenser lens adjustment arm (K-46) and
condenser lens mount (K47)), C) Condenser lens mount bracket (K-48), and D)
Condenser lens mount (K-49).
The HMC condenser (SN 041354) is shown in Figure 54. The condenser unit
integrates into the condenser manipulator assembly via the condenser lens mount.
Final assembly is shown in Figure 55. Hex keys (5.556 mm) are utilized to secure the
condenser lens mount to the manipulator.
The lower section of the condenser lens adjustment arm attaches to the
condenser lens mount bracket using a 4.762 mm hex key. The condenser lens mount
attaches to the mount bracket using 3.175 mm hex keys. Significant play is included in
the condenser unit mounting to allow alignment within the center point of the
75
manipulator range of motion. The mounting point to the rear aft sled can be modified to
vary placement.
The condenser unit is attached to the condenser lens mount using 3.175 mm
screws and nuts. The condenser unit can be placed in a fore or aft position with respect
to the condenser lens mount. Completed condenser unit in the fore position is shown in
Figure 55. The aft configuration is the recommended set-up for electrophysiology
experimentation.
It is critical to verify the rough condenser alignment with the upper optical unit. A
plumb-line mounted to an adaptor was utilized for this task. A mounted laser pointer
was also utilized. Figure 56 shows the slit apertures critical to the function of the HMC
system. When the system is not utilized, the HMC turret is rotated into a configuration
with no slit insert. With placement of the condenser unit and rough alignment of the
condenser completed, the polarized light source can be installed in relationship to the
condenser optics. The polarized light assembly is shown in Figures 57 to 59. Basic
optical concepts are further expanded in K-63.
The unit is mounted in alignment with the condenser unit to minimize the
distance between the circular opening in the condenser lens mount and the polarization
filter. Mounting base plates (L-7) are attached to the lower side of the fore sled
assembly using 6.35 mm hex keys. The fore sled assembly is shown in Figure 14 and
Figure 60 (K-2). Figure 57 shows the assembly. The outer slide block (L-6) is attached
to the mounting base plate using 2.26 mm hex keys. Travel is adjusted using the
various mounting screws.
To connect the assemblies to the lens holder, side extension plates (L-4) in
tandem with the lens holder side mounting block (L-3) are used (various hex keys as
defined in drawing). This allows the motor holder assembly (L-8) to be integrated into
the unit.
With the drive motor installed, the polarizing lens holder (L-2) and gear lens
holder (L-11 and Figure 59) are placed in final alignment. Modifications to the gear
collar limit slippage and the collars fitting to the motor assembly are defined are in
Figure 58 (L-10). The 45 mm lens holder is shown in L-12. Figure 60 defines the final
relationship of the condenser assembly and polarizer mount. Figure 61 shows the final
assembled unit as view from the underside of the fore sled unit.
.
76
Assembly Not In Alignment
77
79
77
Figure 20: MTC-4 Lower Optical Train Assemblies.

The figure shows the placement of the HMC condenser in relationship to the polarizer and light source assembly. The motor is for manipulation of HMC
polarization lens distal to the LED light source but proximal to the condenser turret unit. The condenser unit is not centered with the fore plate in the image. The
LED power source is not attached in the figure. Power source attached to the lower side of the LED assembly.
78
7880
Figure 21: MTC-4 Lower Optical Train Assemblies and Manipulator.

The figure shows the lower optical train manipulator mounted to the adaptor plate between the upper optical train mounting and objective assembly mounting arm.
The condenser manipulator mounting plate (K-44) has been modified to an open slot configuration. This allows for maximal travel and ease of placement. An
Edmonds Optics X-, Y-, and Z-axis manipulator was utilized. Manipulator control is manual. Recommend motorized manipulators in future systems.
Mounting Slots Are Elongated to Allow for

Varying Geometry Configurations
Figure 22: MTC-4 Lower Optical Train Assemblies.

The figure shows the lower optical train components; A) Manual manipulator (Edmund Optics), B) Condenser main
manipulator arm assembly (condenser lens adjustment arm (K-46) and condenser lens mount (K-47)), C) Condenser
lens mount bracket (K-48), and D) Condenser lens mount (K-48). All components have elongated mounting slots at
relevant connection points to optimize system flexibility.
79
Figure 23: MTC-4 Hoffman Modulation Contrast Condenser.

The figure shows the HMC condenser (SN 041354) with HMC slit apertures for 20x and 40x Hoffman modified
Nikon Lens. The condenser is shown in the HMC 40x position. Storage configuration is in blank position.
80
Multiple Screw Positions
Mounting Slots Are Elongated to Allow for

Varying Geometry Configurations
Figure 24: MTC-4 Condenser Manipulator Arm.

The figure shows the final assembly of the condenser manipulator adaptor arm. System components are designed
with significant travel at integration points. There are multiple mounting points to allow for optimal alignment.
81
Figure 25: MTC-4 Condenser Manipulator Arm and Condenser In Fore Position.
The figure shows the condenser turret mounted to the manipulator arm assembly. System is shown in fore mounting
configuration. Aft mounting is the normal configuration for electrophysiology experiments. Configuration was utilized
for imaging.
82
HMC Slit Apertures
83
85
83
Figure 26: MTC-4 HMC Condenser Slit Apertures.

The figure shows the HMC slit apertures as defined in text. Proper alignment with HMC modified objective/s is required. Standard bright field experiments can
be completed with turret turned to the no aperture position. The apertures are not in alignment with the condenser unit to allow for viewing.
E
F
D
84
86
84
B
A
G
C
Figure 27: MTC-4 HMC Polarizer Assembly View One.

The figure shows the HMC polarizer and its associated motorized housing unit; A ) Mounting base plate (L-7), B) Outer slide block (L-6), C) Outer slide block (L5), D) Lens holder extension plate (L-4), E) Lens holder side mounting block (L-3), F) Motor holder (L-6), and G) Polarizing lens holder (L-2).
Figure 28: MTC-4 HMC Polarizer Assembly View Two.

The figure shows a top down view of HMC polarizer assembly with gear collar and modifications visible. Set screws
are required to minimize slippage. High resistance is required to stabilize the polarizer lens. Operation of the polarizer
motor is required for final fit.
85
88
86
86
Figure 1: MTC-4 HMC Polarizer Assembly View Three.
The figure shows the relationship of the (A) motor housing (L-11) to the gear assembly and lens holder. The 45 mm lens holder and gearing is not shown (L-12).
87
HMC Condenser Assembly
HMC Polarizer Assembly
89
87
Figure 1: MTC-4 HMC Condenser and Polarizer Assemblies View Four.
The figure shows the relationship of the HMC condenser assembly and the polarizer assemblies. It is critical that the condenser assembly be mounted to the aft
fore sled while the polarizer unit is mounted to fore sled. Extreme caution is required when manipulating the Fore sled for experiments. Optics may be easily
damaged.
IMAGING
A modified QIClick (QImaging; Surry, British Colombia, CA; SN# Q31817 [137]) digital
charge-coupled device (CCD; 1392 x 1040), as shown in Figures 60 to 65, was utilized
for primary imaging. The camera CCD seal was modified with a windowless CCD. This
modification was completed following the compromise of the stock system on test dives
to 3.457 MPa. Compromise of camera resulted in a catastrophic failure of the imaging
system (Appendix S).
It is hypothesized that the transition rate (P) was exceeded and/or the transition
from a He environment to standard air at chamber opening created a gradient that
compromised the CCD window. The chamber did not exceed the planed 179.164 KPa
min-1 maximal compression rate; rate on system failure was 98.109 KPa min-1.
However, compromise may have been initiated prior to the failure and therefore
pressurization may have been a contributing factor. In summary, we were not able to
definitively define the cause of the fault.
It should be noted that no high resolution stock imaging systems are currently
rated to 3.457 MPa. Removal of the CCD window compromised camera cooling.
Windowless systems are not appropriate for low signal experimentation.
Development of a low signal hyperbaric camera would require integration of a pressure
equilibration system with the camera cooling. The modified QIClick is not a stock
camera. Special attention is required to minimize CCD contamination. Based on our
experience, the camera should be serviced every 2 months during sustained
operations.
Dark current did not significantly differ following modification of window (3.457
MPa maximal, step 101.32 MPa (10 min stabilization per step)). Binning of images is
not utilized. Images were saved as RAW files. A RAW file most accurately represents
the image as defined by the CCD. Compression rate was set to a conservative 48.263
KPa min-1.
The camera is threaded into the filter wheel assembly, or placed on adaptor
tubes, to set the CCD for maximal resolution and image capture. The camera mounting
plate (K-22) is shown at the distal end of the upper optical train. Installation of the filter
is accomplished via the microscope connector body and threaded adaptor (K-11). This
can be seen in Figure 62 to 65. The camera adaptor is mounted on the distal side of the
filter wheel. Varying length threaded adaptors were machined (K-61, and K-68 to K-71).
The camera may be mounted directly to the camera plate or to the filter wheel.
Mounting directly to the camera plate is accomplished by utilizing the 0.7x Nikon DXM
reducing lens adaptor (Figure 66). Figure 67 shows the alternative mounting.
General chamber imagining was accomplished utilizing a PixeLINK (S/N
7410003969 [138]) PL-B741E complementary metal oxide semiconductor (CMOS)
monochrome camera. An Edmunds Optics 16 mm lens (F 1.4) was utilized in
88
conjunction with the PL-B741E. The PixeLINK Capture OEM Software Suite was
utilized for imaging. PL-B741E files were stored as 16 Bit Tagged Image File Format
(TIFF; 0-65,535 gray scale) or Nikon Raw (NEF) files. Compression and
decompression had no significant effect on the camera. The camera is for general
imaging of the sample plate and is not appropriate for primary microscopy requirements.
PixeLINK and QIClick data crossed the primary pressure boundary via PAVE
FireWire and USB penetrators (US15L-SS-150-6-28-IEEE 1394 and US15L-SS-150-1USBAMALE-USBAFEMALE; respectively). USB adaptors were utilized for penetratorcomputer and penetrator-camera connections. Penetrators are installed in the
manufactured SS viewports (Appendix A, B, and F). Machining of the viewports was
completed at NEDU.
Figure 68 shows the front mounted PixeLINK in reference to a single Model
MS314 Marzhauser Wetzlar (Wetzlar, Germany) bi-directional (X/Y/Z- axes) controller
(WPI; SN 95533 O3J & 91009 O6H). In contrast, Figure 69 defines placement of the
PixeLINK when multiple manipulators are utilized. The lens was removed during
assembly. Mounting was limited to two items proximal to the sample sled. This allowed
for ease of access during experimental protocols.
89
Upper Optical
Train
90
90
92
Figure 2: MTC-4 Mounting of Camera View One.
The figure shows the camera mounted at the distal end of the upper horizontal optical train following the Prior Scientific HF108 Motorized Filter Wheel. System is
shown mounted on the chamber geometry mock-up. The camera shown is an earlier model camera suitable for operations at low pressure ( 304.058 KPa).
Mounting is similar to the modified camera.
Figure 3: Mobile Test Chamber 4 (MTC-4) Mounting of Camera View Two (Aft).
The figure shows the camera mounted at the distal end of the upper horizontal optical train. Note that Prior Scientific
HF108 Motorized Filter Wheel is proximal to the camera. Experiments are limited to a compression rate of 48.263
KPa min-1. Decompression rate is defined to minimize condensation and icing. The camera shown is an earlier model
camera suitable for operations at low pressure ( 304.058 KPa). Mounting is similar to the modified camera.
91
Tube
Adaptor
9292
94
Figure 4: MTC-4 Mounting Adaptor for the Modified QIClick.
The figure shows the tube adaptor with UHMW polyethylene set screw (set screw not shown) for mounting of the modified QIClick. The Nikon 0.7x DXM
reduction lens is mounted into the aft face of the filter wheel.
Tube Adaptor
Camera Mounted
Directly to Filter
Wheel
9393
95
Figure 5: MTC-4 Filter Wheel Adaptor Version Two.
The figure shows the tubes assembly for mounting the filter wheel to the upper horizontal optical train. Various lengths were constructed to allow for variations in
camera placement and future imaging requirements. The adaptor (K-22) and optical tube are integrated at the camera mount plate (K-22). Note the placement of
the 0.7X DXM Reducing lens (17.5 mm) in relationship to the filter wheel and optical train. System is shown mounted on the chamber geometry mock-up. The
dashed line defines the optical pathway in relationship to the defined adaptors. Adaptors vary depending on experimental requirements.
94 94
96
Figure 6: MTC-4 Nikon 0.7x DXM to Modified QIClick.
The figure shows the screw mounted 0.7x DXM lens installed into the windowless QIClick camera face. System should be stored in this configuration to
minimize contamination on the CCD. Compression rate for camera was limited to 48.263 KPa min-1.
95 97
95
Figure 7: MTC-4 Modified QIClick With Alternative Mounting.
The figure shows optical tubes mounted to the modified QIClick camera face. This allows for optimal matching of CCD to lens. Additionally, the configuration
allows direct integration of the camera and upper optical train. The set-up is independent of the filter wheel assembly. This configuration is utilized for
experimentation not requiring epifluorescence capabilities.
96 9896
PixeLINK PL-B741E
CMOS (Edmunds Optics
16 mm Lens)
Figure 8: MTC-4 PixeLINK PL-B741E CMOS.
The figure shows a PixeLINK PL-B741E CMOS (S/N 7410003969) camera mounted on the Fore sled. An Edmunds Optics 16 mm lens (F 1.4) was utilized for
general imaging. Camera placement is defined in relationship to the whole-cell pipette manipulator. Final placement varies depending on experimental
requirements.
97
9997
PixeLINK PL-B741E
CMOS (Edmunds Optics
16 mm Lens Not
I t ll d)
Figure 9: MTC-4 PixeLINK PL-B741E CMOS Aft Front Sled Mount.

The figure shows the PixeLINK (S/N 7410003969) PL-B741E CMOS mounted on the aft front sled with an Edmund Optics 16 mm lens. This configuration is an
alternative configuration. This mounting approach is generally utilized when multiple manipulators are installed.
EPIFLUORESCENCE
A Lumencor SPECTRA X light engine [132] was utilized as the excitation source for
the MTC-4 hyperbaric epifluorescence source. Figure 71 shows the light pathway for
the excitation and emission frequencies. The SPECTRA X light engine is controlled
using the LLE 7 Channel Graphical Users Interface via a UT232R-200 cable (232R to
USB connector; Driver CDM 20828_Setup). Illumination and verification of the primary
optical path for the system are shown Figures 75 and 76. The figures show unfocused
and focused light sources (90% excitation power).
Lumencor filter (nm) sets include; 1) 390/28, 2) 438/24, 3) 485/20, 4) 513/17, 5)
560/25, 6) 575/25, and 7) 650/13. Emission filters sets are defined as based upon
experimental requirements. Alternative filter sets may be purchased from the vendor. A
Prior Scientific [124] HF110 Motorized High Speed Filter Wheel System (SN 70958)
allows filtering (band-pass) of the signal proximal to the QIClick CCD (Figures 64).
Emission filters utilized in the filter wheel assembly are Chroma Technologies 86013v2
D/F/R set (Center Wave Length/Full Width; nm); 1) AT350/50x, 2) S457/30, 3)
S484/15x, 4) S517/30, 5) S555/25x, and 6) S605/40. A KG1 protective glass insert was
utilized; 95% band-pass to 600 nm.
The scope assembly and camera are attached to the filter wheel by the adaptor
optical tubes. Varying length adaptors are utilized to allow for future camera systems
and to optimize image placement on the CCD face (K-11; shows basic design). The
adaptor tubes are manufactured in 25.4 mm increments.
The filter wheel is controlled using a Prior Scientific CS100K (SN 70263) Filter
Wheel Keypad in conjunction with the H30V4 ProScan II Controller (SN 68832). Testing
of the filter wheel was complete using the manual configuration. The controller/computer
interface requires modification prior to utilization. Contracting issues delayed installation
and modification.
The excitation source crossed the primary pressure using a 6 m (4 m
atmosphere and 2 m chamber) quartz multimodal fiber optic cable (Lumencor PN 1010618) hermetically sealed in a custom PAVE Technology Co VS18-SS-150-1-FO-9648 penetrator (PN 4335). The assembly is defined in M-5. Final penetrator housing is
shown in M-7 and M-8. The custom housing required 6 months to manufacture. A
secondary (not installed) housing was manufactured. The assembly was bi-directionally
tested (Appendix M) to 5.171 MPa (100% He). Integration of the excitation source into
the optical train was accomplished using a manufactured adaptor (Figures 72 to 74).
Figure 77 shows the final assembly.
During manufactures pressure testing (100% He; 5.171 MPa) of the custom
penetrator it was found that the rate of degassing of the Kevlar sleeve covering the
quartz cable resulted in sleeve compromise. The compromised section of the fiber was
coated with temperature insensitive shrink tubing to limit emission frequency loss and to
protect the fiber from mechanical damage. Extreme caution must be used with the fiber
98
as the modified sleeve allows the user to exceed the recommended bend radius of the
fiber. The cable requires coating with temperature sensitive shrink tubing prior to
installation.
99
G
F
100
101
E
A
D
Figure 10: MTC-4 Epifluorescence Excitation and Emission Pathways.

The figure shows the epifluorescence pathways; A) Excitation source from Lumencor SPECTRA X light engine, B) Filter cube blocks excitation source and
90 bend, C) Excitation source to sample, D) Emission frequency from sample, E) Filter cube band-pass emission frequency, F) 45 silver mirror, and G)
Secondary band-pass (optional filter wheel selection). The filter wheel is mounted on the Aft Front Sled using a manufactured mounting plate (K-37). Filter wheel
assembly legs were cut to fit chamber geometry.
101
102101
Figure 11: MTC-4 Upper Vertical Train Fluorescence Adaptors.

The figure shows the manufactured (A) upper vertical optical train epifluorescence adaptor and the vendor supplied (B) Lumencor Spectra Light Engine 1 mm
quartz fiber adaptor (82-10015). The set screw in quartz fiber adaptor allows focusing of the excitation source in the Z-axis. A screw mounting is utilized to
minimize excitation power loss.
102
102
103
Figure 12: MTC-4 Upper Vertical Fluorescence Adaptors Installation.

The figure shows the quartz fiber adaptor and scope adaptor installed in the upper vertical optical housing (K-50).
Figure 13: MTC-4 Upper Vertical Optical Quartz Multi-Modal Fiber Optic Cable Installation.
The figure shows the quartz fiber and scope adaptor installed in the upper vertical optical train (K-50).
103
104
104
105
Figure 14: MTC-4 Emissions Pathway Initial Alignment.
The figure shows the emission frequency prior to adjustment of fiber adaptor. System is not focused. Figure shows the Z-axis at its maximal upper range.
105
105
106
Figure 15: MTC-4 Emission Pathway Initial Alignment View Two.
The figure shows the green light source with Z-axis at its maximal lower range. System is focused.
Figure 16: MTC-4 Fluorescence Adaptor Installations.

The figure shows the final installation of the fluorescence adaptor (A) in the upper microscope train.
106
SLED
Fore (FS; K-2), Aft Front (AFS; K-3), and Aft Rear (ARS; K-4) sleds are shown in
Figures 14 and 47. Aft sleds are connected by manufactured sled linkers (K-6). The Aft
sled is defined as a single unit (AS). Aft and Fore sleds are independent. Locating
spacers are used to define proper location of the FS and AS (Figure 78). The FS and
AS sleds are mounted to the chamber railing systems as defined in Figure 79; final
mounting is based upon experimental requirements.
Sled mounts are constructed of UHMW polyethylene. Compressive modulus and
deformation are non-significant at the pressure range utilized. Sled mounting holes
allow for locking the respective sled units in position following installation. The Aft sled
riser blocks (K-26) are 50.8 mm in height. Front sled risers are 6.35 mm in height and
are similar to those defined in K-26, with modification for mounting. Sleds are held in
place by rail clamps as shown in Figure 78 to 80 (K-15 and K-32). Railing clamp
location is user dependent.
The AS was modified for placement of the micro-handling pump mounting case
as shown in Figure 78. Sample stage mounting supports are shown in Figure 80.
Fore sample stage supports are fixed (K-5) and the rear supports (K-12) are installed
finger tight. This configuration allows for easy installation of the Prior motorized stage.
The final supports are manufactured of UHWM Polyethylene.
Prior to installing the sample stage, the condenser unit is moved to its maximal
downward deflection and the objective manipulators are moved to their maximal upward
deflection. Failure to adjust the defined units may result in stage and/or objective
damage. The final placement of the Prior H29/30 is defined in Figure 81. The unit must
be energized for movement. Manual travel will damage the motor assembly.
Stage positioning utilizes micron sized metallic positioning strips in the X- and Yaxis. These strips are highly susceptible to damage during placement. Strips must be
cleaned daily with 70% EtOH (ddH 2 O). Salt formations will result in compromise of the
stage positioning strips.
107
FS
AS
108
Micro-Handling
Pump Cut Out
Instrumentatio
n Ground Block
Figure 17: MTC-4 Aft Sled Assemblies.

The figure shows the cut out in the Aft Sled (AS) for the installation of micro-handling pump holder mounting. Fore
(FS) and AS are shown. The instrumentation ground block allows for component grounding independent of MTC-4
primary chamber ground.
108
109 110
109
Figure 1: MTC-4 Sled Clamps.
The figure shows the installed UHMW polyethylene mounts for securing sleds to the chamber railing system. A similar approach is utilized to secure the FS (AS is
shown). Locking of the FS and AS units is required for vibration isolation and mechanical stability.
FS
AS
C
110
111
Figure 2: MTC-4 Front Sled Solution Heater and Stage Assemblies Mounts.
The figure shows the Fore (FS) with the distal Aft (AS) sleds. The placement of Prior H29/30 Motorized stage mounts is defined; A) Fore positioner standoffs (K-5)
and B) Aft Prior stage supports. The FS perfusion solution inline heater unit mount (C) is shown. Various mounts are defined; final variants will depend on
experimental requirements.
112
111
A
Figure 3: MTC-4 Stage Mounting.

The figure shows the final mounting of the motorized sample stage on the stage mounts in relationship to objective/s. Travel locator strips must be treated with
extreme caution and cleaned daily (70% EtOH; ddH 2 0). Single axis travel locator strip is shown (A). Groves are included in mounting tray for incorporation of
heater control lines and temperature probes. Heater block temperature probe must be installed with a coating of mineral oil.
HIGH PRESSURE CELLULAR PERFUSION SYSTEM

In vitro hyperbaric perfusion was accomplished based upon the methodology of Imbert
[139], Moore [140] and Dean [84, 111] with modification. The system is not rated for H 2,
and is limited to O 2 21% (101.43 KPa). It is critical to note that to maintain a constant
partial pressure of a defined gas (Gas X ) in solution (P Solution Gas X ), chamber (P Gas Gas X )
and P Solution Gas X must be in equilibrium. We found significant off-gassing when chamber
and perfusion gases differed. This would be predicted by gas-liquid mass transfer [141]
and Ficks Laws of diffusion [142, 143].
It should be noted that this is in contrast to the article of Dean et al [111]. We
could not replicate their finding in which they reported no transfer of gas out of solution
when chamber environment differed from perfusion gas make-up. Additionally, we found
that when cultured cell lines were included in the perfusion pathway off-gassing was
robust. We conclude that the cell culture slip and the cells disrupt laminar flow.
Additionally, we conclude laminar flow is not possible except in the case of a clean
glass cover. It should be noted that during no flow conditions the effect was less
extreme. However, within 5 min gas concentrations were unstable at the bottom of the
sample plate. This is a critical concern since most electrophysiology experiments
require in excess of 20 min following stabilization. Sample plate depth was standard for
experimental protocols common to electrophysiology.
A schematic of the perfusion system is shown in Figure 82; A) High pressure
supply, B) Low pressure supply, and C) Equilibrated liquid. The solutions of interest
utilized gas and/or pneumatic compression strategies to supply a pressure head.
Gas compression utilized a head pressure that was 68.974 KPa over chamber
pressure. Liquids were equilibrated with calibration gases of interest via manual and/or
tracking regulators. Cylinders had a 750 mL working volume (1 L max volume).
Cylinders designated for agents that were of significant risk were limited to a 40 mL
working volume (50 mL maximal volume). The approach was utilized to minimize
aerosol formation in the event of a catastrophic decompression (Appendix I).
Additionally, the exposure level would be predicted to be non-significant as based upon
the volume of the non-pressurized room.
Cylinder working volume was defined to minimize the chance of a reverse flow
condition thru the cylinder purge valve and regulators. Solution backflow was common
when cylinders were filled to 750 mL or 40 mL; respectively. The cylinders defined
as 750 mL maximal volume are Perfusion Cylinder 1-4 (PC1 to PC4). Cylinders defined
as 40 mL maximal working volume are Perfusion Cylinder 5-6 (PC5 to PC6).
Perfusion cylinders were filled utilizing a negative pressure system ( -20 MPa)
with a inline water trap (Gast Manufacturing [144], Inc.; Laboratory Oilless Diaphragm
Vacuum Pump and Compressor; Model DOA-P704; SN 0410612017). It is critical to run
the pump in an open configuration to allow for moisture and/or trapped water removal
following cylinder fill. Oil base compressors are not be utilized with the system.
112
Atmospheric air was vented with 100% He prior to equilibration of the perfusion
solution with the calibration gases. Directional flow was maintained using a 6.894 KPa
check valve. Cylinders (750 mL perfusion media) required 10 min at 34.373 KPa to
reach 99% He as measured by Mass Spectrometry of the cylinder headspace. Extreme
care must be taken to ensure no liquid flow into the Mass Spectrometer. Experimental
gas mixes were vented through the perfusion media for 10 min at 34.373 KPa. Helium
and/ or N 2 was added to O 2 (20.270 KPa) and CO 2 (5.068 KPa) to the defined test
pressures. Gas shifting at depth required a 34.373 KPa flush over chamber pressure for
10 min. Practice is required to successfully maintain a constant pressure during a
flush at depth. Pressure excursions will compromise cell seal integrity. While flow rates
may vary, it is critical to flush x3 chamber volume. The approach minimizes the gas
gradient out of solution. An emphasis must be made to ensure P Gas Gas X = P Solution Gas X at
test pressures. During chamber travel gas concentration was not a concern based upon
the significant concentration gradient out of solution; i.e., the partial pressure of O 2 and
CO 2 remains constant.
Travel perfusion media head pressure was maintained using a TESCOM SJS
Series Tracking Regulator (Model: SJF565VEBFEFFF09). This produces a constant
pressure head during atmospheric, compression, and decompression conditions.
Tracking regulator pressure was set to 68.947 KPa (maximal bias pressure: 689.47
KPa). The tracking regulator was calibrated using the 1 L SS cylinders and calibration
gases as chamber and pressure; respectively. Bias pressure was set as per
manufacture recommendation. Care must be taken to not over-torque the tracking
regulator set screws, as this will damage the spring. The tracking regulator was serviced
on a 6 month interval by the vendor.
Chamber and bias pressure are defined as; 1) PC2 Gas Mixture Input, 2) PC3
Gas Mixture Output, and 3) PC4 chamber. The system allows real-time tracking of bias
pressure over chamber pressure.
Gas equilibrated solutions flow to a six way selector valve and associated 40 m
particulate filters (Swagelok SS-2FT-40). The flow rate is defined downstream using a
Bronkhorst Cori-Tech BV (USA [145]) Model M13V141-ABD-11-K-S Mini COROFLOW digital mass flow meter/controller (MFC; S/N B12201343AA; functional flow rate
0.20 mL to 10 mL min-1). Manufacture calibration (Cert # BHTLO5/CHK/1363660) was
completed on the MFC. The system was zeroed as per manufacture instruction.
Distal and proximal SS tubing was installed in relationship to the MFC. SS tubing
on the distal and proximal ports of the MFC were 304.8 mm. The tubing inhibited
vibrations in the Teflon tubing and minimized mechanical shock to the MFC. The MFC
had an accuracy of 1.2% at an operational flow rate of 1-5 mL min-1. Maximal MFC
input and output pressure are 34.473 MPa and 31.026 MPa; respectively. Particulate
filters (Swagelok SS-2FT-40; 40 M) were mounted distal and proximal to the MFC. A
2.298 KPa check valve (Swagelok SS-CHM2-5) was mounted distal to the MFC.
113
The MFC is stored in a continuous 1 mL min-1 flow configuration (34.473 KPa

He or N 2 ); 70% EtOH ( dd H 2 O). Continuous flow configuration limits the cleaning
requirements and minimizes clogging issues in the MFC. During system shutdown, or
long-term storage, the system is flushed with 200 mL 70% EtOH (ddH 2 O). Dry N 2 (H 2 0
< 1 ppm) is used to purge the distal and proximal MFC lines (30 min). The lines are
sealed with caps when stored.
Affluent solutions enter the chamber via a 6.35 mm NPT Swagelok penetrator
and flows into a Scientific Systems Inc. (SSI; PN 14-0129) Mini-Pulse Pulse Damper
(3:1 pulsation reduction). The pulse damper volume is 160 L. Perfusion lines are
connected using Swagelok 1.6129 mm to 6.35 mm male to male converters. A SSI
solvent debubbler (P/N 010221) was installed distal to the Mini-Pulse Damper. This
approach was successful to 2 mL min-1; rates in excess of this value were not tested.
The cap must be left in the open configuration with the debubbler acting as an open
solution reserve. The debubbler must be mounted away from electrical and optical
components so that liquid spillage will not impact the system.
All perfusion lines, unless denoted, consisted of 3.175 mm Nylaflow H (Type
6/6) or Stainless Steel (SS) tubing. All perfusion lines must be cleaned on a regular
schedule to minimize contamination. Maintenance of the MFC, and inline filters, are
critical for proper system functionality. The MFC is highly susceptible to clogging when
not maintained. Servicing of the unit requires in excess of 4 months as the service
center is overseas.
Experiments requiring a constant partial pressure of gas in solution utilized a
High Pressure Liquid Chromatography (HPLC) Pump. Integration of the hydraulic and
gas systems resulted in numerous system failures prior to defining perfusion operational
procedures. The hybrid system offers the full range of experimental capabilities, with the
addition of improved control of gases in solution during compression and
decompression.
The HPLC system integration point is distal to the MFC. A Swagelok SS-2FT40 40M particulate filter and a 2.298 KPa Swagelok SS-CHM2-5 check valve are
included in the perfusion pathway. The system utilizes gas equilibrated 750 mL
perfusion bottles (also 40mL) and/or normobaric equilibrated reservoirs. Normobaric
equilibrated reserves are temperature clamped (Julabo TW20 heated water bath). The
system is isolated using Swagelok ball valves (common output).
The HPLC pump was primed per the manufacture recommendation. The HPLC
pump was maintained in a constant flow condition (20% EtOH/ 80% ddH 2 0; 50 L min-1)
during experimental down time. This was completed to minimize purging issues. During
prolonged downtime (i.e., 2 weeks) the HPLC pump was disconnected, purged, and
placed in a storage configuration as per manufacture recommendations. During testing,
the initial HPLC was damaged due to back-pressure. The HPLC system is protected
from back-pressure using varying check valves. Normobaric perfusion flasks were
equilibrated at ambient pressure and stored in a water (37 C) bath during all
114
experiments. System design specifications and technical drawings are provided in

Appendix N. Various check valves and system purge points are included in the design.
The system can be operated in multiple configurations.
The HPLC Perfusion System was built by Swagelok Alabama Custom
Engineering Solutions Department (P/N IS-02-10-091312-REVC; Order # 97328722)
and pressure tested to 5.171 KPa (He; bi-directional). Testing was completed on site.
Front panel controls and overall design are shown in Figure 82 and Figures 86 to 91.
The system allows for directional flow to the chamber at a constant pressure and is
independent of the MFC. The MFC can be plumbed to directly provide solution to the
HPLC. This is an alternative configuration.
The HPLC pump acts as a MFC and pressure head. Bubble traps were included
in the flask/s to limit cavitation in the pump. Cavitation in the HPLC pump may cause
system failure. A variable head resistance was included distal to the HPLC pump. This
allows the pump to act against a head pressure when at ambient pressure. HPLC
pumps require a head resistance for optimal performance.
Cells are maintained in a Warner Instruments (WI) [146] RC-27LD (Cat # 641532) holder. The RC-27LD provides sufficient clearance with respect to the objectives
and pipette. The holders length must be in-line with the pipette to allow for maximal
angle of approach. The pipette should enter from the affluent side of the holder. When
the holder is horizontal to the pipette, holder side walls will not allow for proper angle of
entry for cell attachment.
A modified stage insert was manufactured from clear acrylic to mount the RC27LD. Glass Coverslips (P/N CS-24/60, N0.2 Glass Coverslip; 24 x 60 mm thickness)
are utilized for experiments. Coverslips have a no-slip coating on the RC-27LD
interface. The RC-27LD and stage insert are directly mounted to the Prior Scientific
H101AF1 motorized stage. While not currently functional, the stage may be operated
via manufacture software.
Bath temperature is clamped to 37 C utilizing a Warner Instruments (WI) TC344B Dual Channel Automatic Heater Controller (CC-28 heater cable; WI). The system
utilizes a dual channel feedback loop. This maintains a constant solution temperature in
respect to a defined reference temperature (37 C; temperature range: ambient to 50
C); A) Channel 1: SH-27B In-Line Solution Heater (WI) in automatic mode (loop speed:
fast) and B) Channel 2: PM-1 Series Magnetic Platform (WI) in automatic mode (loop
speed: fast).
Tubing and temperature probes were held in place with magnetic clamps (MCK-1
Magnetic Clamp Kit; WI). Chamber cover slips were mounted utilizing Dow Corning 111
Valve Lubricant and Sealant (WI P/N 64-0378). Excess lubricant was removed prior to
perfusion and the set-up was flushed with PBS (1x; sterile ddH 2 0) for 5 min. The
affluent and effluent perfusion solutions enter the chamber coverslip area via flexible
affluent and effluent perfusion solution lines. Lines were Tygon tubing with SS
115
adaptors. To perform low noise operations, glass effluent and affluent bath entry
adaptors were hand manufactured.
The non-SS tubing must be changed on a regular basis to avoid contamination.
Significant caution must be used when defining which type of Tygon tubing to utilize in
the system. Improper tubing will result in leeching. Stainless Steel tubing must be
autoclaved monthly. The laboratory utilized a Tuttnauer 2340 table-top autoclave [147].
Components that could not be autoclaved were cleaned using a VWR Ultrasonic
cleaner [148]. Failure to maintain the system will result in system compromise. All
perfusion components are washed with 200 mL 70% EtOH ( dd H 2 O) following
experiments. The affluent and effluent perfusion lines are blown dry with dry N 2 or He.
The operating procedures for the perfusion systems are defined in Table 3; 1)
Pressure, 2) HPLC, and 4) HPLC/Pressure.
116
High Pressure
Low Pressure
Gas
Gas
Equilibrated Solution
117
117
Figure
Figure4 82. MTC-4 Perfusion Gas Compression Systems.
The figure shows; A) High pressure supply, B) Low pressure supply, and C) Equilibrated solution.
118
118
118
Figure 5
Figure 83: MTC-4 HPLC Pump Based Perfusion Systems Overall Design.
The figure shows the integration of the HPLC based perfusion system with the standard pressure based system in Figure 82.
C
119 119
119
Figure 6: MTC-4 Perfusion Pressurization System External.

The figure shows the external gas supply for the perfusion system as defined in Figure 82. There are 2 locations to the left of each primary He bank for installation
of K-bottles. It is critical that bottles are mounted level and secured (locked mounting straps). The components are; A) Perfusion Bottle 1, B) Perfusion Bottle 2,
and C) Perfusion Control Panel and Gauges. The bottles can be mounted internally. Multiple check valves are installed inline to isolate the perfusion system
pressurization bank and to control for cross-contamination. Perfusion and chamber pressurization are independent systems. Perfusion bottle placement for He
Bank 2 is similar (not shown).
120
120
120
A
C
B
F
Figure 7: MTC-4 Perfusion Pressurization Systems External Primary Control Panel.

The figure shows the external gas supply panel for the perfusion system. Components defined are; A) Perfusion Bank 1 Exhaust, B) Perfusion Bank 2 Supply, C)
Gas line from Perfusion Bank 4, D) Gas line from Perfusion Bank 3, E) Perfusion Bank 2 gas supply whip, and F) Perfusion Bank 1 gas supply line whip. All
controls utilize similar exhaust and supply design. System is limited to 24.131 MPa with inline pressure relief valves. Valves are tested biannually.
121
121121
Figure 8: MTC-4 HPLC Pump Based Perfusion Systems View One.
The figure shows the completed HPLC system. Top (A) and side (B) view of HPLC based perfusion system as based on the design shown in Figure 83. The
housing allows for mounting of the unit in a standard 482.6 mm equipment rack. Extreme care must be taken as rear connection points and front panel controls are
easily damaged during system mounting or transfer. The completed assembly weighted in excess of 45.36 Kg. A support beam was utilized to stabilize the
housing during installation.
122
122122
Figure 9: MTC-4 HPLC Pump Based Perfusion Systems View Two.
The figure shows the completed HPLC system. Rear view of HPLC based perfusion as based upon the design in Figure 83. The housing allows for mounting of
the unit in a standard 482.6 mm equipment rack. Extreme care must be taken as rear connection points and front panel controls are easily damaged during system
mounting or transfer. The completed assembly weighted in excess of 45.36 Kg. A support beam was utilized to stabilize the housing during installation. Lines are
to be capped or plugged during down time to minimize contamination. The system should be stored at pressure during down time. The design allows for
pressurization of the system for storage independent of the HPLC pump. Store pressurized with dry He or N 2 .
123
123
123
Figure 10: MTC-4 HPLC Pump Based Perfusion Systems View Three.
The figure shows the completed HPLC system. Component view of the HPLC based perfusion as based upon design shown in Figure 83. The housing allows for
mounting of the unit in a standard 482.6 mm equipment rack. Extreme care must be taken as rear connection points and front panel controls are easily damaged
during system mounting or transfer. The complete assembly weighted in excess of 45.36 Kg. A support beam was utilized to stabilize the housing during
installation. Lines are to be capped or plugged during down time to minimize contamination. The system should be stored at pressure during down time. The
design allows for pressurization of the system for storage independent of the HPLC. Store pressurized with dry He or N 2 . The parts are defined in Figures 89 and
90.
124
124124
Figure 11: MTC-4 HPLC Pump Based Perfusion Systems View Four.
The figure shows the completed HPLC system. Side component view of HPLC based perfusion as based upon in Figure 83. The housing allows for mounting of
the unit in a standard 482.6 mm equipment rack. Extreme care must be taken as rear connection points and front panel controls are easily damaged during system
mounting or transfer. The complete assembly weighted in excess of 45.36 Kg. A support beam was utilized to stabilize the housing during installation. Lines are to
be capped or plugged during down time to minimize contamination. The system should be stored at pressure during down time. The design allows for
pressurization of the system for storage independent of the HPLC. Store pressurized with dry He or N 2 .
125
125 125
Figure 12: MTC-4 HPLC Based Perfusion Systems View Five.
Front component view of HPLC perfusion systems as based upon the design shown in Figure 83. The housing allows for mounting of the unit in a standard 482.6
mm equipment rack. Extreme care must be taken as rear connection points and front panel controls are easily damaged during system mounting or transfer. The
completed assembly weighted in excess of 45.36 Kg. A support beam was utilized to stabilize the housing during installation. Lines are to be capped or plugged
during down time to minimize contamination. The system should be stored at pressure during down time. The design allows for pressurization of the system for
storage independent of the HPLC. Store pressurized with dry He or N 2 .
126
126
126
Figure 13: MTC-4 HPLC Pump Based Perfusion Systems View Six.
The figure shows the completed front panel control from the design shown in Figure 83. The design is shown in Swagelok Technical Drawings (Figures 83 to 87).
Sample lines are removed for ease of viewing. Sample lines consisted of SS, Tygon, or Chemflour tubing. Tubing was defined as based upon chemical
reactivity and pressure requirements (R-10 to R-12). Swagelok components were utilized for integration into primary perfusion system and room equilibrated
solutions (37C). System should be flushed following utilization with 70% EtOH (ddH 2 0). The system should be stored at pressure during down time. The design
allows for pressurization of the system for storage independent of the HPLC. Store pressurized with dry He or N 2 . Daily down time should be configured in a
constant perfusion configuration with 70% EtOH (ddH 2 0). It is critical to understand the flow path as defined in Figure 83 for proper operation.
127
127
127
Figure 14: Mobile Test Chamber 4 (MTC-4) Perfusion Bank Internal Supply Panel.
The figure shows the high pressure supply panel. Gases from outside perfusion gas banks as defined in Figure 74 and 85 (Gas-1 and Gas-2). Supply pressure is
dropped to working pressure with a maximal differential across the MFC of 344.73 KPa. The system may be operated in tracking mode (68.947 KPa tracking
bias) or static pressure head mode. Systems utilize a jumper approach. Caution must be taken not to create a negative pressure differential opposite to flow.
Gas From Perfusion Gas

Bank
127
Liquid Side
I
F
Figure 15: Mobile Test Chamber 4 (MTC-4) Perfusion Bank Gas Fluid Integration Control Panel.
The figure shows the gas/fluid integration system for MTC-4 as defined in the text and Figure 82. The components for
perfusion bottle three are: A) Not used, B) Quick connect [149] from P-X or Tracking Out, C) Vent gas line, D) Shutoff gas line, E) Connection to 1 L bottle, G) 1 L Bottle vent/fill, H) Liquid shut-off, I) Lower connect solution line from
bottle, and J) Shut-off. Perfusion Bottles 1, 2, and 4 are the same.
128
A
E
127
Figure 16: MTC-4 Perfusion System Integration Panel Gas and HPLC.
The figure shows the integration point of the perfusion systems defined in Figures 82 and 83. Perfusion system
selector components are defined as: A) MFC, B) Gas-based perfusion selector (Figure 92), C) MFC/HPLC
input/output selectors, D) Not utilized, E) 50 mL cylinders, and F) Spare 1 L cylinders. Ancillary connection points E)
and F) may be connected directly to the perfusion gas system and/or a manual regulator. System must be cleaned on
a regular schedule. System should be stored with a slight positive pressure with all outputs capped or plugged.
129
Notes:
130
Table Three: MTC-4 Pressure Head and HPLC Operating Procedures.
The Operational procedures vary significantly and are dependent on experimental requirements.
Table 3 is designed as a guide only.
The system can run with the external solutions in a normobaric bath and/or via pressure head.
The defined operational procedure is in conjunction with the pressure head system (Steps 1- OPT3).
Pressure Head Based System Operating Procedures:
Prior to system operation, the operator is to place all valves in the perfusion system to the closed/no flow
position as required.
High Pressure (HP), Low Pressure (LP) and Fluid (FL) are defined in Figure 82.
It is critical to flush solution pathways prior to utilization with 200 mL ddH20 prior. This flush is to be flowed
by a flush with perfusion media or ddH20.
System configured for Supply 1 (Supplies 2-4 are similar).
Only one supply can be in the tracking configuration.

INITIAL SET-UP:
Step
Position
Info/ Figure
Notes
1 (HP)
Closed
External perfusion gas panel perfusion
Verify exhaust turn
bank supply and vents closed.
valve/s and ball valve/s
Figure 82 to 85
are in the closed

position.
Attached
Attach the pressure source to the
Do not exceed the
external calibration gas supply.
system high pressure
Do not exceed 3000 psig (20.68 MPa).
input maximal
Pressure relief tripped at 3300 psig
pressure.
(22.75 MPa).
3
Open
Ensure there is sufficient head

pressure.
Open
Verify the volume of gas.
Turn the perfusion off.

CONTINUED:
131
Calibration Gas
Table Three: MTC-4 Pressure Head and HPLC Operating Procedures Continued.
CONTINUED INITIAL SET-UP:
Step
Position
Info/ Figure
Open
HPI-S1 (turn slowly)
Gauge
Supply 1 gas patch panel pressure
Notes
head from calibration bottle.

7
RG-1
Set to 20 psig (137.895 KPa) over

chamber maximal excursion.
METHOLOGICAL CONSIDERATION FOR TRACKING OR HEAD BASED APPROACH

Approach A: Standard Pressure Head
Approach B: Tracking
7-A1
P1 to TRG Input
Tygon Jumper
Ensure jumpers in
proper configuration.
7-A2
TRG Output to QC1-2
Tygon Jumper from upper gas patch
Jumper may be
panel to lower gas patch panel.
attached to any flow

path.
Approach A: Tracking
7-B1
P1 to QC1-2
Tygon Jumper from upper gas patch
The jumper may be
panel to lower gas patch panel.
attached to any of the

flow paths.
Complete the Equilibration and System Set-Up (Figure 88).

750/40mL
Cylinder/s filled as required.
The jumper may be
Cylinder (1 L/
Cylinder/s flushed for 10 min
attached to any of the
50mL maximal
at 5 mL min-1
flow paths.
volume)
Figure 93 750 mL cylinder (1 L).
Figure 94 item E 40 mL cylinder

(50 mL).
PCV-1
Open
CONTINUED:
132
Table Three: MTC-4 Pressure Head and HPLC Operating Procedures Continued Approach A.
Step
Position
Info/ Figure
11
PSV1-2
Open
12
VE1-2
Closed
Notes
Open for venting as

required and used for
filling.
13
PSV1-3
Open
14
PSV1-4
Open
15
To perfusion control panel (FIGURE 88-2).
Verify MFC in the correct position.

MFI/HPLC IN to MFI
Verify that head pressure does not

exceed Mass Flow Controller 100
psig (689.475 KPa).
16
MFO/HPLC OUT to
MFO
17
Mass Flow Controller
to maximal flow rate

for Purging
Set MFC to maximal flow

rate and purge.
Set the MFC to required flow

rate (front panel in g hr-1;
convert to mL min-1).
Target flow rate

-1
to 2mL min
CONTINUED:
133
Table Three: Mobile Test Chamber 4 (MTC-4) Pressure Head and HPLC Operating Procedures Continued.
Step
Position
OPT 2
Info/ Figure
PSV1-4 rear panel attach

jumpers to 40 mL.
OPT 3
Equilibrate 40 mL cylinders.
Run Items 15 to 17.
No flush due to limited

solution.
Verify up-stream turn valves are in open configuration
Place micro-handling pump in mode to match inflow from MFC.
Balance requires significant trial and error.
Notes:
CONTINUED:
134
Notes
Table Three: MTC-4 Pressure Head and HPLC Operating Procedures Continued.
PRESSURE AND HPLC SYSTEM:
Approach B
Verify pump primed and pump connected to to pump and from pump in proper configuration.
Ensure you are familiar with Figure 83 and 94 prior to utilization.
Solution selector valve utilized in external bottle configuration.
Step
Position
Info/ Figure
2-HPLC
MFI/HPLC IN to HPLC
IN
3-HPLC
MFO/HPLC OUT to
HPLC OUT
4-HPLC
HPLC out jumper to
To perfusion
Closed; open as
perfusion solution
selector valve
5-HPLC
Bubble Vent
required during priming

6-HPLC
MFC/HPLC Selection
From HPLC pump
Valve
Notes:
CONTINUED:
135
Notes
Table Three: MTC-4 Pressure Head and HPLC Operating Procedures Approach B Continued.
Step
8-HPLC
Position
Perfusion Output
Info/ Figure
To the HPLC Pump.
To the HPLC Pump.
10 to 20 psig (68.947 to
Selector Valve
9-HPLC
PURGE/HPLC Pump
Selection Valve
10-HPLC
Back Pressure
Regulator
137.895 KPa) head

pressure.
Notes:
END TABLE 3
136
Notes
HIGH PRESSURE CELLULAR PERFUSION SYSTEM 750 ML CYLINDER (1 L)

CHARACTERIZATION
An Extrel Questor [150] QGP Process Mass Spectrometer (QCPMS; ABB Extrel S/N
21915) was utilized to define on-gassing/off-gassing rates of He, O2, N2, CO2 in
standard external perfusion media (SEPM). SEPM media consisted of (mM) 5 KCl, 135
NaCl, 1 MgCl, 2 CaCl, 5.5 D-Glucose Monohydrate; pH 7.5 (1N NaOH).
Figure 95 shows the test configuration. The QCPMS was calibrated as per the
manufacture recommendation. Calibration gases consisted of (Air Gas [151]
(certification number; CERT#)); A) Sensitivity: 20.86% O2, 0.92% Ar, 590 ppm CO2, and
balance N2 (CERT# WP00V325B), B) Fragmentation: 8.9487% CO2 and balance O2
(CERT# 100092017), and C) Background: 100% He (CERT# 23-400082730-1).
The 750 mL SS cylinders were utilized for analysis of off-gassing. Cylinders are
filled with 500 mL of room equilibrated (60 min; RT 20 +/-2 C) SEPM. Cell Culture Air
(4.993% CO2, 20.00% O2, and balanced N2; CERT# 23-40082732-1) and He (100% He,
CERT# 23-400082730-1) were connected via a manufactured gas control panel (Table
4; Appendix T). The gases were bubbled into the distal end of the SS cylinder using
3.175 SS tubing. A bubble stick was utilized in the SS cylinder to ensure gas/liquid
mixing. The gas flow rate was set to 200 mL min-1.
The headspace port was attached to the QCPMS. A water trap was installed
between the QCPMS and 750 mL cylinders. Detailed manufactured gas panel drawings
are provided in Appendix T. All connectors were Swagelok fittings, with Tygon and/or
Swagelok shielded tubing. Tubing was selected to minimize the chance of cross
reactivity and leaching.
Cell Culture Air (5 min) was utilized to establish a headspace baseline as defined
by the certificate of analysis; 4.993% CO2, 20.00% O2, and balance N2. Solutions were
degassed with He (100%) until >99% He saturation. Solutions were stabilized for 10
minutes after reaching baseline. Baseline (stabilization) was defined when CO2, O2 and
N2 intensity reached plateau value 0.2% for 1 min. Solutions were bubbled with Cell
Culture Air until > 99% Cell Culture Air was defined in the headspace. Gas control panel
components are defined in Table 4.
A representative trace of on-gassing and de-gassing rates is shown in Figure 96.
On-gassing and degassing rates (n=10) for He, N2, O2, and CO2 in 500mL SEPM at
101.352 KPa in 1 L cylinders (vacuum filled) were completed. The samples were
defined as degassed when He >100.339 KPa (100.346+/- 0. 291 KPa; +/- 1 min). Cell
Culture Air was defined as equilibrated when He was <0.101 KPa (54.669 +/- 3.091
KPa; +/- 3min). Cell Culture Air equilibration (21.28 KPa O2, 5.07 KPa CO2, and 75.002
KPa) values are (KPa) 20.159+/- 0.00324 O2, +/- 5.239 +/-0.022 CO2, and 75.952 +/0.021 N2. Following He de-gassing (KPa), O2 (0.18243; +/- 0.0000517), CO2 (0.16547;
+/- 0.01743), N2 (0.58784; +/- 0.01540) reached stable values. Gas equilibration was
137
analyzed by reversing the process (KPa); 137.660 +/- 0.0004 O2, 5.533 +/- 0.03474
CO2, & 75.376 +/- 0.0344 N2.
There was no significant (p>0.05) difference between sample ongassing/degassing rates for He, N2, O2, and CO2 in 500 mL of SEPM and
gassing/degassing rates of ddH2O (N=5).
Helium on-gassings (He>100.339 KPa) was accomplished in 16.43 +/-1.22 min.
Helium de-gassing was completed in 17.12+/-0.55 min. There was no significant
difference between He on-gassing and de-gassing rates (p>0.05) in the defined system.
Therefore a stable degassing point can be conservatively defined at 20 min (200 mL
min-1) following He (100%). Equilibration time of test gases can be predicted to be 20
min following He degassing. A total cylinder degassing and test gas equilibration of 40
min is recommended for solutions of physiological significance.
138
A
2
44
5
8
Figure 1: Experimental Set-Up Extrel Questor QGP Process Mass Spectrometer (QCPMS) 750 mL
(1 L) Sample Cylinder De-gassing and On-gassing.
The figure shows the experimental set-up (A) and schematic (B) representation of the 750 mL test system. Schematic
components defined are; 1) Sensitivity Gas (20.86% O2, 0.92% Ar, 590 ppm CO2, and balance N2), 2) Fragmentation
Gas (8.9487% CO2 and balance O2), 3) Helium (100% He), 4) Cell Culture Air (4.993% CO2, 20.00% O2, and
balanced N2), 5) Flow meter, 6) Gas panel, 7) Swagelok CT8079 1 L cylinder, and 8) QCPMS.
139
Figure 2: 750 mL (1 L) Swagelok CT8079 Perfusion Reserve Characterization.

The figure shows the He, N2, O2, and CO2 degassing rates (N=10 runs; RT) as defined using an Extrel Questor QGP
Process Mass Spectrometer (QCPMS). The characterization used a 200 mL min-1 flow rate for Cell Culture Air (21%
O2, 4.993% CO2, 0.92% Ar; balance N2) and He (100%). The solution (500 mL) consisted of (mM); 135 NaCl, 5 KCl,
0.22 NaH2PO4, 1 MgCl, 2 CaCl, 5.5 Glucose & 10 HEPES; pH 7.35 (1N NaOH). A 5 minute equilibration (+ 5:00 min)
was completed using Cell Culture Air. Values shown in the representative figure are from the same sample. He
degassing reached >99% (99.011%) equilibrium at 18:24 min and Cell Culture Air (19.67% O2, 5.4756% CO2, 74.4%
N2) reached equilibrium at 35:28 min. Figure shows; A) He gassing/degassing rate, B) N 2 on-gassing/degassing rate,
C) O2 on-gassing/degassing rates, D) CO2 on-gassing/degassing rate. Scales are not equal between panels. The
time to reach a stable reading prior to the experiment following the switch to He or He to Compressed Air can be
defined by the dead space in the HPLC sample tubing. Summary statistics are provided in the main text with values
converted to KPa.
140
Table Four: MTC-4 Mass Spec Gas Control Panel.

MATERIALS LEGEND:
= Diameter
Component
Technical Drawing
Cellular Gas
123554-01
Control System
Panel
Cellular Gas
123554-02
Control System
Plate- Side
Cellular Gas
123554-03
Control System
Plate- Plan
Cellular Gas
123554-04
Control System
Plate- Lettering
Appendix Page
T-2
T-3
T-4
T-5
Line-Vent
123554-05
T-6
Line-1
123554-06
T-7
Line-2
123554-07
T-8
Line-3
123554-08
T-9
Line-4
123554-09
T-10
Line-5
123554-10
T-11
Line-7
123554-11
T-12
Line-8
123554-12
T-13
Line-9
123554-13
T-14
Line-10
123554-14
T-15
Line-11
123554-15
T-16
Cellular Gas
Control System
Assembly-Front
123554-GA1
T-17
Continued:
141
Info
2.65684 mm SS;
White Powder
Coated
2.65684 mm SS;
White Powder
Coated
2.65684 mm SS;
White Powder
Coated
2.65684 mm SS;
White Powder
Coated// Location of
Lettering
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
3.175 mm SS
Tubing
Various; installation
of front panel
controls
Table Four: Mobile Test Chamber 4 (MTC-4) Mass Spec Gas Control Panel
Continued.
MATERIALS LEGEND:
= Diameter
Component
Technical Drawing
Appendix Page
Info
Cellular Gas
123554-GA3
T-19
Various; overview of
Control System
Lines 1-11
Assembly- Plan
Cellular Gas
123554-GA4
T-20
Overview
Control System
Assembly- Left
Cellular Gas
123554-GA5
T-21
Overview
Control System
Assembly- Left
Notes:
END TABLE FOUR

142
NEGATIVE-PRESSURE SYSTEM (NPS)

Whole-cell recordings require the formation of a G seal between the phospholipid
bilayer and the patch pipette (World Precisions Instruments (WPI) P/N 1B150-6;
borosilicate single barrel configuration, 1.5/0.84 OD/ID (mm)). Pipette resistance is
generally 2 to 3 M for neuronal soma. However, it should be noted that pipette
resistance and geometry is dependent on the cell type.
Following formation of the G seal and membrane stabilization, a negativepressure pulse is applied to compromise the plasma membrane. This configuration
completes the RC circuit. For a detailed explanation of formation of the whole-cell
configuration and considerations related to the membrane/pipette geometry, the authors
recommend the manuscripts Suchyna [152] and Priel [153].
The P1000 and P-97 Pipette Cookbook [154] is an outstanding resource for
pipette theory, design and practical consideration. Pipettes are pulled into the correct
geometry using a Model P-97 Sutter Instruments Co. [155] Flaming/Brown Micropipette
Puller (S/N 9878). The pipettes are fire polished using a MF-830 Micro-Forge (Narishige
[156], Japan; S/N 08087). Pipettes had a length of 38.1 mm.
The formation of a G seal is required. Failure to form a G seal will result in a
low seal resistance (RSeal). This will significantly impact recordings (correct
configuration; R(Seal)>>R(Membrane)>>R(Pippette)) as R(Seal)<<R(Membrane). For detailed
description of the concepts related to the circuitry involved in whole-cell recordings the
authors recommend the text Ion Channels of Excitable Membranes [157] and
Foundations of Cellular Neurophysiology [158]. Additionally, the Axon Guide [159] is
an outstanding review of concepts and amplifier design and theory.
Following placement of the pipette in close geometry to the cell membrane (
to
rad) the chamber is sealed and pressurized to 13.789 to 34.473 KPa over ambient
pressure. If starting with He as the test gas the chamber must be flushed to set a
He/O2/CO2 background. Chamber flushing requires approximately x3 volume of
chamber volume as previously discussed. It is critical that the chamber flush
characteristics (varying rates) be defined via a Mass Spectrometer prior to
experimentation. Chamber pressurization rate will significantly impact whole-cell
success.
3
An Ashcroft [160] Model 3000 Volumetric Controller is utilized to obtain the cellattached and whole-cell configurations. The Model 3000 allows precise pressure control
to +/-1.7 Pa; maximal working pressure 41.368 MPa. Specifics of the tubing and overall
model information are defined in R-19 to R-22.
The Model 3000 has two configurations as installed for MTC-4; 1) Balance Valve
Open System in Pressure Balance (BVOPB) and 2) Balance Valve Closed System in
Operation Mode (BVCOM). In BVOPB the system is equilibrated with chamber
pressure and is the configuration for initial pressurization and chamber travel. The
143
system is placed in BVCOM to form the cell-attached and whole-cell configuration.

Additionally, BVCOM is utilized in the event a small negative pressure is required to reestablish the seal at pressure. Operational procedures for the NPS are defined in Table
5.
During testing it was found that a hysteresis in the dead space between the NPS
and MTC-4 resulted in a pressure pulse when transitioning from BVCOM to BVOPB. To
mitigate the pressure differential a directional control valve was installed proximal to the
pipette holder. The Ingersoll-Rand/ARO (SN L1192) 4 way/2 position 3.175 mm
Solenoid/Spring A211SS-120-A (Grainger; PN 4XT32) was utilized.
The energized state allows the pressure hysteresis inherent in the tubing to be
mitigated at the pipette holder. Therefore the system stays within the 1.034 MPa
differential limit of the A211SS-120-A. When shifted to the de-energized state the
pipette and volumetric controller are equilibrated in either the bias or balanced
configuration. Figures 97 to 100 shows the A211SS-120-A (disconnect) schematic
representation of the external and internal NPS system, the NPS controller panel, and
the A211SS-120-A control box in the final operational configuration.
Stable whole-cell recordings were obtained with a 30% success rate. Defining
criteria for a successful recording are; 1) Obtaining a whole-cell configuration and 2)
Stable whole-cell configuration during compression. Whole-cell configurations were
stable to 3.457 MPa. A controlled dive is critical to successfully maintaining the G
seal. Experiments require significant experience. The operator/s must have an
understanding of the condition of the cells, theoretical aspects, and applied aspects of
the system.
144
TABLE 5: MTC-4 Negative Pressure System (NPS) Procedures.

Step
1
2
3
4
5
6
Component
Procedure
Note
CA1
Closed
Not connected
CA2
Closed
Not connected
CA5
Open
Chamber reference
CA6
Open
Volumetric bias
CA3
Open
Equilibrated
A211SS-120- Unpowered
Solenoid valve
A
System in Ready Configuration (Steps 1-6 must be completed prepressurization).
8
Chamber closed and pressurize to 2 psig (13.789 KPa).
9
CA3
Closed
Bias activated
10
Placement of pipette in proper geometry for cell attached
configuration.
11
Controller
Counter
Gentle negative pressure to
clockwise
form G seal.
12
Stabilization of G seal; + 5 min.
13
Controller
Counter
clockwise
enter whole-cell configuration
14
Whole-cell configuration stabilization and dialization; +10 min.
15
A211SS-120- Powered
Solenoid
A
16
CA3
Open
Equilibrated
System in Dive Configuration; verify A211SS-120-A in powered configuration
prior to travel.
Optional Step (Seal Enhancement at Depth)
Opt 1
CA3
Closed
Bias activated
Opt 2
A211SS-120- Unpowered
Solenoid valve
A
Opt 3
Controller
Counter
clockwise
improve seal.
Opt 4
A211SS-120- Powered
Solenoid valve
A
Opt 5
CA3
Open
Equilibrated
Opt 5
Repeat Opt 1-5 as required; ensure in equilibrated position and
A211SS-120-A powered prior to chamber travel
Notes:
Continued:
145
146
147
147
C
Figure 1: MTC-4 A211SS-120-A 4 Way 2 Position Solenoid Valve and Local Equipment.
The figure shows the locations of; A) Perfusion bath micro-handling pump, B) Negative pressure system solenoid valve, and C) Instrumentation ground block.
Perfusion plate location and connections to TC-344B are critical due to limited space. Associated wiring is shown. Components are disconnected for viewing.
148
148
147
Figure 2: MTC-4 Negative Pressure System (NOS) Schematic Representation.
The figure shows the external schematic diagram of the NPS. Components defined are external to primary pressure boundary.
Figure 3: MTC-4 Negative Pressure System (NPS) A211SS-120-A 4 Way 2 Position Solenoid Valve.
The figure shows the powered and unpowered solenoid valve configurations.
149
150
149
150
Figure 4: MTC-4 Negative Pressure System (NPS) External Controls.
The figure shows the external control panel for the NPS. Operational procedures are defined in Table 5.
ELECTROPHYSIOLOGY METHODS AND EQUIPMENT

The head-stage cable (4.0 m) was sealed in a PAVE PT16-B-150-AXOPATCH200B70-48 (P/N 36618) penetrator. The unit is hermetically sealed in a bi-directional
pressure housing and was pressure tested to 5.171 MPa (100% He bi-directional;
Appendix H). An Axon Instruments CV203BU was utilized. Axon instruments calibrated
the cable and head-stage post construction. The CV203BU was mounted to the pipette
manipulators using manufactured head-stage holder (K-59) and support rods (K-60).
The support rod had a diameter of 6.35 mm and was made of SS. The head-stage
holder was manufactured from 6061 Aluminum.
Membrane currents are measured using an Axopatch 200B amplifier (Axon
Instruments). Analog to digital conversion was accomplished using an Axon Instruments
1322A Digidata. Data was low-pass filtered at 1 kHz and sampled at 10 kHz. The
pClamp 10 suite was utilized for experiments and analysis of data. An ET072 (EDAQ)
leak-free Ag/AgCl electrode was utilized as a reference electrode. The ET072 is a nonleak reference electrode.
Cells were cultured on SecureSlip Glass Coverslips (12 mm/12 mm). The
inserts are mounted in the RC-27LD bath (37 C) for experiments. Cells are perfused for
5 min using external perfusion media prior to starting experiments. Culture protocols will
vary depending on the cell type/s utilized.
Two World Precision Instruments (WPI; Sarasota, Florida) DC3001 L (SN 8309
O3H) and DC3001R (SN 95535 03J) motorized manipulators were utilized for
placement of pipette electrodes and ancillary probes. Final configuration is dependent
on experimental requirements. Model MS314 Marzhauser Wetzlar (Wetzlar, Germany)
bi-directional (X/Y/Z- axes) controllers (WPI; SN 95533 O3J & 91009 O6H) are used for
fine placement (continuous speed; 5 m s-1) of probes. Manipulator backlash was not
compensated. Manipulators were maintained as per the manufacture
recommendation [161]. Proper care of the manipulators post experiments is critical to
minimize manipulator drift. Condensation was a major problem following experiments.
Manipulators were cleaned daily using 70% EtOH (ddH 2 0). Manipulators were serviced
bi-annually.
Figure 101 shows the placement of the pipette in reference to the primary
objective. The pipette and objective are advanced in unison. The manipulators,
adaptors, and head stage holder are shown installed in Figure 102. The figure shows a
dual manipulator configuration.
Figure 103 defines the placement of the reference electrode in relationship to the
perfusion plate cell bath (Figure 104). The reference electrode is installed at the effluent
end of the well. The system allows for accurate placement in the cell-attached
configuration as defined in Figure 105.
151
152
152
152
Figure 5: MTC-4 Pipette Placement.
The figure shows placement of 2-3M pipette in reference to the solution path. The laminar flow well may be removed for placement of the pipette in the opposite
configuration. The plate temperature probe and connections are not shown installed. Latter mounting plates include a depression for the cables and mounting
electrodes.
153
153
153
Figure 6: MTC-4 Manipulator Placement.
The figure shows placement of World Precision Instruments (WPI; Sarasota, Florida) DC3001 L (SN 8309 O3H) and DC3001R (SN 95535 03J).
154
154
154
Solution In
Solution Out
Figure 7: Mobile Test Chamber 4 (MTC-4) Bath Reference.

The figure shows the placement of the ET072 (EDAQ) leak-free Ag/AgCl reference electrode in the bath solution. The reference is mounted on the effluent side.
The electrode is shown in a disconnected status for viewing.
155 155
155
Figure 8: MTC-4 RC-27LD Geometry.
The figure shows the RC-27LD bath utilized for electrophysiology experiments. Bath can vary depending on experiments.
156
156
156
Figure 9: Mobile Test Chamber 4 (MTC-4).

The figure shows trypsin treated large diameter dorsal root ganglia soma following dissociation and plating for whole cell experiments. Scope is in a Bright Field
configuration. Cells were purchased from BrainBits and cultured per manufacture recommendations. Detailed cell culture procedures are not included.
HIGH PRESSURE CELLULAR PERFUSION SYSTEM STANDARD BATH SOLUTION

CHARACTERIZATION
To address the stability of gas in solution the dissolved O 2 (DO; M L-1) concentration
was defined using the NeoFox-GT (SN NFG0379) system (Ocean Optics; Dunedin, FL).
Red Eye probes (4 mm and 8 mm; Foxy coating, calibrated to manufacture standard).
Calibration was verified onsite following delivery of probes using varying partial
pressures O2 with a He background. Random samples (N=4) of individual shipments
were tested. Red Eye probes were cleaned with dd H 2 0 and stored as per manufacture
recommendation. Probes were utilized for a maximal of 24hrs. The signal was
transferred using a RE-BIFBORO-2 optical cable. The assumption is that O 2
characteristics will be similar to other gases with the exception of solubility. At the
defined pressure, and maximal chamber pressure, standard gas laws are assumed.
The probes were calibrated using the manufacture supplied calibration disk. TwoPoint calibration followed by Single Point Reset Control was completed daily.
Data was collected and converted to an Excel Database using the NeoFox Viewer
Software. The logging interval was set to 100 ms. All data columns were exported to
Excel for analysis.
Perfusion bath temperature was defined using a NeoFox-GT temperature probe
(NEOFOX-TP). The RC-27LD chamber was labeled with a gird patter as shown in
Figure 106. Grid pattern was defined by sight and should be treated as an rough array.
Probes have an adhesive layer for mounting. The attachment layer and supporting
polymer layer are non-toxic. The chamber is defined as the Standard Well Geometry
(SWG) for analysis purposes.
Reference (135.826 KPa) was defined as 20% O 2 , 5% CO 2 , and balance N 2 .
The experimental set-up is shown in Figure 106. Analysis of DO was completed at 2 mL
min-1 (37 C; +68.947 KPa ) in a 100% He background. Solution temperature was
maintained at 37 C. Gas temperature was ambient; 22.22 C. Solution temperature
was maintained using a inline heater and heating plate in a balanced feedback
configuration. There were no significant temperature variations in solution temperature
when the heating plate was not operational. Maximal pressure was limited to 25.579
KPa with a 6.894 KPa min-1 compression/ decompression rates. Test solution
consisted of 135 mM NaCl (pH 7.32; 1N NaOH). Gases were run until concentrations
reached a plateau. Pressure effects are assumed to be similar to ambient conditions
with respect to degassing rates.
Oxygen concentrations are normalized to reference value as defined in Figure
107. Reference is position 7. All values are defined as the percentage remaining O 2 .
The measured values are (% +/-SE); 1) 15.23 +/-5.02, 2) 42.06 +/-10.55, 3) 73.01 +/20.23, 4) 24.44 +/-6.23, 5) 37.87+/-10.22, 6) 62.82 +/-11.31, 7) reference, 8) 26.99 +/9.21, 9) 33.45 +/-5.55, and 10) 41.91 +/-2.55. As defined in these measurements there
is significant (p<0.05) degassing when compared to the reference value. Reference is
the location of affluent solution entry into the perfusion well. Tested values were
157
unstable as defined by the large standard error in the recordings. Degassing issues
tested with the culture slip were similar.
Red Eye Probe was verified in solution. Oxygen (100%) was bubbled into a 500
mL beaker (ddH 2 0) and the [O 2 ] was measured. The same approach was utilized for
21% O 2 (5% CO 2 balance N 2 ). Recording time was 30 min and 5 runs were completed.
The system set points are; 1) 42.44 KHz sampling rate, 2) Gain 8x, 3) LED 80%, and 4)
25 s LED cycling rate.
158
Affluent
Effluent
1-3
= 4mm Red
Eye Probe
4-7
8-10
Figure 1: MTC-4 RC-27LD 4mm Red Eye Probes Configuration.

The figure shows the RC-27LD well geometry (Upper) and 4 mm Red Eye probe (Lower) placement. To define the
rate of gas transfer, 4 mm O 2 sensitive probes were mounted to the bottom coverslip of the RC-27LD cell perfusion
chamber. The solution was tested at a 2 mL min-1. The flow rate is a standard flow rate for cell assays. Probe
placement is defined as shown in the figure with respect to the well. Probe placement is not exact. Probes are labeled
as defined in the figure with placement defined left to right. Probe position 7 is inlet reference. Probes (not shown) for
cell culture slips were 1-6 and 8-10. Reference probe is mounted on glass cover slip.
159
A
B
Affluent
Solution
Effluent
Solution
Figure 2: Mobile Test Chamber 4 (MTC-4) RC-27D 4 mm Red Eye Probes Experimental Set-up.
The figure shows the final placement of the A) RE-BIFBORO-2 optical cable and B) NEOFOX-TP. Solution afferent
and efferent lines are installed. The flow rate for experiments is 2 mL min-1. Figure shows the laminar flow guard
removed.
160
CHAMBER ANCILLARY AND ELECTRICAL SYSTEMS

Effluent solution is aspirated using a Valco Instruments [162] Company Inc. VICI AG
International M50 Syringe Free Liquid Handling Pump (CP3-8182-625D) controlled by a
Microlynx-4/7 Controller (CP-CM1-P ) [163]. The M50 is non-pulsatile and has an
operational range of 1-2500 L min-1. Tubing seals were replaced as required.
Replacement cycle did not exceed 14 days. Effluent media is collected in a T-150
(culture flask) mounted under the fore sled. A vented flask should be utilized. The T-150
is connected to an external turn valve to allow for removal (Figure 4) of media. The
chamber must be compressed to reach the appropriate pressure following a media
dumb. Venting of the chamber will result in a pressure drop unless venting and
compression are matched. Utilization of the over-flow external dump resulted in a high
experimental failure.
As based upon chamber volume, de-gassing of effluent media during gas
switching was not significant (i.e., reservoir volume is 150 mL and chamber volume is
6.7 x 105 mL). The gas background was tested using Mass Spectrometer. During
constant flow perfusion (240 min; 2 mL min-1) there was no measurable O 2 , CO 2 , and
N 2 increase in the defined background. A large flask was utilized to test stability.
Solutions were dripped into the flask to maximize degassing. No venting was used to
ensure maximal degassing.
Power was supplied by a (+24 VDC, 2.1 A) GLOGTEK, Inc., power supply
(P/N#GS-1181CR) with a vendor provided motor power cable (I-23688-CE). An
Edgeport 4I74162027-0 USB to Serial Hub (RS-232 ribbon cable; P/N I-24183) is used
to integrate the M50 and I-23688-CE. The M50 is shown mounted in Figure 109, with
the housing assembly defined in Figure 104 to 106. The pump assembly measurements
are defined in K-51.
Wiring is defined in Appendix Q. The equipment pin wiring is shown in Figures
112 to 118. Electrical and equipment requirements will vary from system to system and
therefore are specific for experimental requirements.
161
Figure 3: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Mounted.
The figure shows the placement and mounting of the micro-handling pump. Pump electrical connections are mounted
below the Aft Front Sled. Affluent and effluent solution lines are mounted on the motor. Compression fittings ensure a
firm fit and minimize leakage. The effluent line was set to a value slightly greater than the affluent line.
162
C
B
Figure 4: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Components.
The figure shows the effluent perfusion system; A) Waste receptacle (varies based on experiments), B) Pump lower
housing, C) Perfusion line (Tygon; R-3603) 1.5878 mm (ID) with SS port, D) Tubing connection points, and E) M50
syringe pump installed in manufactured housing. Electrical connections are removed from the pump (connector on
front of housing).
163
165
164
Figure 5: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Mounting Upper View.
The figure shows the lower (A) and upper (B) M50 assemblies. The upper assemblies (M50 and housing) are housed in the lower assembly. The completed unit is
installed in the aft front sled using mounting screw as shown in Figure 103.
165
165 166
Compression
Fitting
Adaptor
Figure 6: MTC-4 VICI AG International M50 Syringe Free Liquid Handling Pump Affluent and Effluent Line Connections.
The figure shows the vendor supplied pump adaptor with compression fitting on the Tygon tubing. Tubing verified to be non-leaching.
167
166
Figure 7: MTC-4 Prior H29/30 Stage Wiring Pin Out.
The figure shows the Prior H29/30 main connector pin out. The cable connectors were modified to be flush with the H29/30.
167 167
168
Figure 8: MTC-4 Prior H29/30 X- and Y-Axis L120/K120 Stage Wiring Pin Out.
The figure shows the location of X- and Y- axis motor cables and associated connector pin out.
169
168
168
Figure 9: MTC-4 Prior MM-3M-F-1.5-HT Stage Wiring Pin Out.
The figure shows the 10 pin motor connector pin out for the MM-3M-F-1.5-HT and the manipulators mounting location.
169
169
170
Figure 10: MTC-4 HF110 10 Position Filter Wheel Pin Out.
A/4th Axis Pin Outs

Pin# Signal
1
A+
2
B+
3
+ Limit
4
n/c
5
Sense
Ground
7
n/c
8
5V
9
A10
B11
- Limit
12
Ground
13
n/c
14
home(L)
15
n/c
Note: 1. Pins 3,4, & 11 Short together for filter
wheel
2. Put 560 ohm resistor between 5 and 12 for
rotary axis no limits
3. Put 1500 ohm resistor between 5 and 12 for
SH-27B
Figure 11: MTC-4 SH-27B Inline Heater.
The figure sows the SH-27B inline perfusion flash heater.
170
Figure 12: MTC-4 Inline-Solution heater Cable wiring (Solder side view).
The figure shows the SH-27B connector cable pin out.
171
Figure 13: MTC-4 CC-28 Cable Assembly Heater Plate Cell Bath.
The figure shows the cell plate heater unit connector cable and associated components and pin out.
172
DISCUSSION
The development of the Hyperbaric Upright Hoffman Contrast Microscope with
epifluorescence capability was supported under Office of Naval Research, Undersea
Medicine Program (Warfighter Protection and Application; Division 342, Code 34) award
numbers N0001412WX20784 [1] and N0001412WX20610 [2].The completion of this
project provides the research community with the first hyperbaric system capable of
whole-cell recordings and multiple band fluorescence recordings to a maximal pressure
of 3.457 MPa. As designed, the system has the potential to unlock secrets that will
result in future pharmacological approaches to diving issues.
The system is limited to a maximal O 2 of 21.284 KPa. Perfusion was modified to
equilibrate media with chamber gas. This modification was required since degassing of
solution was significant when chamber and media were not matched. Varying the flow
rate (0.5 mL to 10 mL min-1) in the well did not mitigate the effect. Flow rates 2 mL
min-1 resulted in increased off-gassing. Rates in excess of 2 ml min-1 resulted in
unstable whole-cell configuration and cell compromise.
This is in contrast to reports in the literature [81-84] in which off-gassing was not
significant. However, we question these finding as the law of mass transfer in a wellmixed gas liquid interface would result in a significant degassing issue. However, we did
not replicate the researchers system perfectly. Therefore our results may be due to
variations in measurement technique or perfusion geometry. This issue requires further
elucidation as degassing is a significant variable. If gas concentration is not stable then
it is impossible to define the variations in cellular physiology and accurately produce
dose (concentration) response curves for individual gases. The issue is not addressed
in the literature. The significant flaw in methodology (i.e., degassing is not significant as
based upon flow rate) raises question on the interpretation of data to date.
The system is designed as a standard Brightfield and Hoffman Contrast
fluorescence capable upright microscope and is housed in a hyperbaric chamber (MTC4). MTC-4 is constructed to permit safe neurophysiological studies while under high
energy conditions. Operating and emergency procedures are defined to provide a
guideline for future researchers. It is critical that personnel operating a similar system
have a firm understanding of the engineering and practical aspects of hyperbaric
operations. All assigned individuals entering this field should have a strong training in
hyperbaric safety and a significant background in electrophysiology.
It is our hope that this nascent system will represent the initial stage in the
elucidation of the cellular aspects of narcosis and HPNS. However, as based on the
design it is capable of addressing a variety of issues related to gas concentration.
Quantitative data is being prepared for submission as multiple manuscripts or in the
development of future projects. Therefore, we have written this technical report as a
build and resource manual. All publishable data has been removed from this TR and is
currently being prepared for peer-reviewed publication.
173
In brief, we have provided a detailed build plan for a hyperbaric electrophysiology

system with epifluorescence capabilities. We hope this system will represent the initial
step in developing the field of hyperbaric pharmacology research. We have included all
documentation and design drawings to facilitate future system.
174
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Wetzlar, M. Marzhauser Wetzlar Products. 2014 03212014]; Available
from: http://www.marzhauser.com/en/products.html.
Lumencor. Lumencor Light for Life Sciences. 2014 03212014]; Available from: http://lumencor.com/.
Chroma. Chroma Technology Corp. 2014 03212014]; Available from: http://www.chroma.com/.
Switronix. Switronix. 2014 02312014]; Available from: http://www.switronix.com/.
Designatronics. Designatronics Inc. 2014 03212014]; Available from: http://www.designatronics.com/.
AGS. AGS Lubricants. 2014 03212014]; Available from: http://www.agsco
MPany.com/lubricants/homehardware/183.
Imaging, Q. QImaging. [Online] 2013 [cited 03212014; Available from: http://www.qimaging.com/.
PixeLink. Machine Vision Camera. 2014 03212014]; Available from: http://www.pixelink.com/.
Imbert, G., et al., A system for saturating in vitro preparations with high pressure O2, He, H2, and
mixtures. Undersea Biomed Res, 1992. 19(1): p. 49-53.
Moore, H.J., et al., HYDROGEN-RATED SYSTMI FOR "IN VIT'On SITJDIES AT PRESSURE:
OPERATING PROCEDURE AND EMERGENCY PROCEDURES. 1993, Naval Medical Research Inst.:
Bethesda, Maryland. p. 75.
Viladsen, J., Nielsen, J., and Liden, G., Gas-Liquid Mass Transfer, in Biomedical Engineering Principles.
2011, Springer US. p. 459-496.
Palmes, E.D., and Lindenboom, R.H., Ohm's law, Fick's law, and diffusion samplers for gases. Analytical
Chemistry, 1979. 51(14): p. 2.
Philibert, J., One and a Half Century of Diffusion: Fick, Einstein, before and beyond. DiffusionFunamentals, 2005. 2: p. 10.
Gast. Gast Precision Air-Moving Products. 2014 03212014]; Available from: http://www.gastmfg.com/.
Bronkhorst. Bronkhorst USA. 2014 03232014]; Available from: http://www.bronkhorstusa.com/.
Co MPany, H.A. Warner Instruments. 2014; Available from: https://www.warneronline.com/.
Tuttnauer, Opertaion and Maintenance Manual MAN205-0007-000E rEV.k ed. Table-Top Autoclaves
Models 1730, 2340, 2540, 3140, 3850, 3870 M and MK 1730MK Valueklave. Hauppauge, NY: Tuttnauer
U.S.A. 47.
International, V., Ultrasonic Cleaners Instruction Manual. West Chester, PA: VWR International. 27.
Swagelok, Quick Connects. 2013: USA. p. 20.
Extrel. Extrel Core Mass Spectrometers. 2014 03242014]; Available from: http://www.extrel.com/.
Gas, A. Air Gas. 2014 03242014]; Available from: http://www.airgas.com/.
179
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
Suchyna, T.M., V.S. Markin, and F. Sachs, Biophysics and structure of the patch and the gigaseal. Biophys
J, 2009. 97(3): p. 738-47.
Priel, A., et al., Ionic requirements for membrane-glass adhesion and giga seal formation in patch-clamp
recording. Biophys J, 2007. 92(11): p. 3893-900.
Oesterle, A., P-1000 & P-97 Pipette Cookbook 2011 (Rev. G). 2011, Novato, CA: Sutter Instrument Co
MPany.
Sutter. Sutter Instruments. 2012 03242014]; Available from: http://www.sutter.com/.
Narishige. Narishige Japan. 2014; Available from: http://www.narishige.co.jp/english/.
Hille, B., Ion Channels of Excitable Membranes. 3 ed. 2001: Sinauer Associates, Inc.
Johston, D.W., Samuel Miao-Sin, Foundations of Cellular Neurophysiology. 1994: Bradford Book.
Devices, M., The Axon Guide: A Guide to Electrophysiology & Biophysical Laboratory Techniques (Rev.
C), R. Herman-Gold, Editor. 2007, MDS Analytical Technologies. p. 286.
Ashcroft. Ashcroft. 2014; Available from: http://www.ashcroft.com/.
WPI, Micrimanipulators Care and Maintenance: Instruction Manuel. 2001, World Precision Instruments.
VICI. Valco Instruments CO. Inc. VICI AG International. 2014 03242014]; Available
from: http://www.vici.com/.
Intelligent Motion Systems, I., MicroLYNX 4/7 QuickManual. REV051205 ed. Hardware Software
Applications. 2006, Marlborough, CT: Intelligent Motion Systems, INC. 106.
180
APPENDIX A
MOBILE TEST CHAMBER 4 (MTC-4) CONTROLLED ASSEMBLY
DOCUMENTATION NOTEBOOK
A-1
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-11
A-12
A-13
A-14
A-15
A-16
A-17
A-18
A-19
A-20
A-21
A-22
A-23
A-24
A-25
A-26
A-27
A-28
A-29
A-30
A-31
A-32
A-33
A-34
A-35
A-36
A-37
A-38
A-39
A-40
A-41
A-42
A-43
A-44
A-45
A-46
A-47
A-48
A-49
A-50
A-51
A-52
A-53
A-54
A-55
A-56
A-57
A-58
A-59
A-60
A-61
A-62
A-63
A-64
A-65
A-66
A-67
A-68
A-69
A-70
A-71
A-72
A-73
A-74
A-75
A-76
A-77
A-78
A-79
A-80
A-81
A-82
A-83
A-84
A-85
A-86
A-87
A-88
A-89
A-90
A-91
A-92
A-93
A-94
A-95
A-96
A-97
A-98
A-99
A-100
A-101
A-102
A-103
A-104
A-105
A-106
A-107
A-108
A-109
A-110
A-111
A-112
A-113
A-114
A-115
A-116
A-117
A-118
A-119
A-120
A-121
A-122
A-123
A-124
A-125
A-126
A-127
A-128
A-129
A-130
A-131
A-132
A-133
A-134
A-135
A-136
A-137
A-138
A-139
A-140
A-141
A-142
A-143
A-144
A-145
A-146
A-147
A-148
A-149
A-150
A-151
A-152
A-153
A-154
A-155
A-156
A-157
A-158
A-159
A-160
A-161
A-162
A-163
A-164
A-165
A-166
A-167
A-168
A-169
A-170
A-171
A-172
A-173
A-174
A-175
A-176
A-177
A-178
A-179
A-180
A-181
A-182
A-183
A-184
A-184
A-185
A-186
A-187
A-188
A-189
A-190
A-191
A-192
A-193
A-194
A-195
A-196
A-197
A-198
A-199
A-200
A-201
A-202
A-203
A-204
A-205
A-206
A-207
A-208
A-209
A-210
A-211
A-212
A-213
A-214
A-215
A-216
A-217
A-218
A-219
A-220
A-221
A-222
A-223
A-224
A-225
A-226
A-227
A-228
A-229
A-230
A-231
A-232
A-233
A-234
A-235
A-236
A-237
A-238
A-239
A-240
A-245
A-246
A-247
Non Destructive Testing Mobile Test Chamber 4 (MTC4) Colomb NEDU
A-248
A-249
A-250
A-251
A-252
A-253
A-254
A-255
A-256
A-257
A-258
A-259
A-260
A-261
A-262
A-263
A-264
A-265
A-266
APPENDIX VII
Recommended Unit Symbols, SI Prefixes, and Abbreviations
A.
TABLE I
Recommended Unit Symbols
SI PREFIXES
The following standards provide the recommended abbreviations,

symbols, and units for IEEE publications.
IEEE Standard Dictionary of Electrical and
Electronic Terms, Sixth Edition
IEEE Std 260.1-1993 American National Standard Letter Symbols
for Units of Measurement (SI Units,
Customary Inch-Pound Units, and
Certain Other Units)
IEEE Std 280-1985
American National Standard for Mathematmatical Signs and Symbols for Use
in Physical Sciences and Technology
IEEE Std 280-1985
IEEE Standard Letter Symbols for
(R1997)
Quantities Used in Electrical Science
and Electrical Engineering
IEEE Std 315-1975
IEEE Graphic Symbols foe Electrical and
(R1993)
Electronics Diagrams (Including
Reference Designation Letters)
(Includes supplement 315A-1986, R1993)
SI 10-1997
(IEEE/ASTM) Standard for Use of the
International System of Units (SI)
The Modern Metric System
When a compound unit is formed by the multiplication of two or

more units, its symbol consists of the symbols of the separate units
joined by a raised dot; for example, N # m for newton meter. When a
compound unit is formed by the division of one unit by another, its
symbol consists of the separate symbols either separated by solidus
(slant) or multiplied using negative powers; for example, either m/s or
m # s-1 for meters per second.
Prefix
Symbol
24
yotta
zetta
exa
peta
tera
giga
mega
kilo
hecto
deka
deci
centi
milli
micro
nano
pico
femto
atto
zepto
yocto
Y
Z
E
P
T
G
M
k
h
da
d
c
m
n
p
f
a
z
y
10
1021
1018
1015
1012
109
106
103
102
10
10-2
10-2
10-3
10-6
10-9
10-12
10-15
10-18
10-21
10-24
IEEE Std 100-1996
The above standards are all available from IEEE, 445 Hoes Lane, P.O.
Box 1331, Piscataway, NJ 08855-1331 USA, Telephone +1-800-678IEEE. Some symbols from these standards are given in Table II of
part C of this appendix. Their form is the same for both singular and
plural usages, and period is not used in their abbreviations. The
distinction between the use of upper-case and lower-case letters should
be carefully observed.
Multiple
B.
Recommended SI Prefixes
Prefixes indicating decimal multiples or submultiples of units and

their symbols are given in Table I. Compound prefixes, such as
micromicro for pico and kilomega for giga are discouraged.
C.
Recommended Abbreviations
In general, most abbreviations of technical terms are capitalized,

but there are notable exceptions such as ac, dc, and rms. In addition
to the unit symbols, Table II lists many common technical
abbreviations in their standard IEEE editorial forms. Note that periods
are not used and the abbreviation is the same regardless of whether it
is used as a noun or an adjective. An abbreviation that is new or not
generally accepted should be defined when first used. In abbreviations
involving a persons name, always capitalize the initial for the persons
name.
TABLE II
ABBREVIATIONS AND LETTER SYMBOLS FOR UNITS
Unit or Term
alternating current
American wire gauge
ampere
ampere # hour
ampere turn
amplitude modulation
antilogarithm
audio frequency
automatic frequency control
automatic gain control
automatic volume control
average
Abbreviation
ac
AWG
A
Ah
A
AM
antilog
AF
AFC
AGC
AVC
avg
Unit or Term
baud
beat-frequency oscillator
binary coded decimal
bit
British thermal unit
calorie
candela
candela per square foot
candela per square meter
cathode-ray oscilloscope
cathode-ray tube
centimeter
-29-
A-267
Abbreviation
Bd
BFO
BCD
b
Btu
cal
cd
cd/ft2
cd/m2
CRO
CRT
cm
TABLE II
(continued)
Unit or Term
circular mil
continuous wave
coulomb
cubic centimeter
cubic foot per minute
cubic meter
cubic meter per second
decibel
degree Celsius
degree Fahrenheit
degree (plane angle)
degree Rankine
degree (temperature interval or difference)
diameter
direct current
electromagnetic compatibility
electromagnetic unit
electromotive force
electronic data processing
electronvolt
electrostatic unit
extra-high voltage
extremely high frequency
extremely low frequency
farad
field-effect transistor
foot
foot per minute
foot per second
foot pound-force
frequency modulation
gallon
gallon per minute
gauss
gigaelectronvolt
gigahertz
gram
henry
hertz
high voltage
hour
inch
inch per second
inductance-capacitance
inertia
infrared
inside diameter
intermediate frequency
joule
joule per degree
kelvin
kiloelectronvolt
kilogram
kilohertz
kilohm
Abbreviation
cmil
CW
C
cm3
ft3/min
m3
m3/s
dB
(C
(F
...(
(R
deg
diam
dc
EMC
EMU
EMF
EDP
eV
ESU
EHV
EHF
ELF
F
FET
ft
ft/min
ft/s
ft # lbf
FM
gal
gal/min
G
GeV
GHz
g
H
Hz
HV
h
in
in/s
LC
kg # m2 or lb # ft2
IR
ID
IF
J
J/deg
K
keV
kg
kHz
k6
Unit or Term
kilojoule
kilometer
kilometer per hour
kilovar
kilovolt
kilovoltampere
kilowatt
kilowatthour
lambert
liter
liter per second
logarithm
logarithm, natural
low frequency
lumen
lumen per square foot
lumen per square meter
lumen per watt
lumen second
lux
magnetohydrodynamics
magnetomotive force
medium frequency
megaelectronvolt
megahertz
megavolt
megawatt
megohm
metal-oxide semiconductor
meter
meter-kilogram-second
microampere
microfarad
microgrm
microhenry
micrometer
micromho
microsecond
microwatt
mile per hour
mile (statute)
milliampere
milligram
millihenry
milliliter
millimeter
millisecond
millivolt
milliwatt
minute (plane angle)
minute (time)
nanofarad
nanometer
nanosecond
nanowatt
-30-
A-268
Abbreviation
kJ
km
km/h
kvar
kV
kVA
kW
kWh
L
L
L/s
log
ln
LF
lm
lm/ft2
lm/m2
lm/W
lm # s
lx
MHD
MMF
MF
MeV
MHZ
MV
MW
M6
MOS
m
MKS
A
F
g
H
m
6-1
s
W
mi/h
mi
mA
mg
mH
ml
mm
ms
mV
mW
...
min
nF
nm
ns
nW
TABLE II
(continued)
Unit or Term
neper
newton
newton meter
newton per square meter
ohm
ounce (avoirdupois)
per unit
phase modulation
picoampere
picofarad
picosecond
picowatt
pound
poundal
pound-force
pound-force foot
pound-force per square inch
pound per square inch
power factor
radian
radio frequency
radio-frequency interference
resistance-capacitance
resistance-inductance-capacitance
revolution per minute
revolution per second
roentgen
root-mean-square
second (plane angle)
second (time)
Abbreviation
Np
N
N#m
N/m2
6
oz
pu
PM
pA
pF
ps
pW
lb
pdl
lbf
lbf # ft
lbf/in2
lb/in2
PF
rad
RF
RFI
RC
RLC
r/min
r/s
R
rms
...
s
Unit or Term
short wave
siemens
signal-to-noise ratio
silicon controlled rectifier
square foot
square inch
square meter
square yard
standing-wave ratio
television interference
tesla
thousand circular mils
transverse electric
transverse electromagnetic
transverse magnetic
traveling-wave tube
vacuum-tube voltmeter
var
variable-frequency oscillator
very high frequency
volt
voltage controlled oscillator
voltage standing-wave ratio
voltampere
watt
watthour
watt per steradian
watt per steradian square meter
weber
yard
Although the use of the abbreviation psi is common, it is not recommended. See pound-force per square inch.
-31-
A-269
Abbreviation
SW
S
SNR
SCR
ft2
in2
m2
yd2
SWR
TVI
T
kcmil
TE
TEM
TM
TWT
VTVM
var
VFO
VHF
V
VCO
VSWR
VA
W
Wh
W/sr
W/(sr # m2)
Wb
yd
APPENDIX B
MOBILE TEST CHAMBER 4 (MTC-4) FIBER OPTIC (VS18-SS-150-1-FO96-48) AND USB (PT-16-SS-150-1-USBAFEMALE-USBAMODE)
B-1
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
B-11
B-12
B-13
B-14
B-15
B-16
B-17
B-18
B-19
B-20
Waiver/Deviation
Report
Record #
Draw #
274
2751 Tbunderhawk Court

Dayton, Ohio 45414-3445
Telephone (937) 890-1100
Fax (937) 8911-5165
Type Deviation
2/21 /2013
Date
Criticality Minor
4335
Phone
Customer Navy Lxperimental Diving Unit

Contact
Fax #
Lt. Dennis Colomb
Quantity
850-230-3100-3118
NCR#
30959
Purchase Order No463A 13POOOI
Affected Non Serialized Assembly NI A

Affected serialized assembly number NI A
Affected serialized assembly numbers from
NI A
Affects products manufactured on dates from
Through N/A
2/21/2013
Explaination of this request for deviation or waiver

Cable jacket sp lit and thc Kevlar stranding ins ide was pushed out of the jacket after the 1000 PS I leak test. Presumably
due to high pressure differencial inside cable when pressure was released in testing tube. Lt. Colomb agreed to our
attempted repair considering the part had passed both the leak test and the optuieal continuity test. We were able to push
the majority of the Kevlar stranding back into the jacket and trimmed off what was left outside. We used Kapton tape to
wrap th e split area (approvimately 2 inches starting at abo ut .6 inch from the pressure side (the thread side) of the housing.
Additional comments
Requested by
Production:
Brad Boomershine
Quality Assurance: Jack Holloway
Date
2/2 1/2013
Date
2/21/2013
Customer Response
Name
Title
Date
Follow-up
Comments
PAVE Technology Inc
F-0014
WID
B-21
Rev - B
3/14/2007
PAVE TECHNOLOGY CO.
DAYTON,OH
QUALITY ASSURANCE DEPT.
By
ACCEPTERB
2 2 2013
Date_ _ _ __
PAV ESEAl Dayton. OH USA
VS l SSS-150-1-FO-96 -Ja
PA E #.135 9
WG 22213
B-22
APPENDIX C
MOBILE TEST CHAMBER 4 (MTC-4) SYSTEM PRESSURE RELIEF
VALVES
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
APPENDIX D
MBILE TEST CHAMBER 4 (MTC-4) MODIFIED REAR FLANGE INSTRUMENT GROUND
AND REAR FLANGE ELECTRICAL
D-1
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
D-10
D-11
D-12
D-13
D-14
D-15
D-16
D-17
D-18
D-19
D-20
D-21
D-22
D-23
D-24
D-25
D-26
D-27
D-28
D-29
D-30
D-31
D-32
D-33
D-34
D-35
D-36
D-37
D-38
D-39
D-40
D-41
D-42
D-43
D-44
D-45
D-46
D-47
D-48
D-49
D-50
D-51
D-52
D-53
D-54
D-55
D-56
D-57
D-58
"'
Job Order No.

organizati~nal
Level Cleaning
Record Por All Gas Systems
Navy Experimen~al Diving Unit

Panama City, PL 32407-7051
(904) 230-3100
9'{)~-,
Drawing No.
Rote: Ail service. performed in accordance with NAVXDIVINGUINST 3900 Series.

3
Requesting Activity
Code
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Pate
7
Pointof Contact
Phone/Ext.
Task No.
2) 'i - l.~ -fb~
10
Date Required
ITEM DESCRIPTION 11
. Description
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4.
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CLEANING - .HYDROCARBON ANALYSIS 12
System
Service
Cleaning
Process' (TSP,
~'PX'
_ReOx -Btc. )
NID~ther)
HYdrocarbon Analysis
Test For
Cleaning
Agent Removed
Method
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CLDNING- PARTICULATE ANALYSIS 13
eI
Pre~
Pass/Fail
Microscopic Analysis
Conducted
Fail
Visual Analysis Conducted
Completed By:
CLBMmG/REPLACBMENT SOPTGOODS 14
Item
Work Performed
Material
Compollition
Cleaning
Agent
(Type)
Cleaned
Analysis
Method
(VIS, U/V,
I/R, Etc.)
Date
Completed By
Replaced
15 DELIVERED BY: _ _- - - - - - - - - - - -
16 RETORNBD TO:
(Date)
tDate)
REMARKS:
....
--
----
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Important Document Forward

to Quality Inspector/Engineer
.'
-
34842
Ilia'
2751 Thunderhawk Court

Dayton, Ohio 45414-3445 U.S.A.
1-937-89O-1100x24 1-937-890-5165 Fax
1509001:2000
KEMA CERTIFIED
e-mail: qa@pavetechnologyco.com
Catalog at www.pavetechnologyco.com
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CERTIFICATE OF CONFORMANCE
Customer~AVY't):PEe.LH9/JAt );eil2iN/r p6# !Jo4(P311-o9-PlOO3Item #_---'-A--'---PAVE Drawing #

3.3d- \
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Material
Serial #
Total
Quantity
_
_
,'------
TEST REPORT
All testing is performed within normal ambient conditions (pressure, temperature and relative humidity.)
Thermal Cycle: (As required by customer specification) All testing occurs after Thermal Cycle
Range
, Number of cycles
, Duration of Cycle
Leak Test: (Allow 1 min. for every 2' of wire length at 80 psi, no visible bubbles after time has expired)
PSI
7;0
Duration
,2Q 5.f?tJo/ds 1J1~
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Compressed Air
Nitrogen
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Bubble fluid
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/) Pressure Side
Helium
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Other
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Tester Initials
5'
/}5
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Helium Leak Test:
Test Sensitivity
cclsec He Air Equiv.
1 atmosphere. Number Tested
Equipment: ASM 142 Calibration Date
Tested By
Electrical Tests: (All electrical testing is performed after leak tests)
Continuity test:
AC Hypot:
DC Hypot:
~ Yes
Max. Q Connector Mateability Checked:

VAC,
(;z ,30
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High Voltage
. {) (
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Other
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seconds,
Maximum Leakage, mA
:TlJi)
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M-[L
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Minimum Resistance
Maximum Leakage, mA
,Q impedance, Capacitance
f.
'---
Tester Initials _----.=..:....1.-'1----.,1-"""'4(4:(",;/-1'>..:>.----------
Optical Test:
Maximum attenuation
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Visual check of Optical Continuity
Number Tested
Visual Workmanship Inspection
nm ( @ wavelength)
Tester Initials
~~~~
t::tt=
Documents Included
Additional notes: _---:...'_'
--------------------
OBJECTIVE EVIDENCE TO SUPPORT THIS CERTIFICATE OF COMPLIANCE IS ON FILE & WILL BE MADE AVAILABLE TO THE BUYER FOR
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Date
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Important Document Forward to Quality Inspector/Engineer
34843
~ ~

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KEMA CERTIFIED
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&I .;:;;:7t:;~.'::;; Cl~

Customer
U5 tl/hJ Y EXPcR
~J.-.g
PAVE Drawing #I
PAVE Order Code or Description

Seri~# ~
CERTIFICATE OF CONFORMANCE
ll1ENtAL LPitivv80#lNo+to3A-Oj -Poct:>3 Item#l _ _
L1
Rev.
Corresponding #I
vS J5 L - 55 - I S=Q -/ - U 5 8RmIJLE - U513a e male.,

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Housing
Ma~ri~
~.
Total
Quan~~
_ _~~/
TEST REPORT
All testing is performed within normal ambient conditions (pressure, temperature and relative
~_ _
humidi~.)
Thermal Cycle: (As required by customer specification) All testing occurs after Thermal Cycle
Range
, Number of cycles
, Duration of Cycle
Leak Test: (Allow 1 min. for every 2' of wire length at 80 psi, no visible bubbles after time has expired)
PSI
'1sD
Duration
<3[) 'y?CCYlclSm; I)
vi
Compressed Air
Nitrogen
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Bubble fluid
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\,L
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Helium
_c/:....-
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J' :::/
Other
"
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Tester Initials
!IJ
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Test Sensitivity
cc/sec He Air Equiv. @ 1 atmosphere. Number Tested
Equ~ment ASM142C~ib~~nD~e
~~edBy
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<...
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AC Hypot:
DC Hypot:
ttt.30
High Voltage
TOR test:
Other_"
VDC,
0 (
"VDC, '
seconds,
aVes
No
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seconds, \,.>
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500 M
&
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Q impedance, Capacitance
Minimum Resistance
Maximum Leakage, mA
Number Tested
Tester Initials
1\..
:..JCi!
VV\...
Optical Test:
Maximum attenuation
dB
Visual check of Optical Continuity
Number Tested
Visual Workmanship Inspection
Addnional notes:
C(jP
nm (
wavelength)
Tester Initials
Additional Quality
Documents Included
U7
OBJECTIVE EVIDENCE TO SUPPORT THIS CERTIFICATE OF COMPLIANCE IS ON FILE & WILL BE MADE AVAILABLE TO THE BUYER FOR REVIEW
UPON REQUEST.
. .
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~ATERIAlS ARE CERTIFIED BY OUR SUPPLIERS. All~~IAl CERTIFICA
Quality Assurance
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~R
LABlE UPON REQUEST.
Date
C of C
D-62
l~
F-0001 Revised 09/04
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3nOHOII108 MO
D-64
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D-66
TABLE 25 HOlO-KROME MECHANICAL PROPERTIES SOCKET HEAD CAP SCREWS-1960 SERIES
Standard Holo-Krome Alloy Steel Socket Head Cap Screws are manu
factured from Halo-Krome analysis high grade alloy steel hardened and
tempered to provide the following mechanical properties:
NOMINAL
SIZES
TENSILE STRENGTH
psi MIN.
YIELD STRENGTH
psi MIN.
ELONGATION
IN 2 INCHES
PER CENT MIN.
REDUCTION OF AREA
PER CENT MIN.
MINIMUM HARDNESS
ROCKWEUC
oto 1/2
Over 1/2
180,000
170,000
162,000
153,000
8
10
35
40
39
37
* The values for elongation and reduction of area apply only to cylindrical test speCImens.
Values are based on ASTM A574
CAUTION: IMPROPER LOADING OF THESE FASTENERS WILL CAUSE FAILURES

TABLE 25 TENSILE, YIELD AND SHEAR STRENGTH
NOMINAL
SIZE
BASIC
SCREW
DIAMETER
THREAD STRESS
AREA
STRAIGHT
TENSILE STRENGTH
(POUNDS MIN.)
UNRC
UNRF
UNRC
UNRF
STRAIGHT YIELD
STRENGTH
(POUNDS MIN.)
UNRC
UNRF
SINGLE SHEAR STRENGTH

(POUNDS MIN.)
THREAD SECTION
BODY
SECTION
475
665
875
320
500
710
940
425
600
790
'290
450
635
845
305
450
625
830
1,090
1,430
1,640
2,520
1,190
1,490
1,825
2,650
975
1,290
1,470
2,270
1,070
1,345
1,645
2,385
0.0200
0.0364
0.0580
0.0878
3,150
5,725
9,430
13,950
3,600
6,550
10,440
15,805
2,835
5,150
8,490
12,555
0.1187
0.1599
0.256
0.373
19,13~
21,365
28,780
43,500
63,400
17,220
25,540
38,400
56,750
78,500
103,000
164,700
238,800
86,500
112,700
182,400
268,800
0
1
2
3
0.0600
0.0730
0.0860
0.0990
0.00263
0.00370
0.00487
0.00180
0.00278
0.00394
0.00523
4
5
6
8
0.1120
0.1250
0.1380
0.1640
' 0.00604
0.00796
0.00909
0.0140
0.00661
0.00830
0.01015
0.01474
10
1/4
5/16
3/8
0.1900
0.2500
0.3125
0.3750
0.0175
0.0318
0.0524
0.On5
7/16
1/2
5/8
3/4
0.4375
0.5000
0.6250
0.7500
0.1063
0.'1419',>,
0.226
0.334
7/8
1
1-1/4
1-1/2
0.8750
1.0000
1.2500
1.5000
0.462
0.606
0.969
1.405
0.509
0.663
1.073
.1.581
UNRC
UNRF
290
405
535
205
315
445
595
1,060
1,325
1,615
2,280
665
875
1,000
1,535
750
940
1,150
1,670
3,240
5,900
9,395
14,225
3,060
5,295
8,285
11,910
1,920
3,495
5,750
8,510
2,270
4,130
6,575
9,950
34,550
51,100
19,230
25,905
39,150
57,050
16,200
21,175
31,300
45,050
11,650
15,565
23,450
34,650
13,445
18,105
27,400
39,950
70,700
92,700
148,250
214,950
77,850
101,450
164,150
241,900
61,350
80,100
125,100
180,200
47,900
62,850
100,500
145,700
54,500
71,000
114,900
169,300
22~990
NOTES FOR TABLE 25
The materials and heat treatment used for standard Holo-Krome Alloy
Socket Head Cap Screws were selected to provide products that would
have optimum tensile strength, impact strength and fatigue life. For
specific applications, screws can be supplied to higher hardness levels
so that their tensile strength will exceed values listed. However, such
screws will have lower ductility, impact strength and fatigue life at higher
stress levels than will standard screws.
The mechanical properties listed are for standard screws tested at

normal room temperatures. In applications where the temperatures
are considerably above or below room temperature the effect of the
temperature on the screw strength must be considered. Short time
tensile, creep and stress relaxation should be considered for applica
tions where the temperature exceeds 400F (204C). Lower ductility,
impact strength and fatigue life must be considered for screws subject
to temperatures below -20F (-29C).
31
D-67
TABLE 26 HOLO-KROME SOCKET HEAD CAP SCREWS - TORQUE DATA -1960 SERIES
TIGHTENING TORQUE DATA
TORQUE-TENSION
NOMINAL
SIZE
BASIC
SCREW
DIAMETER
TENSION INDUCED IN SCREWS

TOROUED AS RECOMMENDED
(POUNDS)
UNRF
UNRC
0
1
2
3
0.0600
0.0730
0.0860
0.0990
280
390
510
190
290
410
550
4
5
6
8
0.1120
0.1250
0.1380
0.1640
630
830
950
1,460
10
1/4
5/16
3/8 '
0.1900
0.2500
0.3125
0.3750
7/16
1/2
5/8
3/4
0.4375
0.5000
0.6250
0.7500
7/8
1
1-1/4
1-1/2
0.8750
1.0000
1.2500
1.5000
RECOMMENDED TIGHTENING TOROUE

(INCH - POUNDS)*
UNRC
UNRF
2.6
4.5
7.5
11.0
4.8
8.0
12.0
690
870
1,070
1,550
16.0
24.0
30.0
55.0
18.0
24.0
34.0
58.0
1,840
3,530
5,820
8,620
2,100
4,040
6,450
9,770
79.0
200.0
415.0
740.0
90.0
230.0
460.0
845.0
11,830
23,740
35,080
13,180
17,800
26,890
39,150
1,190.0
1,800.0
3,400.0
6,000.0
1,305.0
2,065.0
3,800.0
6,750.0
41,590
54,350
87,225
126,450
45,830
59,662
96,600
142,280
8,250.0
12,500.0
25,000.0
43,500.0
9,200.0
13,000.0
27,750.0
49,000.0
,---",""","
]
"--
* These tightening torque values are 75% of the torque required to yield the screw, and apply only for the conditions listed below.
Different percentages of torque-ta-yield values are also commonly used for special conditions.
NOTES FOR TABLE 26
These are average values for standard Holo-Krome 1960 Series Alloy
Steel Socket Cap Screws with black finish, tested with hardened steel
plates, and hardened nuts with the threads and bearing areas lubri
cated with plain, medium viscosity machine oil.
The relationship between the torque and the induced tension (preload)
can be expressed by the empirical formula T=KDP, in which T is the
tightening torque in inch pounds; 0 is the nominal diameter of the
screw; P is the tension (in pounds) induced in the screw; and K is the
torque coefficient. The torque coeffiecient is not constant but varies
with the material, surface finish 'and lubricity of the threads and head
bearing area of the screws and parts fastened.
For the conditions shown above (standard alloy steel black finished
screws clamping hardened steel parts), K will range from 0.19 to 0.25.
For cadmium plated screws with steel parts, Kwill usually fall between
0.13 and 0.17. For zinc plated screws K may fall between 0.30 and
0.34. When the thread and head bearing surfaces are covered with
certain types of lubricants, or with anti-seize compounds, K can drop
as low as 0.05. Atthe other extreme, combinations of certain materials,
such as austenitic stainless steel screws and parts not lubricated or
coated, can result in K values as high as 0.35, or more.
Because the induced tension can vary considerably from one type of
assembly to another for any given torque, the above data should be
sed with caution - particularly in applications where the control of
,reload is critical and must be obtained by the torque wrench method.
For such applications, the relationship between torque and induced
tension should be determined experimentally for the actual parts and

lubrication practice involved.
TIGHTENING TORQUES. At the tightening torques listed standard
Hol~Krome alloy steel 1960 series screws, used under the conditions
described, will be preloadad to apprOXimately 750/0 of the tension
induced at yield. The bearing stress under the head at these preloads
will be approximately 80,000 psi, so indentation should not occur when
the parts clamped are of steel or cast iron with a hardness equal to or
in excess of Rockwell B 85. With softer materials, washers may be
required under the heads of the screws to avoid indentation.
In applications where screws are subject to fatigue from dynamic
loading, the Importance of proper preloading during assembly
cannot be over-emphaslzed. The proper preload is especially
importantfor rigid-type (metal-to-metal) joints, where it has been found
that the use of a preload greater than the extemalload will usually
eliminate the possibility of fatigue failure. Forthis reason, the preferred
practice for such asemblies is to preload to 75k of the induced tension
at yield.
~IN~,HOWEVER,ANDFORASSEMBLIES
~TEMPERATURES, MUCH LOWER

PRELOADS MAY BE REQUIRED. No aeneral recommendations are
possible for such a
Ii s or saRMe cone,hons. bach appiacat,on
a yze .n IV' ua y, because the preload requirements may
vary considerably from one application to another. Users who desire
suggestions forthe preload ortightening torque to be used for a specifIC
application are invited to contact Holo-Krome.
32
D-68
Page 1 of 3
Bolt Preload Calculation
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Bar-Lock Rebar Couplers
Torque Sensing Solutions
Structural Analysis - aCE
Thread Protectors, LL
Easily and quickly splice rebar. No

threading or bar end treatment.
Measure Process Variables: Torque,

Speed, Horsepower, Angle, Force
Thermal, Stress & Vibration Structural

Analysis of Components
Oilfield Thread Protectors

Metal,Plastic,Cast,New, USI
www.daytonsuperior.com/Bar_Lock
www.mdi-sensor.com
www.odonnellconsulting.com
www.threadproducts.com
2002 Garrett D. Euler
2002-04-14, Rev. 2002-04-22.

Question: How is bolt installation preload calculated?
Answer: Bolt pretension, also called preload or prestress, comes from the installation torque T you
apply when you install the bolt. The inclined plane of the bolt thread helix converts torque to bolt
pretension. Bolt preload is computed as follows.
p.1
=T/(K D)
(Eq.l)
where Pi = bolt preload (called Fi in Shigley).

T = bolt installation torque.
K torque coefficient.
D bolt nominal shank diameter (i.e., bolt nominal size).
=
=
Torque coefficient K is a function of thread geometry, thread coefficient of friction lit, and collar
coefficient of friction Ile Look up K for your specific thread interface and collar (bolt head or nut
annulus) interface materials, surface condition, and lubricant (if any). ("Torque specs for screws,"
Shigley, and various other sources discuss various K value estimates.) If you cannot find or obtain K
from credible references or sources for your specific interfaces, then you would need to research to try to
find the coefficients of friction for your specific interfaces, then calculate K yourself using one of the
following two formulas listed below (Shigley, Mechanical Engineering Design, 5 ed., McGraw-Hill,
1989, p. 346, Eq. 8-19, and MIL-HDBK-60, 1990~ Sect. 100.5.1, p. 26, Eq. 100.5.1, respectively), the
latter being far simpler.
K ={[(0.5 dp)(tan A. + J.Lt sec f3)/(1 - ~t tan A. sec f3)] + [0.625 J.Lc D]}ID
K = {[0.5 pITt] + [0.5 Ilt (D - 0.75 P sin a)/sin a] + [0.625 Jlc D]}ID
(Eq.2)
(Eq.3)
where D bolt nominal shank diameter.
p thread pitch (bolt longitudinal distance per thread).
ex thread profile angle 60 (for M, MJ, UN, UNR, and UNJ thread profiles).
~ thread profile half angle 60/2 = 30.
tan A. = thread helix angle tan = p/(1t d p).
=
=
dp
=bolt pitch diameter.
http://euler9.tripod.comlfasteners/preload.html
D-69
7/21/2008
Page 2 of3
Jlt = thread coefficient of friction.

Jl c =collar coefficient of friction.
D and p can be obtained from bolt tables such as Standard Metric and USA Bolt Shank Dimensions.
The three terms in Eq. 3 are axial load component (coefficient) of torque resistance due to (1) thread
helix inclined plane normal force, (2) thread helix inclined plane tangential (thread friction) force, and
(3) bolt head or nut washer face friction force, respectively.
However, whether you look up K in references or calculate it yourself, the engineer must understand that
using theoretical equations and.typical values for K and coefficients of friction merely gives a preload
estimate. Coefficient of friction data in published tables vary widely, are often tenuous, and are often
not specific to your specific interface combinations and lubricants.. Such things as unacknowledged
surface condition variations and ignored dirt in the internal thread can skew the results and produce a
false indication of preload.
The engineer and technician must understand that published K values apply to perfectly clean interfaces
and lubricants (if any). If, for example, the threads of a steel, zinc-plated, K = 0.22, "dry" installation
fastener were not clean, this might cause K to increase to a value of 0.32 or even higher. One should
also note that published K values are intended to be used when applying the torque to the nut The K
values will change in relation to fastener length and assembly running torque if the torque is being read
from the bolt head.
One should measure the nut or assembly "running" torque with an accurate, small-scale torque wrench.
("Running" torque, also called prevailing torque, is defined as the torque when all threads are fully
engaged, fastener is in motion, and washer face has not yet made contact.) The only torque that
generates bolt preload is the torque you apply above running torque.
A few more things to be aware of are as follows. Bolt proof strength Sp is the maximum tensile stress
the bolt material can withstand without encountering permanent deformation. Published bolt yield
strengths are determined at room temperature. Heat will lower the yield strength (and proof strength) of
a fastener. Especially in critical situations, you should never reuse a fastener unless you are certain the
fastener has never been yielded.
1.1 Bolt Preload Measurement

If a more accurate answer for bolt preload is needed than discussed above, the specific combination and
lubricant would have to be measured instead of calculated. Measurement methods are generally
involved, time-consuming, and expensive, and are beyond the scope of this article. But perhaps one of
the simplest and least expensive methods, to test specific combinations and lubricants, is to measure the
installed fastener with a micrometer, if possible, and compute torque coefficient K as follows, per
Shigley,op. cit., p. 345, para. 2.
K
=T L/(E A delta D)
(Eq.4)
where T = bolt installation torque, L bolt grip length, E =bolt modulus of elasticity, A bolt cross
~ectional area, D bolt nominal shank diameter, and delta measured bolt elongation in units of length.
http://euler9.tripod.comlfasteners/preload.html
D-70
7/21/2008
APPENDIX E
MOBILE TEST CHAMBER 4 (MTC-4) VIEWPORT AND UNIVERSAL
SERIAL BUS (USB)
E1
E-2
E-3
E-4
E-5
E-6
E-7
E-8
E-9
E-10
E-11
E-12
E-13
E-14
E-15
E-16
E-17
E-18
E-19
E-20
E-21
E-22
E-23
E-24
E-25
E-26
E-27
E-28
E-29
E-30
E-31
E-32
E-33
E-34
E-35
E-36
E-37
E-38
E-39
E-40
E-41
E-42
E-43
E-44
E-45
E-46
E-47
E-48
E-49
E-50
E-51
E-52
E-53
E-54
E-55
E-56
E-57
E-58
E-59
E-60
E-61
E-62
E-63
APPENDIX F
MOBILE TEST CHAMBER 4 (MTC-4) PRESSURIZATION
MODIFICATION AND IEEE 1394 HIGH SPEED SERIAL BUS
(FIREWIRE)
F-1
F-2
F-3
F-4
F-5
F-6
F-7
F-8
F-9
F-10
F-11
F-12
F-13
F-14
F-15
F-16
F-17
F-18
F-19
F-20
F-21
F-22
F-23
F-24
F-25
F-26
F-27
F-28
F-29
F-30
F-31
F-32
F-33
F-34
F-35
F-36
F-37
F-38
F-39
F-40
F-41
F-42
F-43
F-44
F-45
APPENDIX G
MOBILE TEST CHAMBER 4 (MTC-4) ELECTRICAL
G-1
G2
G3
G4
G5
G6
G7
G8
G9
G10
G11
G12
G13
G14
G15
G16
G17
G18
G19
G20
G21
G22
G23
G24
G25
G26
G27
G28
APPENDIX H
MOBILE TEST CHAMBER 4 (MTC-4) PQS
H-1
QUALIFICATION: MOBILE TEST CHAMBER 4 (MTC4)

CHAMBER OPERATOR MTC4
NAME______________________________ RATE/RANK____________________
This page is to be used as a record of satisfactory completion of designated sections of the

Personnel Qualification Standard (PQS). Only specified supervisors may signify completion of
applicable sections either by written or oral examination, or by observation of performance. The
examination or checkout need not cover every item; however, a sufficient number should be
covered to demonstrate the examinees knowledge. Should supervisors give away their
signatures, unnecessary difficulties can be expected in future routine operations.
A copy of this completed page shall be kept in the individuals training jacket.
The trainee has completed all PQS requirements for this watch station. Recommend
designation as a qualified CHAMBER OPERATOR MTC 4.
RECOMMENDED______________________________________ DATE______________
Department Head
QUALIFIED___________________________________________ DATE______________
Commanding Officer or Designated Representative
SERVICE RECORD ENTRY______________________________ DATE______________
H-2
CHAMBER OPERATOR MTC 4

Estimated completion time: 1 week
1.1
PREREQUISITES
FOR OPTIMUM TRAINING EFFECTIVENESS, THE FOLLOWING ITEMS SHOULD BE
COMPLETED PRIOR TO STARTING YOUR ASSIGNED TASKS BUT SHALL BE
COMPLETED PRIOR TO FINAL WATCHSTATION QUALIFICATION. WATCH
STANDER SHALL SHOW KNOWLEDGE OF RESPECTIVE INSTRUCTIONS.
1.1.1 Familiarization with NAVXDIVINGUINST 6240.1 02 Navy

Experimental Diving Unit Chemical Hygiene Plan (CHP)
1.1.2 Familiarization with NAVXDIVINGUINST 6240.2 02 Molecular Biology
Laboratory Safety Program
1.1.3 Familiarization with NAVXDIVINGUINST xxxxx 02 Navy Experimental
Diving Unit Safety Procedures for Working with Chemicals and Biological
Toxins under Hyperbaric Conditions for In Vitro Experiments
1.1.4 Familiarization with NAVXDIVINGU Notice 3150.2 MTC Operating
Procedures
1.2
TASKS
For the tasks listed below:
A.
B.
C.
D.
E.
F.
G.
1.2.1
What are the steps of this procedure?

What are the reasons for each step?
What control/coordination is required?
What means of communications are used?
What safety precautions must be observed?
What parameters/operating limits must be monitored?
Satisfactorily perform this task.
Define MTC4 system capabilities and limitations
Questions
CDEFG
___________________________________
(Signature and Date)
.2
Line-up MTC4 bottle field for charging

___________________________________
H-3
ABCDEFG
Questions
.3
Dive and Surface MTC4
ABCDEFG
___________________________________
.4
Perform system emergency procedures
ABCDEFG
___________________________________
.5
Secure chamber system
ABCDEFG
___________________________________
.6
Complete entries in the Chamber Log during a treatment
ABCDEFG
___________________________________
1.3
INFREQUENT TASKS
For the infrequent tasks listed below:
A.
B.
C.
D.
E.
F.
G.
What are the steps of this procedure?

What are the reasons for each step?
What control/coordination is required?
What safety precautions must be observed?
What parameters must be monitored?
What conditions require this infrequent task?
Satisfactorily perform or simulate this infrequent task.
1.3.1 Perform air pressure and leak test on chamber

___________________________________
H-4
Questions
ABCDEFG
1.4
ABNORMAL CONDITIONS
For the abnormal conditions listed below:
A.
B.
C.
D.
E.
F.
G.
H.
1.4.1
What indications and alarms are received?

What immediate action is required?
What are the probable causes?
What operating limitations are imposed?
What emergencies or malfunctions may occur if immediate action is
not taken?
How does this condition affect other operations/equipment/watch
stations?
What follow-up action is required?
Satisfactorily perform or simulate the corrective/immediate action for
this abnormal condition.
Questions
ABCDEFGH
Increase of depth
___________________________________
.2
Decrease of depth
ABCDEFGH
___________________________________
.3
Increase of temperature
ABCDEFGH
___________________________________
.4
High CO2
ABCDEFGH
___________________________________
1.5
EMERGENCIES
For the emergencies listed below:
A.
B.
C.
D.
E.
F.
G.
What indications and alarms are received?

What immediate action is required?
What are the probable causes?
What operating limitations are imposed?
What other emergencies or malfunctions may occur if immediate
action is not taken?
How does this emergency affect other operations/equipment/ watch
stations?
Satisfactorily perform or simulate the immediate action for this
H-5
emergency.
1.5.1
Questions
ABCDEFG
Loss of primary air supplies

___________________________________
.2
Fire in the chamber
ABCDEFG
___________________________________
.3
Loss of electrical power
ABCDEFG
___________________________________
.4
Rapid increase/decrease of chamber pressure
ABCDEFG
___________________________________
1.6
WATCHES
1.6.1 STAND THE FOLLOWING WATCHES UNDER QUALIFIED SUPERVISION:
Chamber Operator (5 times)
___________________________________
___________________________________
___________________________________
___________________________________
___________________________________
2 EXAMINATIONS
pass an oral examination board

______________________________
H-6
H-7
H-8
APPENDIXI
MOBILETESTCHAMBER4(MTC4)PROCEDURESFORWORKINGWITH
EXCEPTIONALLYDANGEROUSCHEMICALORBIOLOGICALAGENTS
I-1
I-2
I-3
I-4
I-5
APPENDIX J
MOBILE TEST CHAMBER 4 (MTC-4) OPERATIONAL AND
EMERGENCY PROCEDURES
J-1
J-2
J-3
J-4
J-5
J-6
J-7
J-8
J-9
J-10
J-11
J-12
J-13
J-14
J-15
J-16
J-17
J-18
APPENDIX K
MOBILE TEST CHAMBER 4 (MTC-4) MAIN SCOPE DESIGNED
COMPONENTS
K-1
K-2
K-3
K-4
K-5
K-6
K-7
K-8
K-9
K-10
K-11
K-12
K-13
K-14
K-15
K-16
K-17
K-18
K-19
K-20
K-21
K-22
K-23
K-24
K-25
K-26
K-27
K-28
K-29
K-30
K-31
K-32
K-33
K-34
K-35
K-36
K-37
K-38
K-39
K-40
K-41
K-42
K-43
K-44
K-45
K-46
K-47
K-48
K-49
K-50
K-51
K-52
K-53
K-54
K-55
K-56
K-57
K-58
K-59
K-60
COLOMB DESIGN
Filter Wheel
Set screws to allow centering
To Camera
(Awaiting focal
distance from
rear, this side,
focal plane to
the CCD face)
Threads to allowing mounting
on filter wheel
Reducing Lens Adaptor for Filter Wheel
K-61
COLOMB DESIGN
Tube Lens
100-200mm
151.2mm
??? mm
CCD Focal Plane
CoolsnapEZ
Reducing Lens (0.7x); Nikon DXM
OBJ
Objectives to include ( corrected)

1) Nikon Flour 20x/0.50W (HCM)
2) Nikon Flour 40x/0.80W (HCM)
3) Nikon Plan Apo 2x/0.1
Figure One: Current Optical Path of MTC Microscope System

Point One: Distance from the rear of the objective to the tube lens is 100-200mm
Point Two: Distance to the reducing lens from the rear focal plane of the tube lens is set
at 151.2mm
Point Three: The Distance from the reducing lens to the CCD is set at ????? for the Nikon
0.7xDXM Lens.
Note: Reducing lens will be mounted directly on camera
K-62
COLOMB DESIGN
OBJ
Condenser
Slit Aperture
Polarizer
Collector Lens
(Focal point is slit
aperature)
LED Light Source
Double-Convex Lens, 30mm Dia. x 30mm

FL, Uncoated
Figure Two: Light source path with respect to the specimen and the objective
For a collector lens I would like to utilize Uncoated Double-Convex (DCX) Lens, 30mm
Diameter 30mm FL, from Edmond Optics (part number NT63-556).
K-62
MotorizedStages
MM-3M-F
Folded Motorized
MicroMini Stages
TM
Specifications:
*Repeatability:
*Homing Repeatability:
*Accuracy (linearity):
Speed (max.):
Slider Backlash:
Encoder Conversion (resolution):
(See also: gearhead options)
Standard Slider
AB Slider (anti-backlash)
2m
2m
3m/inch
12mm/second @ 12V
50m
0.49609m/count
with 16:1 gearhead
0.5m
0.5m
1.5m/inch
1.65mm/second @ 12V
3m
0.12406m/count
with 64:1 gearhead
*Encoder resolution must be added , based on the gearhead: 16:1 add 0.5m, 64:1 add 0.12 m
Slider backlash represents maximum overshoot
Runout (max.):
Gearhead Backlash:
Motor:
Vacuum compatiblity:
Load Capacity:
Direct top or side load:
Push:
Pull:
Tilt:
Twist:
3m/25.4mm
1-2m equivalent; can be compensated in software without overshoot
10 mm diameter, 6-12 VDC servo, brush type (see motor specifications)
10-3 Torr, standard,10-6 Torr available
0.5 kg
0.5 kg
0.5 kg
8 inch-ounce (560 gram-centimeter)
4 inch-ounce (280 gram-centimeter)
Note: These stages may be run at twice the specified ratings without damage, but with a loss of accuracy and speed.
Travel Ranges and Dimensions:

Travel Range
Model No.
MM-3M-F-0.5
12.7mm (0.5 inch)
MM-3M-F-1
25.4mm (1.0 inch)
MM-3M-F-1.5
38.1mm (1.5 inch)
MM-3M-F-2
50.8mm (2.0 inch)
MM-3M-F-2.5
63.5mm (2.5 inch)
Stage Body (L x W x H)
58.9 x 19.1 x 16.3 (mm)
2.32 x 0.75 x 0.64 (inch)
71.6 x 19.1 x 16.3 (mm)
2.82 x 0.75 x 0.64 (inch)
84.3 x 19.1 x 16.3 (mm)
3.32 x 0.75 x 0.64 (inch)
97.0 x 19.1 x 16.3 (mm)
3.82 x 0.75 x 0.64 (inch)
109.7 x 19.1 x 16.3 (mm)
4.32 x 0.75 x 0.64 (inch)
Weight
53g
53g
58g
58g
63g
63g
68g
68g
73g
73g
x, xy, xyz, xz configurations available

Specify -AB for Anti-Backlash
The information contained in this data sheet is subject to change without notice. Critical dimensions or specifications should be verified with our technical support staff.
National Aperture, Inc. - 16 Northwestern Dr. - Salem, N.H. 03079-4810
- Tel. (800) 360-4598 - (603) 893-7393 - FAX (603) 893-7857 -
K-63
www.nationalaperture.com/www.naimotion.com
21
Folded MM-3M-F Motorized MicroMini Stage - 0.5 to 2.5 inch Travel

Dimensional Data
Bearing
mount
Rail 1
Preload spring
Rail 2
Lead screw thread module
(AB or non-AB)
0-80 clear x 4 typical
for unbraced X-Y
connection
Gearhead (10mm)
Tapped accessory
hole x 2 typical
(0.254mm)
Set screw
x2
Forward limit
switch
(home)
CL
0.120" (3.05mm) typical
FWD.>>
Motor
(10mm)
0-80 tap x 2
0.428" (10.87mm)
apart
for optional
mounting
0.428"
0.250" typ.(10.87mm)
(6.35mm)
0.54" (a)
Tapped 0-80 x 4
0.0100"
(0 - 80)
Rest point x 4
Coupling
gears
0.45" (11.4mm)
0.05" (1.2mm)
Slider
(see detail)
CL
(13.7mm)
0.45"(b)
(11.4mm)
0.54"
(13.7mm)
Slider detail
(a) x (b) = footprint
Lead
screw
Encoder
(80 TPI)
0.100" (2.54mm) typical
Reverse
limit switch
Reverse limit
0.065" typ.
(1.65mm)
0.130" (3.30mm) typical

Forward limit
cable
LCF-3
Mounting hole x 4
Limit
(0-80 clear)
ground plug
Limit ground
0.75"
(19.0mm)
0.015"
(0.38mm)
0.540"
(13.72mm)
0.71"
(18.0mm)
0.645" (H)
(16.38mm)
0-80 tap x 15
Top view,
brace only
(stage not shown)
0.500"
1.0"
0.500"
(12.7mm)
0.10"
(2.54mm)
0.476"
(12.09mm)
0.595"
(15.11mm)
(12.7mm)
0.705"
(17.91mm)
0.788"
(25.4mm)
(20.02mm)
Note: Symmetry is applicable wherever implied

0.788"
MM-3M-F-0.5
MM-3M-F-1.0
MM-3M-F-1.5
MM-3M-F-2.0
MM-3M-F-2.5
T (Travel)
A
0.5 inch
2.32
12.7mm
58.9
1.0 inch
2.82
25.4mm
71.6
1.5 inch
3.32
38.1mm
84.3
2.0 inch
3.82
50.8 mm
97.0
2.5 inch
4.32
63.5 mm 109.7
B
1.730
43.94
2.230
56.64
2.730
69.34
3.230
82.04
3.730
94.74
C
1.86
47.2
2.36
59.9
2.86
72.6
3.36
85.3
3.86
98.0
D
1.400
35.56
1.900
48.26
2.400
60.96
2.900
73.66
3.400
86.36
(20.02mm)
Mounting surface
0.920"
(23.37mm)
0.755" (HB)
(19.18mm)
0.645" (H)
(16.38mm)
BR-3M-X
Optional
Crash-resistant Slider Brace
Note:
Single axis brace is essential where attachments
are vulnerable to crash.
22
- Tel. (800) 360-4598 - (603) 893-7393 - FAX (603) 893-7857 -
K-63
MotorizedStages
MM-4M-EX
Extended Motorized
MicroMini Stages
TM
(For heavier loads)
Specifications:
*Repeatability:
*Homing Repeatability:
*Accuracy (linearity):
Straightness:
Speed, no load (max.):
Slider Backlash:
Gearhead Backlash:
Encoder Conversion (resolution):
0.50 m
0.50 m
1.00 m per 25mm of travel
2.00 m maximum deviation per 50mm of travel
7 mm/second @ 12V with 14:1 gearhead (other gearheads available)
1m
<2.54m; can be compensated in software without overshoot
0.3595m per count, with 14:1 gearhead
*Encoder resolution must be added, based on the gearhead: 16:1 add 0.0005mm, 64:1 add 0.00012mm
Slider backlash represents maximum overshoot
Motor:
13 mm dia., 6-12 VDC servo, brush type
Slide:
Recirculating ball bearings with four point contact, hardened stainless steel rails
Vacuum compatiblity:
10-3 Torr standard, 10-6 Torr available
Load Capacity:
Horizontal:
3.0 kg
Vertical:
1.0 kg
Side:
1.0 kg
Travel Ranges and Dimensions:
Model No.
Travel Range
Stage Body (L x W x H)
Weight
MM-4M-EX-50
MM-4M-EX-80
MM-4M-EX-110
MM-4M-EX-140
MM-4M-EX-170
MM-4M-EX-200
MM-4M-EX-230
MM-4M-EX-260
48 mm
1.90 inch
78 mm
3.07 inch
108 mm
4.25 inch
138 mm
5.43 inch
168 mm
6.61 inch
198 mm
7.79 inch
228 mm
8.97 inch
258 mm
10.15 inch
138.68 x 31.5 x 25.4 (mm)

5.46 x 1.24 x 1.0 (inch)
168.68 x 31.5 x 25.4 (mm)
6.64 x 1.24 x 1.0 (inch)
198.68 x 31.5 x 25.4 (mm)
7.82 x 1.24 x 1.0 (inch)
228.68 x 31.5 x 25.4 (mm)
9.0 x 1.24 x 1.0 (inch)
258.68 x 31.5 x 25.4 (mm)
10.184 x 1.24 x 1.0 (inch)
288.68 x 31.5 x 25.4 (mm)
11.365 x 1.24 x 1.0 (inch)
318.68 x 31.5 x 25.4 (mm)
12.546 x 1.24 x 1.0 (inch)
348.68 x 31.5 x 25.4 (mm)
13.728 x 1.24 x 1.0 (inch)
330g
11.64oz
345g
12.49 oz
360g
12.70 oz
375g
13.23 oz
390g
13.76 oz
405g
14.29 oz
420g
14.82 oz
435g
15.35 oz
- Tel. (800) 360-4598 - (603) 893-7393 - FAX (603) 893-7857 -
K-64
29
MM-4M-EX Motor Stage

Dimensional Data
Tabulated Data for MM-4M-EX Stages
Travel
48mm
5.460 (138.68mm)
7.210 (183.13mm)
1.181 (30.00mm)
1.855 (47.12mm)
78mm
6.641 (168.68mm)
8.391 (213.13mm)
1.772 (45.00mm)
2.445 (62.12mm)
108mm
7.822 (198.68mm)
9.572 (243.13mm)
2.362 (60.00mm)
3.036 (77.12mm)
138mm
9.003 (228.68mm)
10.753 (273.13mm)
2.953 (75.00mm
3.627 (92.12mm)
168mm
10.184 (258.68mm)
11.934 (303.13mm)
3.543 (90mm)
4.217 (107.12mm)
198mm
11.365 (288.68mm)
13.115 (333.13mm)
4.134 (105mm)
4.808 (122.12mm)
228mm
12.546 (318.68mm)
14.296 (363.13mm)
4.724 (120mm)
5.398 (137.12mm)
258mm
13.728 (348.68mm)
15.478 (393.13mm)
5.315 (135mm)
5.989 (152.12mm)
Standard Carrier
13mm Motor (standard)

0.190-80 UNS-3A Leadscrew
1.000" (25.40mm)
0.906" (23.01mm)
"A"
"B"
Side View
"D"
"C"
"C"
1.240" (31.50mm)
0.787" (20.00mm)
0.492" (12.50mm)
Clearance for M2.5 SHCS Typical 10 places
0.984" (25.00mm)
Bottom View
2.520" (64.01mm)
0.787" (20.00mm) Typical
0.787" (20.00mm)
0.394" (10.00mm)
1.240" (31.50mm)
0.394" (10.00mm) Typical
M2.5 x 0.45 Typ. 8 places
0.512" (13.00mm)
0.984" (25.00mm)
1.260" (32.00mm)
0.512" (13.00mm)
0.984" (25.00mm)
1.240" (31.50mm)
M2.5 x 0.45 Typical 18 places
0.630" (16.00mm) Typical
Standard Carrier
Extended Carrier for dual Carriage Option
Top View
Top View
30
- Tel. (800) 360-4598 - (603) 893-7393 - FAX (603) 893-7857 -
K-65
K-66
K-67
K-68
K-69
Figure 1: Need adaptor to connect from rear of scope to filter wheel, adaptor between
needs to be varying length with a few tubes of different lengths. See below for basic idea.
Also, adaptor filter wheel should be threaded for mounting.
Scope
Side
Adapt
Adaptor Tube
Filter
Side
Adapt
K-70
Filter Wheel Threads
Figure 1: Note threading in rear filter wheel assembly
K-71
Middle adaptors of varying

length to adjust optical distance
Figure 3: Adaptor with current threading for filter wheel
K-72
Filter Wheel and Scope

adaptors approach, filter side
should have threads instead of
screw mounts
Figure 4: Adaptor for rear of scope, older adaptor but would like second adaptor in this
configuration however mounting is via threaded mount on rear; threading required to be
shallow based upon low clearance for filter wheel internal assembly.
K-73
APPENDIX L
MOBILE TEST CHAMBER 4 (MTC-4) LOWER OPTICAL UNIT AND
TRANSMITTED LIGHT SOURCE
L-1
L-2
L-3
L-4
L-5
L-6
L-7
L-8
L-9
L-10
L-11
L-12
AREHART QUOTE LOWER LIGHT SYSTEM

AREHART MODEL MAKERS, LLC
James D. Arehart
4197 Carondelet Dr.
Beavercreek, Ohio 45440-3214
November 3, 2011
Lt. Denis Colomb, Ph.D. (MSC, USN)
Biomedical Department
P1 Hyperbaric Electrophysiology
321 Bullfinch Rd.
Arehart Model Makers LLC wishes to submit the following quotation for your approval
Quote # 113522.
113522-1
113522-2
113522-3
113522-4
113522-5
113522-6
113522-7
113522-8
113522-9
113522-10
113522-11
113522-12
113522-13
113522-14
113522-15
113522-16
113522-17
113522-18
113522-19
113522-20
113522-21
2 DC Motors
@ 134.88
1 Motor mounting bracket
1 Lens mounting housing
1 Lens retaining ring
1 Light mounting housing
1 Modify large drive gear
1 Modify small driver gear with shaft adaptor
1 Bracket for mounting motor & light
Design
Drawings of parts and assembly drawings
Assembly of unit
Metal finishing and Black Anodizing of parts
Cost of drive gears
Unit test fixture
Rack mount box
Power supply for drive motor
Power supply for light
2 P C Board
@ 265.00
Misc. Electrical components
Electrical Assembly
Packaging, Shipping & Insurance
Total
Delivery 40 working days after receiving Purchase Order Number.

Thank you for the opportunity to quote this project,
James D. Arehart
AREHART MODEL MAKERS LLC
937-231-6606
jdarehart@aol.com
H-13
296.76
1320.00
825.00
220.00
440.00
220.00
440.00
1320.00
1100.00
900.00
880.00
350.00
95.00
440.00
197.26
350.00
350.00
530.00
160.00
900.00
450.00
11784.02
APPENDIX M
MOBILE TEST CHAMBER 4 (MTC-4) FLOURSCENCE
M-1
M-2
1.750
1.906
ATMOSPHERE
.38
.62
6
5
4
3
2
1
ITEM
VACUUM
.36

Dayton, OH 45414-3445
U.S.A.
tel (937) 890-1100
fax (937) 8905165
www.pavetechnologyco.com
PROJECTION
REVISION
LEVEL
VS18-SS-150-10-RG178-36-36
DESCRIPTION
.84
.10
INSTALLATION PORT
+.01
1.25
- .00
LUBRICANT O-RING SILICONE BASED

NUT VS18-SS
WIRE COAX M17/93 FEP 7 STRAND
O-RING -219 VITON 75
EPOXY BLACK
HOUSING VS18-SS
DESCRIPTION
36 MINIMUM
OVERFILL .06 BOTH SIDES

NOT INCLUDING MENISCUC ON WIRES
SPACING CONTROL NOT REQUIRED THIS SIDE
1.25-12 UNF
SUPER O-LUBE
1014
RG178
-219 VITON
PAVE-Seal 150
0660
PART NUMBER
(1.88)
SEE NOTE 6
A/R
1
10
1
A/R
1
QTY
PART NUMBER
2915
M-3
.1 TYP
36 MINIMUM
NOTES:
1. HE LEAK TEST @ 1ATM <1X10^-6 CC/S.
2. HYPOT 630 VDC 500 MEGOHMS MINIMUM 0.01 SECOND
MINIMUM WIRE TO WIRE, SHIELD AND HOUSING.
3. CONTINUITY TEST SHIELDS END TO END .5 Ohm MAXIMUM.
4. ALL TESTS ARE PERFORMED AT ROOM TEMPERATURE.
5. PERCENTAGE OF PARTS TESTED WILL BE 100% (MAY BE
A LESSER PERCENTAGE FOR LARGE PRODUCTION
QUANTITIES, UNLESS OTHERWISE NOTED OR REQUIRED).
6. VOIDS SMALLER THAN .035 ARE ACCEPTABLE.
7. DIMENSION IS OVER-ALL-LENGTH EXCLUDING OVERFILL
ON EACH SIDE OF HOUSING.
8. REF-OPERATING TEMPERATURE -20 C TO 150 C.
ALL DIMENSIONS AND TOLERANCES APPLY TO FINISHED PART IN INCHES.

ALLOWABLE TOLERANCES UNLESS SPECIFIED OTHERWISE: NONE 0.5
X.X DECIMAL 0.1 X.XX DECIMAL 0.02 X.XXX DECIMAL 0.005
ANGLES 1 DEGREE SURFACE FINISH 128 microinch RMS
32
Date: 21-May-10
URGENT QUOTATION
Prices are valid for 120 days.

PAVE will confirm all orders by fax or e-mail.
Please Confirm your PO in writing. Thank you!
To: Denis G. Colomb
Tel 850-230-3238
Company: Navy Experimental Diving Unit
Fax:
Ref:
denis.colomb@navy.mil
Email:
From: Walt Wood x27
Area Sales Rep:
Quote #: 2010135
Page 1
PAVE drawing/product number must be on the purchase order as the approved design. All tooling is the property of PAVE. Cancellation or
restocking charges of ~30% will normally apply on any customer's cancellation after the purchase order has been accepted by PAVE Technology
Co. Any blanket orders or large quantities quoted must be shipped complete within 12 months of the first shipment release date. Material
supplied to PAVE by a customer is supplied at the customer's own risk with 10% spares required to cover prototype development. Any below
listed one time non-recurring engineering changes (NRE) are invoiced at 50% upon receipt of the purchase order with the balance due upon the
acceptance of the first article. FOUR weeks minimum notice is required on PO shipment changes.
Otherwise terms are VISA/MasterCard credit cards,
CREDIT TERMS: % 10 NET 30 DAYS ON CREDIT APPROVED ACCOUNTS
prepaid or Fed x COD. New accounts to supply 3 references including bank references or D&B rating. Bank Wires to: NATIONAL CITY
BANK DAYTON OH USA, ACCT# WILL ADVISE, ABA ROUTING #042200279.
SHIPPING: F.O.B. DAYTON, OHIO USA Shipped via UPS, DHL, or FED X. Please state your air courier acct#.
SUMMARIZED CUSTOMER REQUIREMENTS FOR NEW DESIGNS:

DIRECTION:
PRESSURE:
LEAK TEST: 80 psi or see print
OPER. TEMP C: -20 C to 150 C maximum THERM CYCLE:
TEST: 630 VDC 500 MEGOHM
AMPERAGE:
VOLTAGE:
WIRE COLORS:
WIRE TYPE:
SEALING:
SPECIAL: 750 psi leak test hex side
ALL TESTING IS DONE AT ROOM TEMPERATURE ~25C AFTER CYCLING.
Please email any available AutoCAD, SolidWorks, or .IGS format assembly drawing to expedite custom design
delivery. Any NRE charge (Non-Recurring Engineering) includes providing the customer with a detailed assembly
drawing for the customer to approve prior to manufacture. A limited lifetime warranty applies to registered trademark
products PAVE-Seal, PAVE-Mate, PAVE Opti-Seal and PAVE-Flex.
Ship Date Est: 10 weeks ARO

DELIVERY IS SUBJECT TO CONFIRMATION AT TIME OF PURCHASE ORDER RECEIPT. WEEKLY RELEASES FOR LARGE ORDERS.
PAVE Part Numbers*/Description-See catalog on descriptions codes *Call for a PAVE 4 digit part/drawing # TBA=To Be Assigned
Line # PAVE Part

1
1791-2
NRE
Quantity
PAVE Description
Total US$
Price
VS18-SS-150-37-TEE20-48-48, leak
tested to 750 psi on hex o-ring side
$650.00
$1,300.00
One time non-recurring Engineering

charge for design, fixtures, and CAD
drawing. Allow 4-6 weeks from PO
issuance and our receipt for drawings to
be sent for review.
$200.00
$200.00
Total
$1,500.00
Sub-Total
$1,500.00
Tax
M-4
M-5
M-6
M7
M8
Page 1 of 1
COLOMB FLOUR PATHWAY BASIC PATH
M-9
file://C:\Users\denis.COLOMB\Desktop\MTC4 Chamber Build\Final\Final\Appendixes\A... 8/25/2013
Mo t o rize d F i l t er W h eel s
High Speed Filter Wheel System
Features
Visible filter position
numbers for both
the light path filter and
the load position filter
Large selection of adapters

to fit most illuminators
Filter wheels
can be stacked
together for
ND filters
Filter wheel
stand for
added stability
A dual track
for better
support
The Prior Scientific filter wheel

system can be provided as a stand
alone unit or used in conjunction
with stage and focus systems
controlled via the Prior ProScanTMII,
PriorPCI2, or OptiScanTMII stepper
motor controllers.
Features of the Filter Wheel
System include:
The ability to mount on the
illumination or emission side of
the microscope
Available for 25mm (10 position)
or 32mm (8 position) filters
A large selection of adapters to fit
most illuminators
Filter wheel position and shutter
status are controlled via either
RS232 or an optional keypad
Filter wheels can be stacked
together in series for ND filters
Magnetic covers over the filter
load position on both sides of
the filter wheel for easy access
to change filters
Visible filter position numbers for
both the light path filter and the
load position filter
Filter wheel stand for added
stability
Optional dual slide in neutral
density filter holder
Prior Scientific products have an
established reputation for quality
and reliability.They are
manufactured using high-quality
components. For the filter wheel
system and all its products,
Prior provides support and service
through an extensive network of
knowledgeable, professional dealers.
M-10
ISO 9001:2000
FM 83854
General Specifications
Filter Wheels
10 position, 25mm filters
8 position, 32mm filters
Filter Wheel Speed
55ms between adjacent positions
(120ms for OptiScanTMII)
Filter Wheel Weight
3.2 kg (7 lbs)
Dimensions:
A Maximum on stand 540mm
A Minimum on stand 34mm
A Minimum off stand 28mm
B for HF110 - 141mm
B for HF108 - 159mm
C for HF110 - 179mm
C for HF108 - 195mm
Controller
Driven via ProScanTMII, PriorPCI2
or OptiScanTMII Controller
External Control
Optional Filter Wheel Keypad
*Specifications subject to change
without notice
MINIM
(W
mm
LOAD PORT
454 mm
NUMBER OF
FILTER AT
LOAD PORT
NUMBER OF
FILTER IN
LIGHT PATH
High Speed Shutters
Filter Wheel Keypad
The high speed shutter has a

40 Hz*. maximum frequency of
operation.The shutter can be
opened and closed completely in
as little as 10 ms.
The optional filter wheel keypad

allows the user to easily select any
mmeither one or
filter position Bfrom
two filter wheels.This is done by
LOAD PORT
simply selecting
the appropriate filter
position key.
mm
MINIMUM OPTICAL PATH LENGTH

(WHEN USING C MOUNTS)
Dual Slide-In Filter Holder
454 mm
NUMBER OF
FILTER AT
The dual slide-in filter holder LOAD PORT

mounts to the filter wheel and holds
two neutral density or IR filters. NUMBER OF
FILTER IN
LIGHT PATH
*25mm shutters have maximum continuous speed of 10 Hz. Maximum

burst speed of 40 Hz is for a maximum of five cycles per 500ms.
*35mm shutters have maximum continuous speed of 5 Hz. Maximum
burst speed of 20 Hz is for a maximum of five cycles
C per
mm 500ms.
Ordering Information
HF202HT
Shutter, high temperature, 25mm, stand

alone
HF204HT
Shutter, high temperature, 32mm, stand
alone
*Excitation Adapters for Transmitted and Emission
HF210
For Leica pre DM series
HF215
For Leica DM series
HF220
For Nikon Eclipse series
HF230
For Olympus BH series
HF235
For Olympus BX, IX & AX series
HF240
For Zeiss Axio series
HF245
For Zeiss pre Axio series
* Please contact Prior Scientific for a complete list of
excitation adapters that are available.
HF110
HF108
CS100K
Filter wheel 10 pos./25mm

Filter wheel 8 pos./32mm
Keypad for manual control of filter
wheels and shutters
HF200
Shutter, 25mm, mounted to HF110
HF201
Shutter, 32mm, mounted to HF108
HF202
Shutter, 25mm, stand alone
HF204
Shutter, 32mm, stand alone
HF205
Dual slide in filter holder 25mm
High Temperature Shutters
HF200HT
Shutter, high temperature, 25mm,
mounted to HF110
HF201HT
Shutter, high temperature, 32mm,
mounted to HF108
M-11
V ISIT P RIOR O N T H E W E B
AT
P R IO R S C IE N TIFIC I N C .,
80 R E SE RV O IR P A R K D R IV E , R O C KLA N D , MA 02370-1062
T E LE PH O N E 781-878-8442 F A X 781-878-8736
www.prior.com
01/07
P R IO R S CIENTIFIC I NSTRUM ENTS L IM ITED ,

U N IT 4, W IL B R A H AM R OAD , F ULBOURN , C AM BRIDGE CB1 5ET
T E LE PH O N E 01223 881711 F AX 01223 881710
APPENDIX N
MOBILE TEST CHAMBER 4 (MTC-4) PERFUSION
N-1
HPLC SYSTEM COLOMB
N-2
HPLC SYSTEM COLOMB
N-3
HPLC SYSTEM COLOMB
N-4
HPLC SYSTEM COLOMB
N-5
HPLC SYSTEM COLOMB
N-6
HPLC SYSTEM COLOMB
N-7
HPLC SYSTEM COLOMB
N-8
HPLC SYSTEM COLOMB
N-9
HPLC SYSTEM COLOMB
N-10
HPLC SYSTEM COLOMB
N-11
HPLC SYSTEM COLOMB
Swagelok Alabama
Quotation Document
Quotation 97328722-REV C
November 15, 2012
Attention: Denis Colomb
Company: Navy Experimental Diving Unit (Customer)
Phone: 850-230-3118
Prepared By: Dean Owens
We have evaluated your request for a Cellular Perfusion System, (Assembly), and Swagelok Alabama
(Distributor) is pleased to furnish this quotation in response to your submitted design and/or specifications.
Please review this quotation carefully before placing an order. We reserve the right to re-bid if changes are
made to the contents of this quote, including any revised drawing and/or bills of material. We will be happy to process
an order upon receipt of a purchase order and receipt of your signature on this quotation.
Safe Product Selection
When selecting a product, the total system design must be considered to ensure safe, trouble-free performance.
Function, material compatibility, adequate ratings, proper installation, operation, and maintenance are the
responsibility of the system designer and user.
Quote Expires in 60 days
1.0 Product Description

Part Number: IS-02-10-091312-REV-A
Price: $7,346.55 (This includes shipping)
Delivery: 8-9 weeks after customer supplied material
This assembly consists of Swagelok valves, fittings, a regulator, a customer supplied pump, and tubing
mounted inside of a stainless steel box. A representation of the assembly and the associated bill-ofmaterial can be found on the attached drawings.
2.0 Tubing
The tubing used in this Assembly will be 1/8 OD x .028 316/316L stainless steel seamless tubing cleaned to
ASTM G93 standards.
3.0 Customer Supplied Components

The following component(s) will be provided (free-issued) by the customer:
HPLC Pump
4.0 Testing
The Customer has indicated that the finished assembly is to be pressurized to 750psig and then checked for
leaks using Snoop.
5.0 Cleaning / Processing

SC-11 (Swagelok Special Cleaning and Packaging) is provided prior to assembly for all individual Swagelok
manufactured components.
November 15, 2012
97328722 Rev-C
Page 1 of 3
This document, information on it, and any attachments are the confidential property of the Distributor and are provided to you for a limited purpose. Neither may be
copied, exhibited, or furnished to others in any form without the written consent of the Distributor.
SF-07-277, Rev. - , DCN # 08-02657, DCN Date: Jul 03, 2008
N-12
HPLC SYSTEM COLOMB: APPROVED BY COLOMB
Swagelok Alabama
Quotation Document
*The cleaning of the tubing used in this assembly will be performed by Technical Micronics Control in Huntsville,
Alabama. Neither Swagelok nor the distributorship can verify that the cleaned product will meet the cleanliness
requirement of the specification and the cleaning is not covered by the Swagelok warranty (supplier warranty
will apply). By signing this document, the customer approves the use of Technical Micronics for cleaning the tubing,
and also that this assembly will not be used in a system for enriched oxygen or other oxidizer.
6.0 Shipping and Handling

The complete Assembly will be shipped to the Customer site with appropriate packaging to protect from
damage during transport.
7.0 Customer Approval

Please review: this Customer Quotation Document, any catalog data sheet(s), accompanying drawing(s),
specification(s), or Bill of Material(s) incorporated herein by reference, (collectively Assembly
Documentation). Please verify the accuracy of the information provided.
By signing below, Customer accepts and authorizes assembly in accordance with the Assembly
Documentation. It also confirms you acceptance of the non-Swagelok components that you have specified to
be used in this assembly.
This Quotation and your order are subject to the Distributors Integrated Services Terms and Conditions
following this page, and incorporated by reference, unless otherwise specified in writing by the Distributor.
Attachments:
Drawing # IS-02-10-091312-REV-A Sheets 1-6
Company Name:
Customer Name (print):
Customer Signature:
Date:
November 15, 2012
97328722 Rev-C
Page 2 of 3
This document, information on it, and any attachments are the confidential property of the Distributor and are provided to you for a limited purpose. Neither may be
copied, exhibited, or furnished to others in any form without the written consent of the Distributor.
SF-07-277, Rev. - , DCN # 08-02657, DCN Date: Jul 03, 2008
N-13
N-14
N-15
N-16
N-17
N-18
N-19
N-20
SL
&
Nylaflow nylon tubing is high quality pressure tubing, available in several formulations.
For applications that require toughness,
resilience, a small bend radius and high burst
strength, Nylaflow pressure tubing is a high
performance, long-lasting alternative to
copper, rubber, aluminum and other types of
plastic tubing. Nylaflow tubing is available
with custom markings, cut to length and
custom packaging.
Types T and H Nylaflow tubing are thin
and heavy wall, general purpose tubing
made from type 6/6 nylon. These types
offer the highest strength and are FDA compliant for use in food or beverage handling
applications.
Nylaflow LM tubing is a premium high
flex tubing available in two types: natural or
black, which exhibits improved light stability.
Type LM offers excellent chemical resistance
and low moisture absorption.
Standard Nylon 6/6 mechanical grade
tubing is designed for low pressure mechanical application such as fluid and air transmission. It is an extremely tough, low
friction, high flexibility tubing that can be
used for cable protectors or in short pieces as
small bushings and washers. S&L Plastics
standard nylon is ideal for a wide variety of
other mechanical applications where higher
pressure ratings are not required. S&L
Plastics tubing resists abrasion and wear and
is better than aluminum or steel. It has an
extremely smooth inner surface with a very
low coefficient of friction. Nylon 6/6 is
produced on a custom basis and is available
in colors, with custom markings and cut to
length.
Type LP Nylaflow tubing is a low pressure,
general purpose type 6 nylon tubing with
carbon added for UV stability.
Both Nylaflow and Standard tubing can
be cut with a sharp knife and flared hot
or cold. They are odorless, tasteless and
non-corrosive.
PLASTICS, INC.
WHERE PLASTICS TAKE SHAPE
NYLAFLOW PRESSURE TUBING
ENGLISH SPECIFICATIONS
O.D.
I.D.
Wall
Min. Bend
Radius
O.D. Tolerance
Wall Tol.
Coil Length
1/8"
3/16"
1/4"
5/16"
3/8"
.079"
.111"
.150"
.188"
.225"
.023"
.038"
.050"
.062"
.075"
3/8"
5/8"
11/4"
2"
21/2"
+.002".004"
+.002".008"
+.002".008"
+.002".008"
+.002".010"
.003"
.003"
.003"
.003"
.004"
1500'
1000'
500
250'
250'
4TD2-53420*
1/8"
4TD2-53820*
5/32"
4TD2-54420*
3/16"
4TD2-55420*
1/4"
4TD2-56420*
5/16"
4TD2-57420*
3/8"
Type LM Natural
.095"
.106"
.137"
.190"
.242"
.295"
.015"
.025"
.025"
.030"
.035"
.040"
5/8"
3/4"
1"
11/4"
2"
3"
+.002".004"
+.002".004"
+.002".008"
+.002".008"
+.002".008"
+.002".010"
.003"
.003"
.003"
.003"
.003"
.004"
1500'
1000'
1000'
500'
250'
250"
4TE1-03420
4TE1-04420*
4TE1-05420*
4TE1-07420
4TE1-08420*
4TE1-10420*
Type LM Black
1/8"
3/16"
1/4"
5/16"
3/8"
1/2"
.095"
.137"
.180"
.232"
.275"
.375
.015"
.025"
.035"
.040"
.050"
.0625"
5/8"
1"
11/4"
2"
3"
41/2"
+.002".004"
+.002".006"
+.002".008"
+.002".008"
+.002".010"
+.005".010"
.002"
.003"
.003"
.003"
.004"
.004"
1500'
1000'
500'
250'
250'
150'
4TE1-03425
4TE1-04425
4TE1-05425
4TE1-07425
4TE1-08425*
4TE1-10425
Type LP
1/8"
3/16"
1/4"
5/16"
3/8"
1/2"
.095"
.137"
.180"
.232"
.275"
.375"
.015"
.025"
.035"
.040"
.050"
.0625"
5/8"
1"
11/4"
2"
3"
41/2"
+.002".004"
+.002".006"
+.002".008"
+.002".008"
+.002".010"
+.005".010"
.002"
.003"
.003"
.003"
.004"
.004"
1500'
1000'
500'
250'
250'
150'
4TC2-03420
4TC2-04420
4TC2-05420
4TC2-06420
4TC2-07420
4TC2-09420
1/8"
3/16"
1/4"
5/16"
3/8"
1/2"
.095"
.137"
.190"
.242"
.295"
.376"
.015"
.025"
.030"
.035"
.040"
.062"
5/8"
1"
11/4"
2"
3"
41/2"
+.002".008"
+.002".006"
+.003".011"
+.003".011"
+.003".016"
+.003".019"
.003"
.003"
.003"
.003"
.004"
.004"
1500'
1000'
500'
250'
250'
150'
Part No.
Type H
4TD2-03420*
4TD2-04420*
4TD2-05420*
4TD2-05420*
4TD2-07420*
Type T
*Stock sizes. All other sizes will have minimum quantities and set-up charges.
S&L PLASTICS, INC 2860 Bath Pike Nazareth, PA 18064 610.759.0280 800.361.5887 Fax: 610.759.0650 mktg@slpinc.cc
N-20
PROPERTIES OF NYLAFLOW AND STANDARD NYLON TUBING

Nylaflow T
(Type 6/6)
Natural
Nylaflow H
(Type 6/6)
Natural
Nylaflow LM
(Type 11 or 12)
Natural
Nylaflow LM
(Type 11 or 12)
Black
Nylaflow LP
(Type 6)
Black
Nylaflow
Tubing (Type 6/6)
Natural
500 5F
500 5F
Type11-365 10F
Type 12-365 10F
420 13F
500 5F
2.50
8.0
2.50
8.0
.9
1.9
.9
1.9
3.50
11.0
2.50
8.0
Suggest Temp.
Range (F)
-65 to +150
-65 to +150
-80 to +200
-80 to +200
-40 to +150
-65 to +150
Light Stabilized
No
No
No
Yes
Yes
No
Hoop Stress at
73F Bone Dry (psi)
7,500
7,500
2,500
2,500
6,000
7,500
Hoop Stress
at 73F 50% R.H. (psi)
4,500
4,500
2,000
2,000
2,600
4,500
Hoop Stress at 73F

Full Saturation (psi)
3,100
3,100
1,850
1,850
2,100
3,100
Materials Flexural
Elastic Modulus
at 73F. 50% R.H. (psi)
175,000
(conditioned)
175,000
(conditioned)
105,000
105,000
130,000
175,000
(conditioned)
Operating Pressure
at 73F. 50% R.H. (psi)
250
625
250
250
175
Not Pressure
Rated
Bursting Pressure
at 73F 50% R.H. (psi)
1,000
Minimum
2,500
Minimum
1,000
Minimum
1,000
Minimum
700
Minimum
Important Facts
Moderate cost.
General Purpose
nylon. Highest
strength. Stiffest
of all nylons. FDA
compliant. Meets 3A
Sanitary Standards.
Carried in stock.
Moderate cost.
General Purpose
nylon. Highest
strength. Stiffest
of all nylons. FDA
compliant. Meets 3A
Sanitary Standards.
Carried in stock.
Premium material.
Excellent Flexibility.
Best chemical
resistance including
resistance to ZnCl2:
(zinc chloride). Lowest
moisture pickup. Wide
temperature range
usage. Carried in stock.
Same as Natural
except light
stability improved.
General Purpose.
Light stability.
Moderate Cost.
General purpose
nylon. Highest
strength. Stiffest
of all nylons.
Meets 3A
Sanitary Standards.
Typical
Applications
Air lines, grease

lines, vacuum lines,
hydraulic lines, high
pressure gases.
Air lines, grease

lines, vacuum lines,
hydraulic lines, high
pressure gases.
Automotive fuel
lines, lubrication
lines, vacuum
lines, air lines.
Automotive Fuel
lines, lubrication
lines, vacuum
lines, air lines.
General purpose
tubing. Excellent for
farm machinery.
Mechanical applications, such as conduit,

small sleeve bearings,
busings, insulators.
Good to pH-5
Good to pH-11
Excellent
Excellent
Excellent
Excellent
Good
Excellent
Excellent
Fair
Poor
Good to pH-5
Good to pH-11
Excellent
Excellent
Excellent
Excellent
Good
Excellent
Excellent
Fair
Poor
Good to pH-5
Good to pH-11
Excellent
Excellent
Excellent
Excellent
Good
Excellent
Excellent
Fair
Good
Good to pH-5
Good to pH-11
Excellent
Excellent
Excellent
Excellent
Good
Excellent
Excellent
Good
Good
Good to pH-5
Good to pH-11
Excellent
Excellent
Excellent
Excellent
Good
Excellent
Excellent
Good
Poor
Good to pH-5
Good to pH-11
Excellent
Excellent
Excellent
Excellent
Good
Excellent
Excellent
Fair
Poor
Color
Melting Point
Water Absorption
at Equilbrium (%)
at Saturation (%)
Chemical
Resistance at 73F
Acids
Alkalies
Hydrocarbons-aromatic
Hydrocarbons-aliphatic
Ketones
Ethers
Alcohols
Salts, neutral
Freons
Sunlight
Zinc chloride
Notes:
Formula for calculating hoop stress of any nylon tube:

S = P(d+t) / 2t
S=hoop stress strength (psi)
d=inside diameter of tube (inches)
P=burst strength (psi)
t=wall thickness of tube (inches)
N-21
NYLATRON GS PROFILE SHAPES
S&L Plastics Inc. stocks a variety of Standard Nylatron GS

Profile shapes. These shapes are used as wear surfaces on conveyers, guide rails and as edge protectors on metal parts and
sides to replace more expensive beading. In addition to the
instock profile shapes, similar custom extruded shapes can be
produced in other materials such as type 6, 6/6, 6/12, 11 and 12
Nylons, Polycarbonate, Ultem, Polysulfone, Polypropylene,
HDPE, LDPE, Styrene, ABS, Buterate, PVC, Celcon, Acrylics,
Elastomers, Noryl, K-Resin, PETG and SAN. Requests for special requirements are custom quoted.
S&L Plastics has more than fifty years experience in the
design and production of custom precision extrusions.
Profiles can be used as lower cost alternatives to injection molding. They are extruded in continuous lengths and cut to your specific requirements, coiled or put on reels.
SNAP-ON PROFILE OPTIONS
DWG NO.
40-922
40-923
40-924
40-925
40-926
40-927
40-928
Size
1/16
1/8
1/4
3/8
1/2
3/4
1
A
.050
.065
.075
.085
.097
.118
.130
B
.175
.270
.425
.565
.750
1.025
1.330
C
.067
.130
.265
.385
.535
.780
1.050
D
.280
.310
.370
.435
.500
.610
.725
E
.055
.095
.225
.305
.475
.675
.900
Length
500' coil
500' coil
12ft.
12ft.
12ft.
12ft.
12ft.
N-22
PROPERTY COMPARISON
Product Description
MECHANICAL
Specific Gravity 73F.
Tensile Strength (at break), 73F.
Tensile Modulus of Elasticity 73F.
Tensile Elongation (at break) 73F.
Flexural Strength, 73F.
Flexural Modulus of Elasticity, 73F.
Shear Strength, 73F.
Compressive Strength, 10% Deformation, 73F.
Compressive Modulus of Elasticity, 73F.
Hardness, Rockwell, Scale as noted, 73F.
Izod Impact (notched), 73F.
Coefficient of Friction (Dry vs. Steel) Dynamic
THERMAL
Coefficient of Linear Thermal Expansion (-40F to 300F.)
Heat Deflection Temperature 264 psi
Melting Point (crystalline) peak
Continous Service Temperature in Air (Max.) (1)
Thermal Conducitivity
Unfilled
Type 6/6
Nylon
Nylatron
GS Nylon
Unfilled
Acetal
Copolymer
Unfilled
Polycarbonate
High
Impact
Polystyrene
1.16
12,500
480,000
25
17,000
460,000
10,500
16,000
420,000
M85 (R115)
0.5
0.20
1.41
8,800
410,000
30
13,000
375,000
7,700
16,000
410,000
M80
1.5
0.15
1.20
10,000
315,000
135
14,200
340,000
6,000
12,500
345,000
M70
17.0
1.04
3,500
270,000
45
7,000
310,000
3,000
6,400
270,00
M65 (R95)
2.0
0.40
Units
Test Method
ASTM
psi
psi
%
psi
psi
psi
psi
psi
D7592
D638
D638
D638
D790
D790
D732
D695
D695
D785
ft.lb./in. of notch
D256 TypeA
D1894
1.15
11,500
425,000
50
15,000
450,000
10,000
12,500
420,000
M85 (R115)
0.6
0.25
in./in./F
F
F
F
5.5x10-5
200
500
210
1.7
4.7x10-5
200
500
220
1.7
4.7x10-5
230
329
180
1.6
3.8x10-5
265
N/A
225
1.3
4.5x105
200
270
125
1.0
BTU in./(hr.Ft2F)
D696
D648
D3418
ELECTRICAL
Dielectric Strength, Short Term
Volume Resistivity
Dielectric Constant, 106 Hz
Dissipation Factor, 106 Hz
Flammability @ 3.1 mm(1/8 in.) (5)
Volts/mil
ohm-cm
D149
D257
D150
D150
UL 94
400
4.5x1013
3.6
0.02
V-2
350
2.5x1013
V-2
500
1.0x1014
3.7
0.006
94 HB
380
1.0x1017
2.96
0.010
V-2
550
1.0x1016
2.5
0.0005
94 HB
CHEMICAL(3)
Water Absorption Immersion 24 Hours
Water Absorption Immersion, Saturation
Acids, Weak, 73F., acetic acid dilute hydrochloric or sulfuric acid
Acids, Strong, 73F., conc. hydrochloric or sulferic acid
Alkalies, Weak, 73F., dilute ammonia or sodium hydroxide
Alkalies, Weak, 73F., strong ammonia or sodium hydroxide
Hydrocarbons-Aromatic, 73F.,benzene, toluene
Hydrocarbons-Aliphatic, 73F., gasoline hexane grease
Ketones, Esters 73F., acetone methyl ethy, ketone
Ethers 73F., diethyl ether tetrahydrofuran
Chlorinated Solvents 73F., methylene chloride, chloroform
Alchohols, 73F., methanol, ethynol, anti-freeze
Inorganic Salt Solutions, 73F., sodium chloride, potassium cyanate
Continuous Sunlight 73F.
% by wt.
% by wt.
D570 (2)
D570 (2)
0.3
7.0
L
U
L
U
A
A
A
A
A
A
A
L
0.3
7.0
L
U
L
U
A
A
A
A
A
A
A
L
0.22
0.80
L
U
A
U
A
A
A
A
A
A
0.15
0.35
A
A
A
U
U
A
U
A
U
A
A
L
0.00
0.00
A
A
L
L
U
U
U
U
U
A
A
U
$$
1
$$
1
$$
1
$$$
3
$
3
OTHER
Relative Cost (4)
Relative Machinability (1-10, 1=Easier to Machine)
(1) Data represents estimated maximum long term service temperature based on practical field experience.
(2) Specimens 1/8 thick x 2 dia. or square.
(3) Chemical resistance data are for little or no applied stress. Increased stress, especially localized may result in more severe attack. Examples of common chemicals also included.
(4) Relative cost of material profiled in this brochure ($ = Least Expensive and $$$$$$ = Most Expensive)
(5) Estimated rating based on available data. The UL 94 Test is a laboratory test and does not relate to actual fire hazard.
(6) Values are typical averages and will vary with resin lots and by size and shape of product. The data shown is based on dry as manufactured test samples.
Key: A = Acceptable Service
L = Limited Service
U = Unacceptable
S&L PLASTICS, INC 2860 Bath Pike Nazareth,

2860 Bath
PAPike
18064
Nazareth,
610.759.0280
PA 18064 800.361.5887
610.759.0280 800.361.5887
Fax: 610.759.0650
Fax:610.759.0650
mktg@slpinc.cc
mktg@slpinc.cc
N-23
N-24
Series 1500 HPLC Pump
The Series 1500 HPLC Pump has been designed for

analytical to small-scale preparative chromatography
applications. The patented low dead-volume pulse
damper and dual piston mechanism make this
pump virtually pulse-free, and provide unmatched
performance for the most demanding applications.
The unit provides flow rates from 0.001 to 12.0 mL/
min., and is typically suitable for columns from 2 mm
to 22 mm ID.
The Series 1500 drive features a linear cam and
pressure compensation to provide accurate gradients
in the high- or low-pressure mode.
Standard controls include RS-232 serial communication port and Run/Stop inputs, remote frequency and remote voltage
controls, real-time pressure monitoring, and constant flow. The high-performance mechanical design provides accurate
pressures to 6000 psi. All LabAlliance pumps include a 3 year warranty.
Standard Features & Controls

Dual self flushing pump heads with overlapping cams for
reduced pulsation
Ruby ball/sapphire seat check valves for consistent performance
Pulse damper for reduced dead volume
Prime purge valve
Real-time pressure monitoring with upper/lower pressure

limits
RS-232 serial com port for complete control & status

monitoring
Motor stall detector
Run/stop inputs (5 volt TTL type)
Remote 0-10 volt & remote 0-10 kHz flow rate control input
Simple front panel key pad controls with LED display
Autoprimeone button toggles flowrate to maximum for

rapid solvent change
Stainless steel or PEEK fluid path
Series 1500 Pump Specifications

Flow Rates....................... 0.001 - 12.0 mL/min. (each pump)
Pressure........................... Stainless Steel: 0 6,000 psi

PEEK: 0 5,000 psi
Pulsation.......................... 0.5% @ 1mL/min. and 1,000 psi
Pressure Accuracy......... 1% of full scale pressure
Flow Accuracy................. 2%
Flow Precision................. 0.2% RSD
Physical............................ 5.5 H x 10 W x 17 D

(14 x 25.4 x 43.2 cm)
Weight.............................. 30 lbs. (14 kg)
Ordering Information
Description
Part No.
Series 1500 Pump, Stainless Steel

Self-Flush, 0.001-12 mL/min., User-Selectable
Voltage, Pulse Damper w/
pressure-monitoring and RS-232 Control
F05SFD01
Series 1500 Pump, PEEK

Self-Flush, 0.001-12 mL/min., User-Selectable
Voltage, Pulse Damper w/
pressure-monitoring and RS-232 Control
F05PFD01
Rev.1 - 12/19/08
TM
www.ssihplc.com
www.laballiance.com
Scientific Systems, Inc. 349 N. Science Park Road State College, PA 16803
Phone: 800-441-4752
Fax: 814-238-7352 Email: sales@ssihplc.com
P-25
N26

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N28

N29

N30

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N33

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N36

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N40

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N-59
VALCO INSTRUMENTS CO.,INC.

P.O. BOX 55603
HOUSTON, TX 77255-5603
713-688-9345
Page 1 of 1
12/01/2010 11:26:01
Quotation
Sold-To-Party
US NAVY EXPERIMENTAL DIVING UNIT
321 BULLFINCH ROAD
PANAMA CITY FL 32407
USA
Purchase Order No.

Payment Term
Gross Weight:
Item
NT30 Net due in 30 days

1.722 KG
Material/Description
Information
Document Number
Document Date
Customer No.
Validity Start Date
Validity End Date
Created By
Ship Via
20002917
12/01/2010
92314
12/01/2010
02/01/2011
SHAGERMAN
UPS Ground
Purchase Order Date

Currency
Net Weight
USD
0 KG
Quantity
Unit Price
Amount
1.00 EA
2,920.00
2,920.00
1.00 EA
25.00
25.00
Denis Colomb
850.230.3238
Freight is estimated 2nd day air, lead time for shipment is
approximately two weeks from receipt of PO
1
2
CP3-8182-625M2
PUMP, M50: CER/88,625 ul,DSM2,CONT,COMPL
FREIGHT
CHARGES: FREIGHT
____________________
Sub Total
2,945.00
____________________
Total Amount
2,945.00
Signature __________________________________________________ Date __________________________

Country of Origin: USA
SALES (800) 367-8424
FAX
(713) 688-8106
www.vici.com
N-60
N-61
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N-63
Acknowledgement A0920164
Company:
Department:
Attention:
Fax:
Date:
Page:
Mr. Boone
850 230-3123
May 06, 2009

Page 1 of 2
Dear Mr. Boone:

Would you please check this order confirmation carefully. Technical data can be verified with the end user or
the engineer responsible.
Invoice address:
Delivery address:

321 Bullfinch Road

321 Bullfinch Road
We will proceed with production according to the specifications as outlined here. If any changes are
necessary, please let us know as soon as possible.
Your order
Order date
Item
001
: PO# N0463A-09-P-0039
: April 21, 2009
Quantity
1
Your contact
Our contact
: Mr. Boone
: Ms./Mr. Regal-Brown
Incorporated
Expected Del. Date
Description
Model
Classification
Material
Range (flow)
Calibration
Pressure P1
Pressure P2
Temperature
Seals
Connections
Purge adapter
Output
Setpoint
Supply
S E NS ORS
:
:
:
:
:
:
:
:
:
:
:
:
:
:
M52C2I-ABD-11-K-C
IP-65
AISI 316L
15...750 g/h NaCl (Sodium Chloride
C: 1% RD plus 0-stability
500 psi (g)
400 psi (g)
37 C
Kalrez-6375 with Kalrez-6375 plunger
1/8" OD compression
1/8" OD compression
RS-232 0...100% ( 0...10 Vdc )
RS-232 0...100%
+15...24 Vdc
F OR
MA S S
F L OW A ND
May 28, 2009
P RE S S URE
BRONKHORST USA Inc. 57 S. Commerce Way, Suite 120 Bethlehem PA 18017 USA
T +1 (610) 866-6750 F +1 (610) 866-9212 I www.bronkhorstusa.com E sales@bronkhorstusa.com
N-64
Amount USD
8,477.00
Acknowledgement A0920164
Company:
Department:
Attention:
Fax:
Item
Quantity
002

Mr. Boone
850 230-3123
Payment:
May 06, 2009

Page 2 of 2
Expected Del. Date
Description
Model
Housing
Module:
:
:
:
:
:
:
:
:
10
Instrument Output
External Signal
External Setpoint
Supply
003
Date:
Page:
E-7600-10-BBB
16 TE Panel mount cassette (96x144 mm)
R/C panel with power supply, mains entry and
Sub-D9 analogue communication
0...10 Vdc
0...10 Vdc
0...10 Vdc
100...240 Vac
Connenction cable
Net 30 days
Amount USD
May 28, 2009
1,295.00
May 28, 2009
113.00
Sub Total USD
Shipping & Packing USD
Total USD
9,885.00
245.00
10,130.00
Delivery:
CIF Panama City
Above mentioned prices are excluding tax.
S E NS ORS
V16 DUS03 proc V1
F OR
MA S S
F L OW A ND
P RE S S URE
N-65
Service Receipt A1220240

Company:
Department:
Attn:
Fax:
Date:
Page:
Mr. Colomb
March 29, 2012

Page 1 of 1
Dear Mr. Colomb:

We received below mentioned items, please check the technical information.
Your order
Order date
Item
001
:
: March 29, 2012
Quantity
1
Your contact
Our contact
: Mr. Colomb
: Mr. Regal-Brown Incorporated
Description
Serial Number
Model
Valve Function
Classification
Material
Range (flow)
Pressure P1
Pressure P2
Temperature
Seals
Connections
Purge adapter
Output
Setpoint
Supply
Expected del. week
:
:
:
:
:
:
:
:
:
:
:
:
:
:
:
B9200267A
M52C2I-ABD-11-K-C
Normally Closed
IP-65
AISI 316L
15...750 g/h Water
500 psi (g)
400 psi (g)
37 C
Kalrez-6375 with Kalrez-6375 plunger
1/8" OD compression
1/8" OD compression
RS-232 0...100% ( 1...5 Vdc )
RS-232 0...100%
+15...24 Vdc
Reason of return: Flow blocked. Possible clog in sensor.
Payment term: Net 30 days

V8 DUS07
S E NS ORS
F OR
MA S S
F L OW A ND
P RE S S URE
N-66
N-67
N-68
N-69
N-70
N-71
N-72
N-73
N-74
N-75
N-76
N-77
N-78
Pressure-Reducing Regulators
KPRSeries Maintenance Instructions
Kit Contents
Tool Requirements
Filter (1)
Filter ring (1)
Retaining ring (1)
Poppet damper (1)
Poppet spring (1)
Poppet (1)
Seat (1)
Seat retainer (1)
Diaphragm (2 for 0 to 250 psig and 0 to 500 psig
Needle-nose pliers
15 ftlb (20 Nm) torque wrench
117 ftlb (160 Nm) torque wrench
5/8 in. socket wrench
2 in. wrench
regulator, 1 for all others)
Lubricant tube with MSDS (1)
CAUTION
Before servicing any installed regulator, you must depressurize the system and purge the regulator.
Body Disassembly
1. Turn handle counterclockwise until it stops in
order to release the spring load.
2. Loosen cap ring with a 2 in. wrench, using the
flats to grip the cap ring. Remove cap assembly
and handle as one piece. In 0 to 500 psig
regulator assemblies, the range spring and spring
button will fall from the cap when it is lifted. Set
aside the range spring and spring button for later
use during reassembly.
3. Remove stop plate and diaphragm from body.
It may be necessary to tap the regulator at
an angle, by hand, to remove the diaphragm.
Set aside stop plate for later use. Discard the
diaphragm.
4. Remove seat retainer and seat (as one piece)
using a 5/8 in. socket wrench and discard both
components.
5. Pull poppet, poppet spring, and poppet
damper from body and discard. If poppet
damper remains in the body, invert the body and
use the poppet to loosen and remove the poppet
damper. Be careful not to scratch the inside of
the body.
Filter Disassembly
Cap assembly
Cap ring
Stop plate
Diaphragm
Seat retainer
Seat
Poppet
Poppet spring
1. Use needle-nose pliers or similar tool to remove

the old retaining ring from the inlet port
(marked HP) and discard. Be careful not to
scratch the sides of the inlet port with the pliers.
2. Remove the old filter ring and filter and discard.
Note: If necessary, tap the regulator by hand to
allow the filter ring to fall out.
Note: If necessary, prod the filter with the needlenose pliers to tip it until the filter falls out.
Be careful not to scratch the sides of the
inlet port with the pliers.
MS-CRD- KPRMAINT
Revision R1 03-05
Handle
Poppet damper
Body
Inlet port (marked HP)
Filter
N-79
Filter
ring Retaining
ring
www.swagelok.com
Body Reassembly
Note: Ensure the regulator body and all parts are
clean and free of damage.
1. Place the new seat into the new seat retainer
with the ID groove or boss pointing away from the
seat retainer. Apply a very light coat of provided
lubricant to the seat retainer threads.
Handle
Cap assembly
Cap ring
Lubricate
threads
ID groove or boss on seat
Seat in seat retainer
2. Slip the new poppet spring and new poppet

damper onto the thicker end of the new poppet.
Fit the flat face of the poppet damper against the
poppet spring.
3. Invert the seat retainer/seat assembly and place
the thinner end of the poppet in the center of the
plastic seat.
4. Keeping the poppet/seat retainer assembly
inverted, invert the body and thread the new seat
retainer into the body. Tighten to 15 ftlb (20Nm)
torque with a 5/8 in. socket on the torque wrench.
5. Install the new diaphragm
into the body with the large
convolution facing down into
the body. For 0 to 250 psig
and 0 to 500 psig regulators,
Convolution on
diaphragm
which use two diaphragms,
both diaphragms have the
same orientation.
Boss on
6. Install the stop plate onto
stop plate
the new diaphragm with
the boss on the stop plate
facing up, away from the
diaphragm.
7. For 0 to 500 psig
regulators, place the
spring button into the
Boss on
Range spring
cap assembly with
spring button
the boss facing away
from the handle,
coned side up. Place the range spring into the
cap.
8. Place the cap assembly on the body.
9. Thread the cap ring onto the body. Tighten cap
ring to 117 ft.lb (160 Nm) torque with a 2 in.
wrench adapter on the torque wrench using the
flats to grip the cap ring.
10. Test the regulator for proper operation.
Stop plate
Diaphragm
Seat retainer
Seat
Poppet
Poppet spring
Poppet damper
Body
Inlet port (marked HP)
Filter
Filter
ring
Retaining
ring
Filter Reassembly
1. Place the new filter in the
inlet port (marked HP) with
the rough face toward the
inside of the regulator and Smooth face Rough face
the smooth face out.
2. Place new filter ring and retaining ring into
the inlet port. Ensure that the retaining ring is
fully seated into the groove inside the inlet port.
If necessary, use a blunt tool to fully seat the
retaining ring.
SwagelokTM Swagelok Company
2004, 2005 Swagelok Company
N-80
MS-CRD-KPRMAINT
Revision R1 03-05
N-81
N-82
N-83
N-84
N-85
N-86
N-87
N-88
N-89
N-90
N-91
APPENDIX O
MOBILE TEST CHAMBER 4 (MTC-4) O2 QUANTIFICATION
O-1
O-2
O-3
APPENDIX P
MOBILE TEST CHAMBER 4 (MTC-4) ELECTROPHYSIOLOGY
P-1
Date: 28-Jan-10
URGENT QUOTATION
Prices are valid for 120 days.

PAVE will confirm all orders by fax or e-mail.
Please Confirm your PO in writing. Thank you!
To: LT Denis G. Colomb,
Tel 850-230-3238
Company: Navy Experimental Diving Unit
Fax:
Ref: email
Email:
From: Walt Wood x27
Area Sales Rep:
Quote #: 2010107
Page 1
PAVE drawing/product number must be on the purchase order as the approved design. All tooling is the property of PAVE. Cancellation or
restocking charges of ~30% will normally apply on any customer's cancellation after the purchase order has been accepted by PAVE Technology
Co. Any blanket orders or large quantities quoted must be shipped complete within 12 months of the first shipment release date. Material
supplied to PAVE by a customer is supplied at the customer's own risk with 10% spares required to cover prototype development. Any below
listed one time non-recurring engineering changes (NRE) are invoiced at 50% upon receipt of the purchase order with the balance due upon the
acceptance of the first article. FOUR weeks minimum notice is required on PO shipment changes.
Otherwise terms are VISA/MasterCard credit cards,
CREDIT TERMS: % 10 NET 30 DAYS ON CREDIT APPROVED ACCOUNTS
prepaid or Fed x COD. New accounts to supply 3 references including bank references or D&B rating. Bank Wires to: NATIONAL CITY
BANK DAYTON OH USA, ACCT# WILL ADVISE, ABA ROUTING #042200279.
SHIPPING: F.O.B. DAYTON, OHIO USA Shipped via UPS, DHL, or FED X. Please state your air courier acct#.
SUMMARIZED CUSTOMER REQUIREMENTS FOR NEW DESIGNS:

DIRECTION:
PRESSURE: 750 psi
LEAK TEST: 80 psi or see print
OPER. TEMP C: -20 C to 150 C maximum THERM CYCLE:
TEST: 630 VDC 500 MEGOHM
AMPERAGE:
VOLTAGE:
WIRE COLORS:
WIRE TYPE:
SEALING:
SPECIAL: customer supplied fiber optic cables
ALL TESTING IS DONE AT ROOM TEMPERATURE ~25C AFTER CYCLING.
Please email any available AutoCAD, SolidWorks, or .IGS format assembly drawing to expedite custom design
delivery. Any NRE charge (Non-Recurring Engineering) includes providing the customer with a detailed assembly
drawing for the customer to approve prior to manufacture. A limited lifetime warranty applies to registered trademark
products PAVE-Seal, PAVE-Mate, PAVE Opti-Seal and PAVE-Flex.
Ship Date Est: 10 weeks AROM

DELIVERY IS SUBJECT TO CONFIRMATION AT TIME OF PURCHASE ORDER RECEIPT. WEEKLY RELEASES FOR LARGE ORDERS.
PAVE Part Numbers*/Description-See catalog on descriptions codes *Call for a PAVE 4 digit part/drawing # TBA=To Be Assigned
Line # PAVE Part
Quantity
PAVE Description
Total US$
Price
3815
VS18-SS-150-10-FO-20-5FT, customer
supplied fiber cable, no terminations,
nitrile o-ring. Material certs supplied.
$2,500.00
$5,000.00
NRE
One time non-recurring Engineering

charge for design, fixtures, and CAD
drawing. Allow 4-6 weeks from PO
issuance and our receipt for drawings to
be sent for review.
$900.00
$900.00
Total
$5,900.00
Sub-Total
$5,900.00
P-2
Tax
**** SALES ORDER ****

ACKNOWLEDGEMENT
PAVE Technology Co., Inc.
Dayton, OH 45414-3445
U.S.A
(937) 890-1100
SOLD TO:
NAVY EXPERIMENTAL DIVING UNIT
321 BULLFINCH RD.
ATTN: ACCOUNTS PAYABLE
PAGE:
ORDER NUMBER:
ORDER DATE:
0012165
02/19/10
SALESPERSON:
CUSTOMER NO:
HOUS
00-NAVDIV
SHIP TO:
321 BULLFINCH RD.
RECEIVING OFFICER
7015
Panama City FL 32407-7015
Panama CityFL 32407-7015
United States
United States
-------------------------------------------------------------------------------CUSTOMER P.O.
SHIP VIA
F.O.B
TERMS
N0463A10P0022
UPS GROUND
1/2%10, NET 30
-------------------------------------------------------------------------------ITEM NO.
UNIT ORDERED
SHIPPED
BACK ORD
PRICE
AMOUNT
-------------------------------------------------------------------------------3815
EACH
2.00
0.00
0.00
2,500.00
5,000.00
VS18-SS-150-10-1000UMFO-240-60
*NRE
EACH
1.00
0.00
NON-RECURRING ENGINEERING CHAR
NON-RECURRING ENGINEERING CHARGE FOR
DESIGN, TOOLING AND CAD DRAWING
0.00
SHIP DATE:05/18/10
900.00
900.00
SHIP DATE:05/18/10
NET ORDER:
LESS DISCOUNT:
FREIGHT:
SALES TAX:
ORDER TOTAL:
BOM CHECK HOUSINGS________ CONNECTORS_______
P-3
WIRE_______
WDW___________ DATE____________
-----------5,900.00
.00
.00
.00
-----------5,900.00
Wire Conductor
Amperage
Gage (AWG) O.D.
Rating
30
0,30 mm
2
28
2
0,40
26
0,50
3
24
0,60
3
22
0,78
4
20
0,90
6
18
1,21
8
16
1,50
10
14
1,85
18
12
2,40
25
10
2,94
40
8
3,73
60
6
4,67
80
Nominal Insulation
Overall Diameter (O.D.)
.03
0,8 mm
.04
1,0
.05
1,3
1,5
.06
.06
1,5
.07
1,8
2,0
.08
.09
2,3
.11
2,8
.12
3,0
.16
4,1
.21
5,3
.26
6,6
Also available on-line in Design Assistance section.
Stranded, plated copper wires, 600 V (105 C PVC or 200 C Teflon Insulation)
Any type or size of wire or cable

may be specified including
shielded cables, coaxial cables,
thermocouple wire pairs, high
voltage wires, fiber optic cable,
flat cables, etc. for any length.
NO DETECTABLE LEAKAGE
THRU CONDUCTOR OR
INSULATION.
High moisture or submerged seals in electrically conductive fluids should use non-Teflon
insulated wire or cross linked Teflon to avoid electrical shorts.
CSA
Ratings
Available
Please send the following information with your request for a quote or sales engineering design recommendations:
Your Name_______________________________________Title_________________________ Department_________________________
Company Name_____________________________________________ Street_________________________________________________
City____________________ State/Province_____________________ Mail Code_______________________ Country_______________
Tel:______________________ Fax:________________________ E-Mail ______________________________Mail catalog?____________
How did you become aware of PAVE ________________________________________________________________________________
Operating Conditions
Describe equipment our product will be used in ________________________________________________________________________
Describe media to be sealed (e.g., gas, fluids)___________________________________________________________________________
Maximum Pressure and/or Vacuum ______________________________________ Temperature Extremes (C)____________________
Voltage (Indicate AC or DC)______________________________________ Amperage duty cycle_________________________________
Special Requirements_______________________________________________________________________________________________
Leak Rate Test ____________________________________________________________________________________________________
MAXIMUM DIAMETER
OPTIONAL
DISCONNECT
STYLE
SIZE
MATERIAL
O-RING
BULKHEAD
THICKNESS
BULKHEAD
MATERIAL
PISTON
FACE
OPTIONAL
DISCONNECT
RANGE
FIXED
CHECK CIRCLE TO INDICATE

TEST DIRECTION & AMOUNT
VACUUM LEVEL
PRESSURE LEVEL
OPTIONAL
(E) EN D STRIP OR
(T) TERMINATION?
1 bar=14.5 psi=100 k Pa=34 ft or 10 m of H
THREAD SIDE WIRE LENGTH

OR DISCONNECT TYPE?
2O
Design Parameters
Number of Electrical/Fiber-Optic Conductors By Size/Type.
PAVE-Seal Style with cable both ends (______) Cable Gage(s)____________________ Insulation type____________________________
PAVE-Mate I Stylewith pin disconnects both ends. Number of Pins_______ Gage or Size?___________________________________
PAVE-Mate II Stylewith pin disconnect one side only, Number of Pins (______) Gage _______________________________________
Number of pins/wires?________________________________ Gage or Size?_________________________________________________
PAVE Optic-SealStyle with fiber-optic cable both ends (______) __________________________________________________________
Cable Specification including insulation type and size___________________Define any high pressure cycling, direction, and duration
_________________________________________________________________________________________________________________
Preferred Method of Installation? (Threaded type, O-ring gland or face seal or TIG or laser welded)_____________________________
Quantities to quote_________________________Target/budget prices? ______________________Needed ship date?______________
Please fill in the blocks and
check a ppropriate ci rcles.
CHECK CIRCLE TO INDICATE

PRESSURE DIRECTION & AMOUNT
PRESSURE LEVEL
VACUUM LEVEL
(E) END STRIP OR

(T) TERMINATION ?
HEX SIDE WIRE LENGTH

OR DISCONNECT TYPE?
Quick-shipping standard connectors and rush shipment or prototyping available! Simple or detailed drawings sent along
with your quotation request are encouraged to help clarify your minimum critical mechanical and electrical requirements
so we can help direct you to the most cost effective, fast solution.
Product drawings at: PAVEtechnologyCo.com Phone 1.937.890.1100 Fax: 1.937.890.5165
P-4
BATH QUOTE COLOMB
QUOTATION
207573
Warner Instruments LLC

1125 Dixwell Ave, Hamden, CT 06514
Toll-Free: 800-599-4203
Phone: 203-776-0664
Fax: 203-776-1278
WEB Site: www.warneronline.com
Holliston, 12-16-2011
Customer:
USN003
Quotation:
US NAVY
FISCAL OFFICE
321 BULLFINCH ROAD
PANAMA CITY FL
32407
207573
Quotation Date:
Ref.:
12-16-11
Denis Colomb
Phone #: 850 230 3111

Email: denis.colomb@navy.mil
We thank you for your inquiry.
Quantity
UOM Item Code
Price
UOM
Tax
Discount
Amount
EA
1389.00
EA
443.00
Quote issued to
Lt Denis Colomb Jr
850-230-3238
denis.colmb@navy.mil
SHIPPING IS ESTIMATED.
QUOTE EXPIRES 30 JAN 2011.
ESTIMATED SHIP DATE IS 2 WEEKS AFTER RECEIPT OF ORDER.
3.00
EA
641532
463.0000
CHMBR RC27LD DRIP ULTRA QUIET

RC-27LD
supplied with:
MAG-2 magnetic clamp
magnetic perfusion tube holder
package of CS-24/60 coverglass
package of PE-160/10 tubing
SS-4-500H slice support
NOTE: this chamber is compatible with the PM-7D platform.
It is also compatible with all the SHD-27L slice anchors.
1.00
EA
641548
443.0000
CHMBR RC27D DRIP ULTRA QUIET

RC-27D
supplied with:
MAG-2 magnetic clamp
magnetic perfusion tube holder
package of CS-24/60 coverglass
package of PE-160/10 tubing
NOTE: this chamber is compatible with the PM-7 platform.

Carry forward:
P-5
1832.00
BATH QUOTE COLOMB

Customer:

Toll-Free: 800-599-4203
Phone: 203-776-0664
Fax: 203-776-1278
Quantity
QUOTATION
USN003
Quotation:
207573
Quotation Date:
Ref.:
UOM Item Code
Price
12-16-11
Denis Colomb
UOM
Tax
Discount
Carry forward:
Amount
1832.00
It is also compatible with all the SHD-27L slice anchors.

**this chamber is not supplied with a slice support**
1.00
EA
FRT
32.0000
EA
32.00
Freight
ESTIMATED Shipping charge
UPS 2nd Day from factory (Hamden CT) to
Panama City FL 32407
Estimate Dimension:
2 lbs, 12x9x6 inch
2.00Terms of Delivery:
Prepaid-UPS
Costs:
Goods:
Discount:
Tax:
1832.00
Terms of Payment: Net 30

Forwarding Agent:
1 UPS Ground
Representative:
100 Betty Ball
This quotation expires on:
WARRANTY:
PAYMENT TERMS:
RETURNS:
FOB Point:
SHIPPING:
Total ( $ ):
01-30-12
One year parts and labor.

Domestic: Net 30 on preapproved credit or Mastercard, VISA or American Express International: Bank
transfer or prepayment by check drawn in US funds on US banks.
Returns for credit must be made within 30 days of invoice date. No returns will be accepted after this
period. All returns must be accompanied by a copy of our return authorization form referencing a valid
RMA# from our Customer Service Dept. To be eligible for full credit, merchandise must be in original
shipping cartons and must be complete with all packing materials. ALL Returns MAY be subject to a 15%
restocking fee. NO RETURNS OR CANCELLATIONS ON CUSTOM ORDERS.
Hamden, CT.
Domestic: Freight is prepaid and added to invoice; method is UPS ground unless otherwise specified
International: Orders are shipped best way freight collect. Delivery times are based upon available stock
at time of quote and can not be guaranteed.
P-6
1864.00
BATH HEATER COLOMB
QUOTATION
205042

Toll-Free: 800-599-4203
Phone: 203-776-0664
Fax: 203-776-1278
Holliston, 03-24-2009
Customer:
USN003
Quotation:
US NAVY
FISCAL OFFICE
321 BULLFINCH ROAD
PANAMA CITY FL
32407
205042
Quotation Date:
Ref.:
03-24-09
Denis Colomb
Phone #: 850 230 3111

Email: denis.colomb@navy.mil
We thank you for your inquiry.
Quantity
UOM Item Code
Price
UOM
Tax
EA
Discount
Amount
Quote requested for

Denis Colomb, Lt
Biomedical
Tel: 850-230-3238
Email: denis.colmb@navy.mil
***NOTE***********
QUOTE EXPIRES 8 MAY 2009.
FREIGHT CHARGES ARE NOT INCLUDED WITH THIS
QUOTE.
ESTIMATED SHIP DATE IS APPROXIMATELY 1 WEEK
OR LESS AFTER RECEIPT OF ORDER (ITEM CURRENTLY
IN STOCK)
******************
2.00
EA
640109
70.0000
140.00
CABLE ASSEMBLY FOR HEATER CONTROLLERS

CC-35
Carry forward:
P-7
140.00
BATH HEATER COLOMB

Customer:

Toll-Free: 800-599-4203
Phone: 203-776-0664
Fax: 203-776-1278
Quantity
QUOTATION
USN003
Quotation:
205042
Quotation Date:
Ref.:
UOM Item Code
Price
03-24-09
Denis Colomb
UOM
Tax
Discount
Carry forward:
Terms of Delivery:
Prepaid-UPS
Costs:
Goods:
Discount:
Tax:
Amount
140.00
140.00
Terms of Payment: Net 30

Forwarding Agent:
1 UPS Ground
Representative:
100 Betty Ball
This quotation expires on:
WARRANTY:
PAYMENT TERMS:
RETURNS:
FOB Point:
SHIPPING:
Total ( $ ):
05-08-09
One year parts and labor.

Domestic: Net 30 on preapproved credit or Mastercard, VISA or American Express International: Bank
transfer or prepayment by check drawn in US funds on US banks.
Returns for credit must be made within 30 days of invoice date. No returns will be accepted after this
period. All returns must be accompanied by a copy of our return authorization form referencing a valid
RMA# from our Customer Service Dept. To be eligible for full credit, merchandise must be in original
shipping cartons and must be complete with all packing materials. ALL Returns MAY be subject to a 15%
restocking fee. NO RETURNS OR CANCELLATIONS ON CUSTOM ORDERS.
Hamden, CT.
Domestic: Freight is prepaid and added to invoice; method is UPS ground unless otherwise specified
International: Orders are shipped best way freight collect. Delivery times are based upon available stock
at time of quote and can not be guaranteed.
P-8
140.00
PIPETTE HOLDER COLOMB
ORDER ACKNOWLEDGEMENT
Customer:

Toll-Free: 800-599-4203
Phone: 203-776-0664
Fax: 203-776-1278
Bill to:
Order Date:
Q207026
Ship to:
US NAVY
EXPERIMENTAL DIVING UNIT/NEDU
RECEIVING OFFICER
321 BULLFINCH RD
PANAMA CITY FL
32407
FISCAL OFFICE
NAVY EXPERIMENTAL DIVING U
321 BULLFINCH ROAD
PANAMA CITY FL
32407
Attention: JAMES CALDWELL -PO# 4520259397
Dear Customer, we thank you for your order.

UOM Item Code
04-06-11
Ref.: 4520259397
US NAVY
Quantity
USN003
Order Number: 838840
Unit List
Discount
Net Amount UOM
Tax
Estimated
Ship Date
Extended Price
PHONE ORDER
JAMES CALDWELL
PHONE: 850 230 2317
EMAIL: JAMES.M.CALDWELL@NAVY.MIL
DEPT CONTACT: Lt Denis Colomb Jr PhD
850-230-3238
QUOTE 207026
**CREDIT CARD ORDER - VISA**

PLEASE EMAIL INVOICE: JAMES.M.CALDWELL@NAVY.MIL
AUTH CODE: 086510
*************************************************
NOTE: THESE ITEMS ARE CUSTOM MADE PRODUCTS;
NO RETURN OR CANCELLATION ALLOWED ON CUSTOM
PRODUCT ORDERS.
*************************************************
4.00
EA
991038
120.0000
120.0000
EA
CUSTOM QSP-T15P ELE HLDR
04-28-11
480.0000
04-28-11
520.0000
0.00
CUSTOM ELECTRODE HOLDER QSW-T SERIES WITH PORT STRAIGHT 1.5MM A
QSP-T15P CUSTOM HOLDER
NOTE: THIS IS A CUSTOM PRODUCT; NO RETURN OR

CANCELLATION ALLOWED ON CUSTOM PRODUCT ORDERS.
4.00
EA
991039
130.0000
130.0000
CUSTOM QRP-T15P ELE HLDR
EA
N
0.00
CUSTOM ELECTRODE HOLDER QR-T SERIES 90 STYLE 1.5MM AG PELLET
P-9
PIPETTE HOLDER COLOMB
ORDER ACKNOWLEDGEMENT
Customer:

Toll-Free: 800-599-4203
Phone: 203-776-0664
Fax: 203-776-1278
Quantity
UOM Item Code
USN003
Order Number: 838840

Order Date:
Unit List
Discount
Net Amount UOM
04-06-11
Tax
Estimated
Ship Date
Extended Price
QRP-T15P CUSTOM HOLDER

NOTE: THIS IS A CUSTOM PRODUCT; NO RETURN OR
CANCELLATION ALLOWED ON CUSTOM PRODUCT ORDERS.
1.00
EA
FRT
11.0000
11.0000
EA
Freight
04-28-11
11.0000
0.00
UPS GROUND SERVICE
Costs:
Delivery: Prepaid-UPS
11.00
Goods:
1000.00
Forwarding Agent: 1 UPS Ground

Representative: 100 Betty Ball
Discount:
Tax:
Payment: Paid by Credit Card

Credit Card Number:
Total ( $ ):
********************
1011.00
WARRANTY: One year all parts and labor.

RETURNS:
Returns for credit must be made within 30 days of invoice date. No returns will be accepted after this period. All
returns must be accompanied by a copy of our return authorization form referencing a valid RMA# from our Customer
Service Dept. at Warner Instruments. To obtain a return authorization call 800-599-4203. All items are to be
returned to Warner Instruments, 1125 Dixwell Ave, Hamden, CT 06514. To be eligible for full credit, merchandise
must be in original shipping cartons and must be complete with all packing materials. All returns MAY be subject to a
15% restocking fee.
NO RETURNS OR CANCELLATIONS ON CUSTOM ORDERS.
FOB Point:
Origin
SHIPPING:
Domestic: Freight is prepaid and added to invoice, method is UPS.
International: Orders are shipped best way freight collect.
P-10
40
1.59
34.9
1.38
NOTES:
1. LEAK TEST: 750 PSI, NO BUBBLES 1 MINUTE MINIMUM.
2. HYPOT 630 VDC 500 MEGOHMS MINIMUM 0.01
SECOND MINIMUM WIRE TO WIRE AND HOUSING.
3. ALL TESTS ARE PERFORMED AT ROOM TEMPERATURE.
4. ALL PARTS MUST PASS ALL TESTS.
5. VOIDS LARGER THAN .035 [.89] ARE NOT
ACCEPTABLE.
REF-OPERATING TEMPERATURE -20 C TO 125 C.
DIMENSIONS ARE INCHES [millimeters].
6.
7.
ALL DIMENSIONS AND TOLERANCES APPLY TO FINISHED PART IN INCHES.

ALLOWABLE TOLERANCES UNLESS SPECIFIED OTHERWISE: NONE 0.5
X.X DECIMAL 0.1 X.XX DECIMAL 0.02 X.XXX DECIMAL 0.005
ANGLES 1 DEGREE SURFACE FINISH 128 microinch RMS
.06 [1.5] MAXIMUM

OVER/UNDERFILL
BOTH SIDES
1778
70 MINIMUM
3
1 2100-260
2
1 PAVE-Seal 150
1
1 1625-B
ITEM QTY PART NUMBER
ATMOSPHERE
1
34.7
1.37
(1" NPT)
PRESSURE
CABLE AXOPATCH 200B

EPOXY BLACK
HOUSING PT16-B
DESCRIPTION
6
.3
TYP
70
2.8
1219
48 MINIMUM
REVISION
LEVEL
MATERIAL
NOTED
25
1.0

Dayton, OH 45414-3445
U.S.A.
tel (937) 890-1100
fax (937) 8905165
www.pavetechnologyco.com
PROJECTION
PT16-B-150-1-AXOPATCH200B-70-48
DESCRIPTION
PART NUMBER
3618
P-11
P-12
HEADSTAGE APPROACH COLOMB
P-13
HEADSTAGE APPROACH COLOMB
P-14
BATH INFO
P-15
BATH INFO
P-16
BATH INFO
P-17
BATH INFO
P-18
APPOVAL HEADSTAGE MODIFICATION COLOMB
P-19
COC PAVE HEADSTAGE MOD
P-20
P-21
SECONDARY HOLDER COLOMB
P-22
SECONDARY HOLDER
P-23
P-24
P-25
P-26
Controller
With the MS 314 controller all three axes of the manipulator DC-3K or DC-3KS can be operated bi-directional, but not simultaneously, via six push buttons on the front panel. A brief (< 1 sec.) push of a button activates the single step mode, as defined in "step size" (the same for all three axes).
The backlash (caused by gearing ratio) that usually occurs with change of direction, is virtually eliminated
with this controller. With each change of direction additional current impulses are sent to the motors, causing them to turn almost exactly as many times as needed to compensate the gearbox play. This is especially helpful in the single step mode, as it prevents "skipping" (the motor turns, but the micromanipulator
does not move).
MS 314
push button controller
for DC-3K / DC-3KS
On the rear the MS 314 is equipped with an earthing socket and it includes shielded cables. Using the
optional remote control, all three axes can be directed for forward and reverse movement.
MS 314 with DC - 3K
Specifications
Order information
Step size:
adjustable from 0,5 to 10 m
MS 314
Part No.: 00-75-220-0814
Continuous speed:
continuous velocity
adjustable from 0 to 200 m/s
Accessories
Power supply:
115/230 volt 50-60 Hz
Fuses:
115 volt T 0.1A / 230 volt T

0.05A (5x20mm)
Thermal fuse:
98C
Operating temperature:
5C to 40C
Storage temperature:
-20C to 60C
Dimensions:
190 x 47 x 138 mm
Mrzhuser GmbH & Co. KG
Weight:
900 g
In der Murch 15
Remote control
Part No.: 00-75-220-1814
35579 Wetzlar-Steindorf
Tel.: +49 (0) 64 41/91 16-0
Fax: +49 (0) 64 41/91 16-40
For more detailed information, please visit our web-site at: www.marzhauser.com
2004-02-22
P-27
eMail: info@marzhauser.com
www.marzhauser.com
APPENDIX Q
MOBILE TEST CHAMBER 4 (MTC-4) ELECTRICAL AND ANSCILLARY
COMPONENTS
Q-1
O-2
O-3
Q-4
Q-5
Q-6
Q-7
Q-8
Q-9
Q-10
Q-11
Q-12
Q-13
Q-14
Q-15
Q-16
Q-17
Q-18
Q-19
Q-20
Q-21
Q-22
Q-23
Q-24
Q-25
Q-26
Swagelok Company
29500 Solon Rd
Solon, OH 44139 U.S.A
440.349.5600
440.519.4997 fax
Certificate of Compliance/Typical Material Certification (EN 10204-2.2)

Distributor
Alabama Fluid System Technologies Inc.
26280 Publuc Works Road, Suite A-5
Daphne, AL 36526
Customer
NEDU
321 BULLFINCH RD
PANAMA CITY BEACH, FL 32407
No. Part Number

1 B-2-A
2 B-4-A
Customer PO#
CREDIT CARD ORDER
Qty
8
8
Information contained in the above customer address column (marked as "Customer") and area marked "Reference" (when
applicable) of this certification are for reference purposes only. Swagelok Company makes no stipulations, nor takes
responsibility, for the accuracy or reliability of such information.
Swagelok products referenced above are manufactured from material purchased and certified as being in accordance with
the specification(s) listed below.
Swagelok products are manufactured under conditions which are free from mercury. No Mercury bearing components have
been used in the products of your order and no Mercury bearing instruments or other equipment have been used in their
manufacture, assembly, or testing in such a manner as might cause contamination.
No asbestos or asbestos-containing components are used in Swagelok brand products.
The material stipulations included in this certification do not include such components as snap rings, springs, balls, o-rings,
gaskets, jam nuts, space collars, seals, locking dogs, lanyards, or sleeves.
All parts were cleaned and packaged in accordance with Swagelok Specifications.
Typical mechanical and/or chemical analysis of the material used in the manufacture of the Swagelok products involved are
listed below. These values are average values determined from a sample of certified material test reports. Actual values for
a material heat may vary from those indicated.
MATERIAL STANDARDS
Components
Brass 345 or 353 Barstock
Brass 360 Barstock
Brass 377 Forging
Material
Brass 345 or 353 Bar
Brass 360 Bar
Brass forgings (Type 377)
Components / Materials
Brass 360 Barstock
Brass 377 Forging
Yield Strength
(ksi/MPa)
36/248
45/310
32/221
Standards
ASTM B453
ASTM B16, ASTM B249
ASTM B283, ASTM B249
MECHANICAL PROPERTIES
Brass 360 Barstock
Brass 377 Forging
PC090416406-001
Cu
63.35
60.64
59.20
Tensile Strength
(ksi/MPa)
55/379
60/414
64/441
Elongation
(%)
33
24
34
Hardness
HRB 62
HRB 65
CHEMICAL ANALYSIS
Fe
0.03
0.11
0.08
Pb
1.85
3.31
1.83
Zn
36.00
35.81
38.89
Page:
Q-27
of
04/16/2009
Swagelok Company
29500 Solon Rd
440.349.5600
440.519.4997 fax
The Swagelok product specified above were manufactured in accordance with Swagelok Companys Quality Assurance
Manual (latest revision, revision H, dated December 10, 2007). Swagelok Companys Quality System is approved to ISO
9001 (BSI Certificate # FM01729).
Certifications Supervisor
Jonathan Seewald
PC090416406-001
Page:
Q-28
of
04/16/2009
Swagelok Company
29500 Solon Rd
440.349.5600
440.519.4997 fax
Certificate of Compliance/Typical Material Certification (EN 10204-2.2)

Distributor
26280 Publuc Works Road, Suite A-5
Daphne, AL 36526
Customer
NEDU
321 BULLFINCH RD
PANAMA CITY BEACH, FL 32407
No. Part Number

1 B-16-A
Customer PO#
CREDIT CARD ORDER
Qty
8
Swagelok products referenced above are manufactured from material purchased and certified as being in accordance with
the specification(s) listed below.
The material stipulations included in this certification do not include such components as snap rings, springs, balls, o-rings,
gaskets, jam nuts, space collars, seals, locking dogs, lanyards, or sleeves.
All parts were cleaned and packaged in accordance with Swagelok Specifications.
Typical mechanical and/or chemical analysis of the material used in the manufacture of the Swagelok products involved are
listed below. These values are average values determined from a sample of certified material test reports. Actual values for
a material heat may vary from those indicated.
Components
Brass 360 Barstock
Brass 377 Forging
MATERIAL STANDARDS
Material
Brass 345 or 353 Bar
Brass 360 Bar
Brass forgings (Type 377)
Standards
ASTM B453
ASTM B16, ASTM B249
ASTM B283, ASTM B249
MECHANICAL PROPERTIES
Brass 360 Barstock
Brass 377 Forging
Brass 360 Barstock
Brass 377 Forging
Yield Strength
(ksi/MPa)
36/248
45/310
32/221
Cu
63.35
60.64
59.20
Tensile Strength
(ksi/MPa)
55/379
60/414
64/441
Elongation
(%)
33
24
34
Hardness
HRB 62
HRB 65
CHEMICAL ANALYSIS
Fe
0.03
0.11
0.08
Pb
1.85
3.31
1.83
Zn
36.00
35.81
38.89
The Swagelok product specified above were manufactured in accordance with Swagelok Companys Quality Assurance
Manual (latest revision, revision H, dated December 10, 2007). Swagelok Companys Quality System is approved to ISO
9001 (BSI Certificate # FM01729).
Certifications Supervisor
Jonathan Seewald
PC090416406-002
Page:
Q-29
of
04/16/2009
Q-30
Swagelok Alabama
237 Cahaba Valley Parkway
Pelham, AL 35124
205-988-4812
9/16/2013 12:35:14 PM
www.swagelok.com
Pipe Adapters
Part No.
B-2-A
Part Description:
Brass Pipe Fitting, Adapter, 1/8 in. Female NPT x 1/8 in. Male NPT
Product Specifications
General
Body Material
Brass
Cleaning Process
Standard Cleaning and Packaging (SC-10)
Connection 1 Size
1/8 in.
Connection 1 Type
Female NPT
Connection 2 Size
1/8 in.
Connection 2 Type
Male NPT
eClass (4.1)
37030708
eClass (6.0)
37-02-05-90
UNSPSC (11.0501)
40142300
UNSPSC (4.03)
40141719
UNSPSC (SWG01)
40141719
The complete catalog contents must be reviewed to ensure that the system designer and user make a safe product selection. When
selecting products, the total system design must be considered to ensure safe, trouble-free performance. Function, material compatibility,
adequate ratings, proper installation, operation, and maintenance are the responsibilities of the system designer and user.
Caution: Do not mix or interchange valve components with those of other manufacturers.
2013 Swagelok Company
Q-31
APPENDIX R
MOBILE TEST CHAMBER 4 (MTC-4) ANCILLARY
R-1
3/3/2013 1:49:38 PM
www.swagelok.com
Caps and Plugs

Part No.
B-1610-C
Part Description:
Brass Cap for 1 in. OD Tubing
General
Body Material
Brass
Cleaning Process
Configuration
Cap
Connection 1 Size
1 in.
Connection 1 Type
Swagelok Tube Fitting
eClass (4.1)
37030703
eClass (6.0)
22-56-02-07
UNSPSC (11.0501)
40142607
UNSPSC (4.03)
40141720
UNSPSC (SWG01)
40141720
R-2
3/3/2013 1:48:04 PM
www.swagelok.com
Caps and Plugs

Part No.
B-400-C
Part Description:
Brass Cap for 1/4 in. OD Tubing
General
Body Material
Brass
Cleaning Process
Configuration
Cap
Connection 1 Size
1/4 in.
Connection 1 Type
eClass (4.1)
37030703
eClass (6.0)
22-56-02-07
UNSPSC (11.0501)
40142607
UNSPSC (4.03)
27121703
UNSPSC (SWG01)
27121703
R-3
3/3/2013 1:50:59 PM
www.swagelok.com
Caps and Plugs

Part No.
B-200-C
Part Description:
General
Body Material
Brass
Cleaning Process
Configuration
Cap
Connection 1 Size
1/8 in.
Connection 1 Type
eClass (4.1)
37030703
eClass (6.0)
22-56-02-07
UNSPSC (11.0501)
40142607
UNSPSC (4.03)
40141706
UNSPSC (SWG01)
40141706
R-4
3/3/2013 1:44:19 PM
www.swagelok.com
Caps and Plugs

Part No.
B-1210-C
Part Description:
General
Body Material
Brass
Cleaning Process
Configuration
Cap
Connection 1 Size
3/4 in.
Connection 1 Type
eClass (4.1)
37030703
eClass (6.0)
22-56-02-07
UNSPSC (11.0501)
40142607
UNSPSC (4.03)
40141600
UNSPSC (SWG01)
40141600
R-5
CUSTOMER DRAWING
1/8'' NPT
- DRAWING NOT TO SCALE

- DIMENSION ARE INCHES NEXT TO [MILIMETRES]
B-2-A
Adapter
TITLE
PART
NO.
1/8'' NPT
PART
NO.
B-2-A
R-6
1.10[27.9]
9/16'' HEX
- DRAWING IS SUBJECT TO CHANGE WITHOUT NOTICE.

- ALL ASSEMBLED NUTS AND FERRULES ARE SHOWN AT FINGER TIGHT DIMENSIONS.
- ALL HEX CALL-OUT ARE ACROSS FLATS.
0.19[4.8]
CUSTOMER DRAWING
1/4'' NPT
- DRAWING NOT TO SCALE
3/4'' HEX
- DIMENSION ARE INCHES NEXT TO [MILIMETRES]

- DRAWING IS SUBJECT TO CHANGE WITHOUT NOTICE.
1.40[35.5]
- ALL ASSEMBLED NUTS AND FERRULES ARE SHOWN AT FINGER TIGHT DIMENSIONS.
- ALL HEX CALL-OUT ARE ACROSS FLATS.
B-4-A
Adapter
TITLE
PART
NO.
1/4'' NPT
B-4-A
R-7
PART
NO.
0.28[7.1]
4/17/2009 11:21:18 AM

26280 Public Works Road, Suite A-5
Daphne, AL 36526
251-625-2949
www.swagelok.com
Pipe Adapters
Ordering #:
B-4-A
Description:
Brass Pipe Fitting, Adapter, 1/4 in. Female NPT x 1/4 in. Male NPT
Specification Summary
General
Body Material
Cleaning Process
Connection 1 Size
Connection 1 Type
Connection 2 Size
Connection 2 Type
eClass (4.1)
eClass (6.0)
UNSPSC (11.0501)
UNSPSC (4.03)
Brass
1/4 in.
Female NPT
1/4 in.
Male NPT
37030708
37-02-05-90
40142300
40141719
REVIEW PRODUCT CATALOG FOR COMPLETE SPECIFICATIONS INCLUDING WARNINGS AND CAUTIONS.
Safe Product Selection: When selecting a product, the total system design must be considered to ensure safe, trouble-free performance. Function, material compatibility,
2006 Swagelok Company, Elgiloy - TM Elgiloy Specialty Metals, SAF 2507 - TM Sandvik AB
All trademarks shown are owned and registered by Swagelok Company, unless otherwise noted.
R-8
R-9
R-10
R-11
R-12
R-13
R-14
R-14
R-15
R-16
R-17
R-18
Type AVC-1000 & AVC-3000

Pressure Volume Controller
APPLICATION
For applications requiring extremely
fine pressure adjustment
FEATURES
Precisely set a pressure in a closed
pneumatic system
Pneumatic Volume controller
Vacuum - 1,000 or 3000psi
Piston/cylinder device provides fine
adjustment in closed pneumatic
systems
Ashcroft Pressure Volume Controllers

provide an easy-to-use means of rapidly
and accurately adjusting pressure in a
closed system. These pressure controllers
provide the ability to precisely adjust pressure within 0.00025 psi. When used with
digital pressure gauges, dial gauges,
transducers, pressure switches, air deadweight testers, manometers and other similar devices they provide a fast and accurate means for setting a precise pressure.
STANDARD SPECIFICATIONS
Model......................................................
AVC-1000
Range (psi)...............................................
Vacuum-1000
Proof (psi).................................................
2000
Burst (psi).................................................
6000 min.
Resolution (psi)..........................................
0.00025
Volume Change (cubic inches).........................
3.5
Mechanical Rotation (total turns).....................
31
Connection................................................
1/8 NPTF
Operating Temperature. Range........................
20-120 F
Construction Aluminium body, Stainless Steel, Brass, Teflon, Delrin and Buna-N
Vent Valve
Inlet Valve
Balance
Valve
AVC-3000
Vacuum-3000
6000
12,000 min.
0.0005
2.5
61
1/8 NPTF
20-120 F
Vent Valve
Inlet Valve
Balance
Valve
Balance Valve Open System in

Pressure Balance or Vent Mode
DIMENSIONS
Balance Valve Closed

System in Test Mode
1.22 CLOSED (31.0)

1.37 OPEN (34.8)
.88 DIA
(22.35)
1.50
(38.1)
INLET PORT
VENT PORT
6-32 UNC-2B THREAD
TYP. (4 PLACES)
1.75
(44.45)
2.25 DIA
(57.15)
2.00 SQ.
(50.8)
3.34
(84.84)
1.00 SQ.
(25.4)
ASHCROFT
3.5
(88.9)
2.50 SQ.
(63.5)
7.16
(181.86)
BULLETIN AVC-1000
All specifications are subject to change without notice.

All sales subject to standard terms and conditions.
Ashcroft Inc. 2007
R-19
Ashcroft Inc., 250 East Main Street, Stratford, CT 06614 USA

Tel: 203-378-8281 Fax: 203-385-0408
email: info@ashcroft.com www.ashcroft .com
R-20
Volume Controller
Type AVC-1000 & 3000
Type AVC-1000 & 3000
1.22 CLOSED (31.0)
1.37 OPEN (34.8)
.88 DIA
(22.35)
1.50
(38.1)
2.25 DIA
(57.15)
3.34
(84.84)
7.16
(181.86)
INLET PORT
VENT PORT
6-32 UNC-2B THREAD
TYP. (4 PLACES)
1.75
(44.45)
2.00 SQ.
(50.8)
1.00 SQ.
(25.4)
Volume Controller
Type AVC-1000 & 3000 Dim. Dwg.
ASHCROFT
3.5
(88.9)
2.50 SQ.
(63.5)
Ashcroft Inc 2006., 250 East Main St., Stratford, CT 06614-5145 USA, Tel: 203-378-8281, Fax: 203-385-0499, www.ashcroft.com
R-21
67
Volume Controller
Type AVC-1000 & 3000
Ashcroft precision-pressure
volume controllers provide a quickand-easy method for precisely setting a pressure in a closed pneumatic
system. They are ideal for use with
Ashcroft test gauges for the calibration of other pressure-measurement
and control devices.
The AVC unit consists of a volume
chamber with an internal piston
assembly. The piston seals across
the diameter of the chamber. Once
the AVC unit is connected to a pneumatic system, the volume of the
chamber becomes part of the volume
of the system. The pressure-adjust
knob at the front of the unit repositions the piston within the chamber
through interaction with a precisionmachined lead screw. Piston movement within the chamber increases or
decreases the volume of the system,
depending on the direction of movement. In a closed system where gas
cannot leak out upon compression
or be drawn in upon expansion, this
volume change results in a change in
the internal pressure. Increasing the
volume by moving the piston toward
the front of the AVC unit will decrease
the pressure. Conversely, decreasing
the volume by moving the piston toward the rear of the unit will increase
the pressure. The pressure change
generated by a given amount of

piston travel is proportional to the
change in volume as compared to the
total system volume.
AVC units are available for pressures up to 3000 psi. The AVC-1000
can be used to set pressures from
vacuum through 1000 psi while the
AVC-3000 can be used for pressures
from vacuum through 3000 psi.
An integral balance valve provides
a means for equalizing pressure on
both sides of the piston prior to
making the nal adjustments when
setting the pressure. This minimizes
the resistance encountered when
repositioning the piston and assures
ease of pressure setting, even at
3000 psi. The balance valve also
serves as a pressure-relief valve,
assuring that the differential pressure
across the piston does not reach
unsafe levels.
AVC units can also be used without
a compressed air source for the generation of moderate levels of positive
pressure and vacuum. The high resolution of the AVC, combined with
the ability to generate pressure and
vacuum, make it an ideal tool for lowpressure (below 1 psi) calibration and
test as well as higher pressure calibration and test activities.
GENERAL SPECIFICATIONS
Type
Range (psi)
Resolution (psi)
Volume Change (cubic inches)
Mechanical Rotation (turns)
Proof Pressure (psi)
Burst Pressure (psi)
Operating Temperature Range
Operating Media
Construction
AVC-1000
AVC-3000
vacuum-1000
vacuum-3000
0.00025
0.0005
3.5
2.5
31
61
2000
6000
6000 min
12,000 min
20-120F
20-120F
Clean, dry noncorrosive gas such as
compressed air or nitrogen
Aluminum body, stainless steel, brass
Teon, Delrin and Buna N
Consult factory for guidance in product selection
Phone (203) 385-0217, Fax (203) 385-0602 or
visit our web site at www.ashcroft.com
R-22
INTEGRATED MICROSTEPPING MOTOR

DRIVER AND HIGH PERFORMANCE
MACHINE/PROCESS CONTROLLER
FEATURES
Integrated Driver and Controller
in One Package
Low Cost
Ultra Small Size (2.4 x 3.5 x 2.9 inches)
(60.96 x 88.9 x 73.66 mm)
2 Drive Output Selections:
+12 to +48 VDC, 3A RMS - 4A peak
+24 to +75 VDC, 5A RMS - 7A peak
Single Supply Operation
No Minimum Motor Inductance
Microstep Resolution up to 51,200
Steps per Rev. (1.8 Motor)
Programmable Current Settings Boost Acceleration and Allow Motor to Run Cooler
Electronic Gearing*
Open or Closed Loop* Control
Motion Values Scalable to any Units
Six +5 to +24 VDC Isolated I/O Lines
(Expandable to 24)
Programmable Digital Filtering for Inputs
32 Bit Floating Point Math, Logic and
Conditional Functions
Simple 2 to 5 Letter Programming
Instructions Similar to Basic
7 Hardware and 62 Software Addresses
for Multi-Drop Communications
Isolated Independent RS-232 and RS-485
with Selectable BAUD Rate to 38.4K, Full
or Half Duplex or CAN Bus
0 to 5MHz Step Clock Rate, Selectable
in 0.005Hz Increments
4 Pre-Defined and 1 User Defined
Acceleration/Deceleration Curves
Easy to Wire Removable Terminal Strips
or Optional Pin Headers
3 Expansion Slots for Optional Accessory
Modules
Short Circuit and Over Temp Protection
Power and Fault LED
* Requires High-Speed Differential I/O Module

RS-232 or RS-485 Available on Port 2 with
Optional Expansion Module
I/O, Communication and Expansion Modules Only
ACTUAL SIZE
DESCRIPTION
The MicroLYNX is a powerful machine control system
in te grat ing a bi po lar microstepping driver and expandable
programmable controller into
a com pact panel mounted
assembly.
Available in two output power
ranges, MicroLYNX fits a variety
of motor sizes. Features such
as +5 to +24 VDC isolated
I/O, multiple communication
types and numerous expansion
options make the MicroLYNX an
effective and powerful machine
control solution.
Optional differential I/O modules give the MicroLYNX the
ca pa bil i ty of driving two additional axes sequentially or
allowing electronic gearing
by following a rotary or linear
axis, or outputting a second
clock at an electronically controlled ratio.
The MicroLYNX provides for
two fully independent communication ports. It will accept
commands from either port and
direct output to either as well. A
RoHS compliant products may be indicated by a -G1 part number and/or RoHS Compliant label.
system could be configured to

use COMM port one to communicate to a host PC or PLC while
the second communicates with
an operator interface or additional MicroLYNX.
EXPANDABILITY
The MicroLYNX can be used
to con trol both sim ple and
complex systems. With plug-in
accessory modules OEMs have
the option of only purchasing
the features they need for any
given system design, reducing
overall cost. The MicroLYNX
can be field upgraded by simply removing its side cover and
adding or changing an expansion module, minimizing system
down time.
UPGRADABILITY
Software for the MicroLYNX is
upgradable. Updates are posted on the IMS web site and can
be downloaded and installed using the IMS Terminal/Upgrade
soft ware provided with the
MicroLYNX. This allows older
units to use new features and
expansion modules as they become available.
REV012407
GENERAL SPECIFICATIONS
ELECTRICAL
MOTION
Power Supply Requirements
Counters
Voltage1
Type . . . . . . . . . . . . . . . . . . . . . . . . . . Position, Encoder1, Encoder2: 32 Bits

Edge Rate (Max) . . . . . . . . . . . . . . 5 MHz
4 version
7 version
......................
......................
+12 to +48 VDC

+24 to +75 VDC
1Includes Motor Back EMF, Ripple and Hi Line.
Current2
4 version
7 version
......................
......................
2 Amps typical
3 Amps typical
2Actual requirements depend on application and programmable current
settings.
Electronic Gearing
External Clock In
Range* . . . . . . . . . . . . . . . . . . . . .
Resolution . . . . . . . . . . . . . . . . . .
Secondary Clock Out
Range* . . . . . . . . . . . . . . . . . . . . .
Resolution . . . . . . . . . . . . . . . . . .
-1 to 1
32 Bits
-2 to 2
16 Bits
Requires Differential I/O Expansion Module.

*Adjusting the microstep resolution of the driver can increase the range.
Recommended supply: ISP200 ( 4/7)
Velocity
Motor Drive
Motor Type . . . . . . . . . . . . . . . . . . . . . . 2/4 phase bipolar stepper
Motor Current (software programmable)
4 version . . . . . . . . . . . . . . . . . . . . . to 4 Amps peak
7 version . . . . . . . . . . . . . . . . . . . . . to 7 Amps peak
Resolution
Number of settings. . . . . . . . . . . . 14
Steps per Revolution
(1.8 Motor) . . . . . . . . . . . . . . . . . . 400, 800, 1000, 1600, 2000,
3200, 5000, 6400, 10000,
12800, 25000, 25600,
50000, 51200
Range . . . . . . . . . . . . . . . . . . . . . . . . 5,000,000 steps/sec

Resolution . . . . . . . . . . . . . . . . . . . . 0.005 steps/sec
Update Period . . . . . . . . . . . . . . . . 25.6 Microseconds
Acceleration/Deceleration
Range . . . . . . . . . . . . . . . . . . . . . . . . 1,530,000,000 steps/sec2
Resolution . . . . . . . . . . . . . . . . . . . . 0.711 steps/sec2
Types . . . . . . . . . . . . . . . . . . . . . . . . . Linear, triangle s-curve, parabolic,
sinusoidal s-curve, user defined
SOFTWARE
User Program Space . . . . . . . . . 8175 Bytes

Number of User Definable
Labels, Variables and Flags... 291
Program and Data Storage. . . Flash
General Purpose I/O
Math, Logic and Conditional
Number of I/O . . . . . . . . . . . . . . . . . . 6
Functions . . . . . . . . . . . . . . . . . . 32 Bit Floating Point Math IEEE
Input Voltage. . . . . . . . . . . . . . . . . . . . . +5 to +24 VDC
Format Add, Subtract, Multiply,
Output Current Sink . . . . . . . . . . . . . 350 mA
Divide, Sine, Cosine, Tangent, Arc
Input Filter Range . . . . . . . . . . . . . . . . 215Hz to 21.5 kHz (programmable)
Sine, Arc Cosine, Arc Tangent,
Pull-ups . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 k ohm individually switchable
AND, OR, XOR, NOT, Less Than,
Pull-up Voltage . . . . . . . . . . . . . . . . . . . +5 VDC on-board or externally
Greater Than, Equal, Square
up to +24 VDC
Root, Absolute, Integer Part,
Protection . . . . . . . . . . . . . . . . . . . . . . . Over Temp, Short Circuit,
Fractional Part
Inductive Clamp
Acceleration & Deceleration . . . Separate Variables and Flags
Isolated Ground . . . . . . . . . . . . . . . . . . Common to 6 I/O
4 Pre-Defined Types and
1 User Defined
Limit Switch . . . . . . . . . . . . . . . . . . Definable: Deceleration and Type
COMMUNICATION
Isolated I/O Line . . . . . . . . . . . . . . Software Selectable as
Dedicated or General Purpose
Predefined I/O Functions . . . . . 25 (Limit, Home, etc.)
Program Trip Functions . . . . . . 13 4 I/O Input Trips,
Asynchronous
4 Timer Trips, 4 Position Trips,
Interface Type . . . . . . . . . . . . . . . . . . . COMM 1: RS-232
1 Velocity Trip
COMM 2: RS-485
User Programs . . . . . . . . . . . . . . . 2 Executed Simultaneously
Number of Bits/Character . . . . . . 8
1 Foreground, 1 Background
Parity . . . . . . . . . . . . . . . . . . . . . . . . . . . . none
Party Mode Names . . . . . . . . . . . 62
Handshake. . . . . . . . . . . . . . . . . . . . . . . none
Communication Modes . . . . . . . 2 ASCII, Binary
Baud Rate . . . . . . . . . . . . . . . . . . . . . . . 4.8 to 38.4kbps selectable
Mechanical Compensation . . . . Backlash
Error Checking. . . . . . . . . . . . . . . . . . . 16 bit check sum (binary mode)
Encoder Functions . . . . . . . . . . . . Stall Detection and
Position Maintenance
ASCII Text or Binary Communication Modes
CAN
CAN Communications replaces Asynchronous Communications in
Base System (uses COMM 1 internally)
CAN compliance . . . . . . . . . . . . . . . . . Version 2.0B Active
2 receive message frames
1 transmit frame
Isolated Ground . . . . . . . . . . . . . . . . . . Common to COMM1 and COMM2
ENVIRONMENTAL
Ambient Operating Temp . . . . . 0 to 50 C*
Storage Temperature . . . . . . . . . 20 to 70 C
Humidity . . . . . . . . . . . . . . . . . . . . . . 0 to 90% non-condensing
* Can be duty cycle dependent.
MECHANICAL
Dimensions . . . . . . . . . . . . . . . . . . .
# of Expansion Modules . . . . . .
Cooling. . . . . . . . . . . . . . . . . . . . . . . .
Mounting . . . . . . . . . . . . . . . . . . . . .
Mounting Screw Torque . . . . . .
(see Mechanical Specs)

up to 3
Built-in fan
2 #6 (or M3.5) machine screws
5.0 to 7.0 lb-in
REV012407
MECHANICAL
SPECIFICATIONS
Dimensions in Inches (mm)
CONNECTORS
Power and Motor
CAN Version
6 position pluggable terminal block connector.
7 position pluggable terminal block connector

or optional 10 pin pin-header.
PIN
1
2
3
4
5
6
SIGNAL NAME
Phase A
Phase /A
Phase B
Phase /B
+V
Ground
FUNCTION
PIN
Motor Connections
Input Power
Power and Motor Connections
Communication
Dual COMM Version
CONNECTOR OPTION
TERMINAL BLOCK
PIN HEADER
Isolated GND
NO CONNECT
CAN_L
CAN_L
SHIELD
Isolated GND
CAN_H
SHIELD
NO CONNECT
SHIELD
/CONFIG
NO CONNECT
NO CONNECT
CAN_H
NO CONNECT
NO CONNECT
10
/CONFIG
CAN Comm1 Communications Connections

(Comm2 Available with Expansion Modules)
PIN
CONNECTOR OPTION
TERMINAL BLOCK
232RX
232TX
485RX
485RX+
485TX
C GROUND
485TX+
1
2
3
4
5
6
7
8
9
10
PIN HEADER
NO CONNECT
232TX
232RX
NO CONNECT
C GROUND
485RX+
485RX
485TX
485TX+
C GROUND
Asynchronous Communications Connections
SWITCHES
SWITCH
SIGNAL NAME
16
I/O 2126
79
Address 2 0
10
Upgrade
FUNCTION
Pull-up on/off Switches
for I/O Lines 21 26
Multi-drop Communication
Address (also settable by
software)
Firmware Upgrade
General Purpose I/O

PIN
CONNECTOR OPTION
TERMINAL BLOCK
VPULLUP
PIN HEADER
I/O 21
I/O 21
I/O 22
3
4
5
6
7
8
9
10
I/O 22
I/O 23
I/O 24
I/O 25
I/O 26
I/O GROUND
VPULLUP
I/O 23
FAULT INPUT +
I/O 24
FAULT INPUT
I/O 25
I/O GROUND
I/O 26
General Purpose I/O Connections
REV012407
EXPANSION MODULES
Up to three expansion modules can be added
to fill the MicroLYNX expansion slots. Module
combinations are limited to 2 Analog Input/
Joystick Modules, 2 Analog Output Modules,
1 Twelve Channel I/O Module, up to 2 HighSpeed Differential I/O Modules, and up to 3
General Purpose I/O Modules per system.
By simply removing two screws on the
MicroLYNX side cover, expansion modules can
be added, removed or reconfigured. No additional hardware is required. Modules snap easily into place, making even field change quick
and easy.
Expansion Modules
snap easily into place.
DEVICENET MicroLYNX
The DeviceNet MicroLYNX is specifically designed to conform with ODVA
Volume II, Release 2.0 Errata 3 as a
Position Controller (Device Type 16).
Node Address MSD and LSD

switch-selectable on front panel.
Data Rate switch-selectable on
front panel.
Conforms to the Predefined
Master/Slave Connection Set as
a group 2 Slave.
Supports Poll I/O and Explicit
Messaging.
Included GUI to aid in configuration and EDS file.
No support for UCMM.
* High Speed Differential I/O

Board Optional for Encoder
Functions. Board must be
placed in Expansion Slot #2.
Note that the High Speed Differential I/O Expansion is the ONLY
Expansion Module available for
the DeviceNet MicroLYNX.
Communication
5 Pin DeviceNet Micro, Male
V-
CAN_H
CAN_L
V+
Drain
DeviceNet I/O
8 position pluggable terminal block
connector or optional 10 pin pin-header.
PIN
CONNECTOR OPTION
TERMINAL BLOCK
PIN HEADER
VPULLUP
Home Input
Home Input
CW Limit Input
CW Limit Input
VPULLUP
CWW Limit Input
CWW Limit Input
Fault Input
No Connect
Brake Output
Fault Input
General Purpose
No Connect
I/O Ground
Brake Output
9
10
I/O Ground
General Purpose
DeviceNET I/O Connections
REV012407
ORDER INFORMATION
Standard Product Shown in Bold Italics. Longer Lead Times May Apply to Other Versions.
Note: Terminal Block Mating Connector Supplied.
Pin Header Mating Connector Not Supplied, order information at www.imshome.com/quick.html.
MICROLYNX CONTROL SYSTEMS

TYPE
DESCRIPTION
Control System with

Asynchronous
Communication
[6] +5 to +24 VDC Isolated I/O, RS-232 and

RS-485 Communication,
4 or 7 Amp Motor Drive
Control System with

CAN Communication
[6] +5 to +24 VDC

Isolated I/O, CAN
Communication,
Control System with

DeviceNet
Communication
[6] +5 to +24 VDC

Isolated I/O, DeviceNet
Communication,
CONNECTOR TYPE
COMMUNICATION
I/O
PART NUMBER
4 AMP DRIVER
7 AMP DRIVER
Pin Header**
Terminal Block
Pin Header**
Terminal Block
Pin Header
Terminal Block
Terminal Block
Pin Header
Pin Header
Terminal Block
Terminal Block
Terminal Block
MX-CS201-401
MX-CS201-701
Pin Header
Pin Header
MX-CS202-401
MX-CS202-701
Terminal Block
DeviceNet Micro,
Male
MX-CS100-401
MX-CS101-401
MX-CS102-401
MX-CS103-401
MX-CS200-401
MX-CS100-701
MX-CS101-701
MX-CS102-701
MX-CS103-701
MX-CS200-701
Pin Header
MX-CS203-401
MX-CS203-701
Terminal Block
Pin Header
MX-CS300-401
MX-CS302-401
MX-CS300-701
MX-CS302-701
**Communication cable accessory

recommended with first order.
EXPANSION MODULES
TYPE
(max # per system)
Isolated Digital I/O

Module (3)
DESCRIPTION
PART NUMBER
TERMINAL
PIN HEADER
BLOCK
[6] +5 to +24 VDC General Purpose Isolated Digital I/O
Analog Input/Joystick
Module (2)
[3] High-Speed Differential (or Single Ended) Lines for

General Purpose I/O, Encoder Feedback, Electronic
Gearing, or Driving an Additional Axis Sequentially
[2] Channels of 0 to +5 VDC 12 Bit
Resolution Analog Input or Single Axis Joystick
Analog Output Module

(2)
[2] Channels (Up to 4 total) of 0 to +5 VDC

12 Bit Resolution Analog Output
RS-232 Communication
Module (1)
RS-485 Communication
Module (1)
RS-232 Port 2 Communication Module used only with CAN

Based Control Sytem cannot combine with RS-485 Module
RS-485 Port 2 Communication Module used only with CAN
Based Control System cannot combine with RS-232 Module
12 Channel I/0
Module (1)
General Purpose I/O Module with

Twelve +5 to +24 VDC Isolated Digital I/O
High-Speed Differential
I/O Module (2)
MX-DI100-000
MX-DI200-000
MX-DD100-000
MX-DD200-000
MX-AJ100-000
MX-AJ200-000
MX-DA100-000
MX-DA200-000
MX-CM102-000
MX-CM202-000
MX-CM104-000
MX-CM204-000
Not Available
PIN & RECEPTACLE

MX-DI401-000
MX-DI400-000
Refer to 12 Channel I/O page for details.
ACCESSORIES
TYPE
DESCRIPTION
PART NUMBER
Human Machine
Interface (HMI)
A Programmable User Interface with 20 Character by 4 Line Display,

6 Function Keys, Numeric Keypad
LX-HI100-000
Operating Manual
10 Pin Pin-Receptacle to 9 Pin Sub D

Provides Easy Connection for RS-232 Communication
[2] 10 Pin Pin-Receptacle to 9 Pin Sub D (For Party Line Communication with Two
MicroLYNX) Provides Easy Connection for RS-232 Communication to First MicroLYNX
and RS-485 Communication from First to Second MicroLYNX
Operating Instruction Manual for MicroLYNX Systems - Paper Version*
6 Pin Terminal Block
[6] Pin Screw-Type Terminal Block Motor and Power

[7] Pin Spring Clamp Terminal Block 0.1" Center MicroLYNX Communications
MX-CN007
[8] Pin Spring Clamp Terminal Block 0.1" Center, MicroLYNX I/O and Expansion Modules
MX-CN008
CAN Dongle
Communication Cable for MicroLYNX CAN Version
MX-CC500-000
DeviceNet Programmer
Cable for use in Upgrading MicroLYNX DeviceNet Firmware
MX-CC600-000
Communication Cable
Communication Cable
(For Party Line)
MX-CC100-000
MX-CC200-000
LX-OM200-000
MX-CN006
*Manual on CD provided with system shipment.
REV012407
APPENDIX S
MOBILE TEST CHAMBER 4 (MTC-4) CCD FAILURE
S-1
M-2
Whatisleftfrom
CCD
S-2
M-3
S-3
CCDlocationfor
Camera
M-4
S-4
M5
S-5
Notetherubbergasket,andfrontwindow..Chip
shatterered;cannottellifrearcrushedorifHe
rushingincausingittoexplode.Engineers
believeHeinfluxcaused
M6
S-6
APPENDIX T
MOBILE TEST CHAMBER 4 (MTC-4) GAS PANEL FOR 1L CYLINDER
CHARACTERIZATION
T-1
T-2
T-3
T-4
T-5
T-6
T-7
T-8
T-9
T-10
T-11
T-12
T-13
T-14
T-15
T-16
T-17
T-18
T-19
T-20
T-21
APPENDIX U
MOBILE TEST CHAMBER 4 (MTC-4) PRESSURE RELIEF CHECK
U1
U-2
U-3
U-4
U-5
U-6
U-7
U-8
U-9
U-10
U-11
U-12
U-13
U-14
U-15
U-16

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Navy Experimental Diving Unit

DEVELOPMENT OF A HYPERBARIC UPRIGHT EPIFLUORESCENCE HOFFMAN

REPORT DOCUMENTATION PAGE

1. REPORT DATE (DD-MM-YYYY)

The Development of a Hyperbaric Upright Epifluorescence Hoffman Contrast Microscope for

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)

3. DATES COVERED (From - To)

12. DISTRIBUTION / AVAILABILITY STATEMENT

15. SUBJECT TERMS

Figure 61: MTC-4 HMC Polarizer Assembly View Three. ..................................................................................... 86

provide a blueprint of the development and construction of a cellular system for

although no detailed cellular analysis of the difference between hydrostatic and

SYSTEM DESIGN AND BUILD

4) Appendix E: MTC-4 Viewport and USB),

Figure 2 : MTC-4 Internal View (Fore).

Figure 3: MTC-4 Chamber Banks 1 and 2.

Gas System MTC-4

Gas Flow Chamber

He Bottles 6-10: Bank 2

He Bottles 1-5: Bank 1

Figure 5: MTC-4 Chamber Bank One.

He Bottles 6-10: Bank 2

Figure 7: MTC-4 Chamber Control Panel.

Figure 8: MTC-4 Port View.

Figure 9: MTC-4 Starboard View.

Figure 10: MTC-4 Aft View Rear Flange.

Figure 11: MTC-4 External Electrical Panel.

Fore Chamber Electrical Panel

Figure 12: MTC-4 Internal Fore Sled Electrical Panel.

Aft Chamber Electrical Panel

Figure 13: MTC-4 Internal Aft Sled Electrical Panel.

Table One: MTC-4 Electrical Penetrations and Drawings

Microscope Stage Info

MICROSCOPE DESIGN AND CONSTRUCTION

For a general review of alignment procedures and theory related to brightfield

Figure 15 (Item C) shows the placement of the objective in relationship to the

The objective manipulator system is utilized to place the microscope objectives in

The completed manipulator mounting plate, manipulator system, and wiring

Figure 14: MTC-4 Port Microscope Components.

Figure 15: MTC-4 Starboard Main Components.

Figure 16: MTC-4 Upper Optical Pathways.

Set Screw Head

Set Screw to Define

Optical Pathway Horizontal

Figure 19: MTC-4 Tube Len Assemblies.

Aft (CCD Side)

Figure 20: MTC-4 Upper Horizontal Optical Assemblies.

Aft (CCD Side)

Figure 21: MTC-4 Upper Horizontal Optical Assemblies View Two.

Figure 22: MTC-4 Upper Horizontal Optical Assemblies View Three.

Figure 23: MTC-4 Upper Horizontal Optical Assemblies View Four.

Figure 29: MTC-4 Upper Vertical Optical Assembly Fore View.

CCD Side (Aft)

Figure 30: MTC-4 Upper Vertical and Horizontal Assembly.

Figure 31: MTC-4 Objective Manipulator Assemblies.

Figure 32: MTC-4 Objective Manipulator View One.

Figure 1: MTC-4 Course Objective Manipulator View Two.

Figure 2: MTC-4 NAI MM-4M-F-50-HT Z- Axis Manipulator Unit.

Figure 4: MTC-4 NAI MM-4M-F-50-HT Z- Axis Unit Manipulator Mounting.

Figure 5: MTC-4 Objective Mount (Upper View).

Objective Lock Screws/ Balance Adjustments

Figure 6: MTC-4 Lens Mount (Lower View).