analySIS®

MegaViewII

User’s
Guide
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A note on the following documentation
The following documentation describes the software for operating the
MegaView II TEM camera by the Soft Imaging System GmbH. That
the hardware has been correctly installed is assumed, as well as that
the necessary fundamental settings have been undertaken such that
the camera is capable of interfacing with your microscope. Infor-
mation on installation and on technical data are located in the Instal-
lation Guide. The MegaView II software is fully integrated into the
analySIS® image-analytical software. Please refer to the analySIS ®
User’s Guide with regard to any questions you may have concerning
analySIS® or have a look at Getting Started - our introductory manual.
 MegaView II

What is MegaView II? 5

MegaView II Hardware Components ...................................................5

Software Camera Control 6

MegaView II Input Configuration 10

The Magnification tab............................................................12

The Input tab.........................................................................13
The Display tab .....................................................................15
The Format tab .....................................................................17

Shading-Correction Set-up 19

Image Acquisition 25

Real-time functions .......................................................26

Frequently-used-channels button bar ...........................29
Common image-acquisition steps .................................31
MVII1099E
 MegaView II

What is MegaView II?

Definition MegaView II is a fast, high-resolution CCD-Camera of 12-bit depth
for biomedical and materials science applications. It can be flange-
mounted laterally on the wide-angle port of all standard Transmission
High dynamic Electron Microscopes (TEM). Its high dynamic range provides you
range not only with diffraction pattern acquisition capability but also imaging
of extremely radiosensitive specimens.
Rapid frame rate The rapid frame rate of more than 20 images per second permits you
to use the live image to effortlessly search for suitable sites within the
Real-time specimen and for TEM adjustment. Integrated real-time functions
functions such as the Real-time FFT (Fast Fourier Transformation) aid you in
interactive focussing and adjustment of the TEM.
analySIS® Software operation of the MegaView II is completely integrated with
integration the analySIS ® image-analytical software. This gives you direct
access to all functions analySIS ® offers for digital acquisition,
processing, analyzing, documentation and archiving of microscopic
Simple images. Easy-to-access buttons (on the PC screen) as well as a
operation convenient manual switchbox are available for the pneumatic
positioning of the camera in the ray path.

Hardware Components
Hardware necessary for the operation of the camera consists of the
following components:
• The wide-angle adapter with the built-in MegaView II camera and
reflecting prism is flange-mounted on the wide-angle port of the
TEM.
• The MegaView II Controller Box supplies the wide-angle adapter
with compressed air in order to position the reflecting prism, and the
camera (rigid connection to the prism) in the ray path, and to retract
it.
• The analySIS® PC reads in the images acquired by the camera via
the Digital Input Board (which is linked to the GrabBit Frame-
grabber), and via a serial PC interface, and processes them fur-
ther. The PC can also drive the MegaView II Controller via the TTL
Board: simply click on either "I" and "O" (two software buttons) to
move in or retract the camera.
• The camera can also be positioned using the manual switchbox:
the green button moves the camera into the ray path; the red button
retracts it.
• The individual components are connected to one another via
electrical cables and compressed-air hoses. The hose leading to
the TEM compressed-air line has been equipped with a quick
coupling. An additional electrical cable will be necessary for PC
remote-control of the TEM.
Warning Never connect or disconnect the cable between the computer and
MegaView II when the computer is on. The camera can be damaged
as a result.

5
 MegaView II

Block diagram The block diagram provides you with a schematic overview of how the
individual hardware components interact. It is not an installation aid.
A detailed block diagram for simplifying installation can be found in
the MegaView II Installation Guide.

Software Camera Control

Logical input The MegaView II software fits in with analySIS®’s own logical-input
channels concept. What this means: once camera and the corresponding
hardware have been hooked up, and the software has been installed
and configured on the computer, the camera is as easy to use as a
regular video camera. No other menus will appear in the menu bar of
the analySIS® Graphical User Interface (GUI). You first have to select
the Set Input... command (in the Image menu) - then you will observe
that further logical input channels are available, and that these can be
set up as best suits your needs. One or two channels are pre-
configured generally. For step-by-step instructions on how to set and
configure a new input channel, see the end of this chapter.

6
 MegaView II

Using tabs for Carry out configuration by entering appropriate values in several tabs.
configuration The four tabs relevant to MegaView II are explained in detail in the
chapter on "MegaView II Input Configuration". Proposed entries for See also
three sample channels are shown to the left of the illustrations of the MegaView II Input
four tabs. These channels are called: "MegaView II Search", "Mega- Configuration 10
View II Focus" and "MegaView II Snapshot". We recommend you set
up three input channels for searching, adjusting and acquisition -
according to our proposed values.
Logical input channels contain, for example, information on what type
of image signal is present, how high magnification is, and what is to
be done with the image signal before it reaches the analySIS ® image
buffer. Each channel must be calibrated separately. A channel for
image acquisition via MegaView II would typically be called "Mega-
View II ..., GrabBit Mega View II". For a more comprehensive See also
description of the logical input channel concept, we refer you to the Set Input, Image menu,
description of the Set Input... command (of the Image menu) in the User’s Guide
User’s Guide.
MV button bar After installing MegaView II you’ll see the MV button bar for the wide- See also
angle adapter in the analySIS ® Graphical User Interface (GUI). Click Image Acquisition 25
on the "I" button to move the camera into the ray path; and on the "O" Frequently-used-chan-
button to retract it from the ray path. More details on this in the "Image nels button bar 29
Acquisition" chapter.
How to... If your current analySIS ® configuration does not provide you with the
MV button bar, you’ll have to carry out the following steps in order to
activate the corresponding "MVCtrl" module:
1) Open the C-Modul menu.
2) Select the Module Manager... command.
→ The Module Manager dialog box will be opened.
3) Check and see whether the MVCtrl entry is located in either the
Loaded or in the Other list.
4) If it is located in the Other list, select it (using the mouse) and click on
the Load button.
→ The MVCtrl entry will now appear in the Loaded list.
5) If this entry is already in the Loaded list, then you need do nothing.
6) Click on the Close button.
If the MV button bar is still not visible, you can make it visible by doing
the following:
7) Open the Special menu.
8) Select the Edit Button Bars... command.
→ The Edit Button Bars dialog box will be opened.
9) Select the check box next to the MV entry in the Button bars list.
10) Close the dialog box by clicking on OK.
Manual Instead of using the MV button bar, you can use the red and green
switchbox buttons of the manual switchbox for positioning the camera. More on
this is also found in the "Image Acquisition" chapter.
Turning on the Make it a habit to turn on the MegaView II Controller Box before you
hardware start up analySIS ®. If the MegaView II Controller Box has not yet
been turned on, you’ll get an error message that it could not be recog-
nized.

7
 MegaView II

If the cable link between the camera and the PC is defective, you’ll get
this error message:

Click on the Retry button after you’ve turned on the equipment, and
checked the cable connection to re-try starting things up.
Click on the Disable button if you’d like to work with analySIS ®
without acquiring any images via MegaView II, or if you’d like to
operate the camera not via analySIS®.
Click on the Change... button to indicate to the system that another
serial interface is going to be used for the cable connection from
camera to PC.
How to... How to select MegaView II for use as acquisition device and to
configure a corresponding logical input channel:
1) Open the Image menu.
2) Select the Set Input... command.
→ The Set Input dialog box will be opened:

→ Depending on how your system is configured, you may find other

entries in the list in this dialog box as are depicted in the above
diagram.
→ If the input channel desired is already in this list, then you will not
need to click on the New... button. You can thus skip the next two
steps in this case.
3) Click on the New... button to set up a new input channel.
→ The Select Device dialog box will be opened:

8
 MegaView II

4) Select the "GrabBit MegaView II" entry from the Available devices
list and click on OK.
→ You’ll be returned to the Set Input dialog box.
→ If this is the first time you’ve selected the "GrabBit MegaView II"
entry, the MegaView II Remote Interface dialog box will be
opened first:

Select the serial interface of your PC (from the COM port list)
which the camera is hooked up to. After clicking on OK you’ll be
returned to the Set Input dialog box.
5) Select the input channel desired for MegaView II from the list of input
channels.
→ Select the input channel desired by leftclicking on it.
6) If you wish to duplicate the input channel selected, including all its
properties (also including its calibration) so that you can edit the
duplicate without altering the original, then click on the Duplicate
button.
7) Click on the Configure... button.
→ The Configure Input dialog box with its 8 tabs will be opened.
→ To be able to open this dialog directly the next time you wish to
access this dialog box, you can instead simply select the Config-
ure Input... command (in the Image menu).
8) You can now make the settings of the various tabs according to your
application needs.
→ Four of these tabs look exactly the same as for other input chan-
nels. For a more detailed explanation of these tabs we refer you
to the User’s Guide: have a look at the description of the Config- See also
ure Input... command (in the Image menu). Configure Input, Image
→ The Magnification, Input, Display and Format tabs contain the menu, User’s Guide;
settings specifically for MegaView II. These are all described ex- MegaView II Input
Configuration 10

9
 MegaView II

haustively subsequent to this introduction in the chapter on

"MegaView II Input Configuration".
9) You then click on OK.
→ You’ll be returned to the Set Input dialog box.
10) Click on the Close button.
→ The input channel selected will be shown in the channel field of the
status bar - slightly to the lower-right. This is now the active input
channel - ie, the next acquisition will be made according to the
settings you’ve made for this channel.

MegaView II Input Configuration

Tabs The Configure Input dialog box contains 8 tabs for setting the
properties of input channels. What follows are explanations of each
of these tabs: Magnification, Input, Display and Format. These
tabs contain the settings specifically for MegaView II. For descrip-
tions of the other tabs we refer you to the User’s Guide: have a look
at the description of the Configure Input... command (in the Image
menu).
Viewing channel When you acquire an image via a MegaView II input channel its
data properties will automatically be saved along with the image. This data
can be viewed at a later point in time. To do so, load the image into
an analySIS® image buffer and doubleclick on that image buffer in the
image manager. The Image Information dialog box will be opened.
Channel properties are recorded in the Channel Data tab. There are
other ways to open this dialog box. These are described in the User’s
Guide.
3 sample Up to 32 different input channels can be set up. We recommend you
channels set up at least 3 for running MegaView II:
• Here’s one channel for locating relevant parts of a specimen quickly
and easily. In this case, high resolution is of lesser importance than
a rapid frame rate. A name for this channel would be "MegaView
II Search".
• A channel with which you can interactively adjust and focus your
TEM. The real-time Fast Fourier Transformation (RTFFT) will aid
you in doing so. A name for this channel would be "MegaView II
Focus".
• A channel for actually making image acquisitions. This channel
should have the highest resolution available. You could call it
"MegaView II Snapshot".
Tab entries necessary for setting up these three sample channels are
located in the edge column to the left of the diagram of the three
respective tabs.
Another 2 We recommend setting up another input channel for the acquisition of
channels: MIA MIA images - you can call it "MegaView II MIA". MIA is short for
and Fast Multiple Image Alignment. What it does is take several images of
conjunct areas of a specimen and then make them into one complete
high-resolution image of the whole specimen. To do this, select the
Multiple Image Alignment... command (in the Image menu).

10
 MegaView II

The "MegaView II MIA" channel should be given the same settings as

the "MegaView II Snapshot" channel. The only difference between
the two is that (for the former) you select the pre-defined "640 x 512
Pixel, Binning 1 x 1" standard format (in the Format tab, in the Image
format list). This will result in the Clipping border automatically
being set to values of 320 or 256. This means that borders will be
clipped right when you acquire images and it is in these borders that
the most obvious distortions occur - due to aberrations in the TEM
projective-lens system. If you do not clip these areas, these distort-
ions render a seamless MIA placement of the individual component
images adjacent to one another much more difficult.
If your top priority is high-performance, and you wish to operate
MegaView II at the highest frame-rate available, then set up a fifth
channel - call it "MegaView II Fast".
This "MegaView II Fast" channel should be given the same settings
as the "MegaView II Search" channel. There are however two
differences: enter a value of only 30000 µs into the Exposure field in
the Input tab and clear both check boxes of the Shading correction
group.
Overview of To begin with, the following table provides you with an brief overview
channel of the most important properties of the five proposed sample chan-
properties nels:
Name of
Exposure time (in ms)

Shading correction

Pixel clock (in MHz)

Real-time FFT

Image format (in pixels)

Binning

Signal/noise ratio

(eg, Dual Pentium 500 MHz)

Frame rate (images/s)
channel

MegaView II 50 yes 20 no 640 x 2x2 low ca. 16

Search 512
MegaView II 100 yes 20 yes 640 x 2x2 low ca. 10
Focus 512
MegaView II 500 yes 10 no 1280 x 1x1 high ca. 2
Snapshot 1024
MegaView II 500 yes 10 no 640 x 1x1 high ca. 2
MIA 512
MegaView II 30 no 20 no 640 x 2x2 low ca. 22
Fast 512

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 MegaView II

The Magnification tab

Entries for the three sample
channels:
Enter the magnifcation level
desired in this tab.
As far as the sample
channels are concerned, we
recommend you use the
same magnification level for
all three sample channels.

Magnification Enter the current microscope magnification level into the Magnifi-
cation field, or select a magnification level from the list. This value
remains unchanged for all images acquired with this channel.
• Be sure that you adjust magnification in the Magnification tab if the
microscope’s magnification changes. If you have a version of
analySIS ® that runs macros you can program your system such
that this tab will always be opened when you select either the
Acquire or the Snapshot commands (in the Image menu).
• Before beginning acquisition make sure that the input channel has
been calibrated correctly. The corresponding XY Calibration and
Z Calibration tabs are described in the User’s Guide. Getting Star-
ted contains step-by-step instructions on these tabs. If the channel
is not calibrated beforehand, the image itself will also be uncali-
brated and thus any magnification selection you might make is
meaningless.
Device If your analySIS® version supports the remote function, the Device
list will contain the names of several types of microscopes which
analySIS® can operate per remote.
Remote Select the On check box to activate or deactivate remote control.
Once the remote has been turned on, you can use the Read button to
read in the current magnification automatically.
Set Unit... Clicking on the Set Unit... button enables you to determine the unit
per pixel.
Automatic When you select the Automatic check box, the values of the resulting
calibration will be automatically shown in the appropriate scaling unit.
Resulting The Resulting calibration group shows the values resulting from
calibration magnification - ie, pixel width, pixel height and the X/Y ratio.

12
 MegaView II

The Input tab

Entries for the three sample
channels:
MegaView II Search:
Exposure 50000 µs,
Pixel clock 20 MHz,
"Offset" and "Gain" check
boxes selected.
MegaView II Focus:
Exposure 100000 µs,
Pixel clock 20 MHz,
"Offset" and "Gain" check
boxes selected.
MegaView II Snapshot:
Exposure 500000 µs,
Pixel clock 10 MHz,
"Offset" and "Gain" check
boxes selected.

Exposure Enter the number of micro-seconds the CCD chip of the camera is to
be exposed into the Exposure field. You can also use the arrow keys
located to the right of this field to set this value. You can also set the
value in this field interactively while viewing the live image with the aid
of the Camera Control... command. This is described in the "Image See also
Acquisition" chapter later on under the "Camera Control" margin Camera Control 26
heading.
The lowest Exposure time available is 100 µs and the greatest 160 s.
This tremendous dynamic range going from extremely brief to extra-
ordinarily lengthy exposure times enables you to acquire images of
both extremely radiosensitive specimens as well as the acquisition of
images that have both very bright and very dark regions.
Warning When acquiring diffraction patterns always use the beam blanker for
the primary beam. In a worst-case scenario the primary beam can
be so great in intensity that the luminescent screen of the TEM, or the
luminescent screen of the MegaView II reflecting prism is damaged
at the impact point of the beam.
Options Select the Bad pixel correction check box in the Options group, to
Bad pixel have defective pixels of the camera or defective columns replaced
correction with the values of neighboring gray values right at image acquisition.
Prerequisite for doing this is that you indicate the precise position of See also
the defective pixels and columns. To do this, use the Define Bad Define Bad Pixel List,
Pixel List... command (in the Image command). Image menu, User’s
Guide
Calculate focus To have a focus value calculated during image acquisition you can
value normally simply select the Calculate focus value check box. To
date, this focus value is only accessible for use via macros especially
designed for this purpose.

13
 MegaView II

For MegaView II the Calculate focus value check box is not

available.
8 bit mode Select the 8 bit mode check box if your images are to be saved in 8-
bit depth instead of 16 bit.
At image acquisition MegaView II uses 12 bits for the intensity value
of each pixel. The image is then usually saved as a 16-bit image by
analySIS®. In the 8-bit mode the lower 4 bits (of the 12 bits) are
clipped before the image is saved. If you’ve selected the Activate
check box in the Automatic gain display group (in the Display tab),
an optimization of the gray-value display (of the generated 8-bit
images onscreen) will take place following the clipping of these lower
4 bits.
Application Use of the 8-bit mode is recommended in cases where you wish - at
a later point - to make use of your images in application programs
which can only process 8-bit images. This is also a way to save
memory space. Please keep in mind however that a reduction in bit
depth means that image information is lost. The image acquisition
itself will not be any faster by selecting the 8 bit mode check box.
MegaView II images can be converted to 8 bits (from 16 bits) at a later
time using the analySIS ® Change Bit Depth, To 8-Bit command (in
the Oper menu).
Pixel clock Select the frequency with which the pixels of the camera’s CCD chip
are to be read out from the Pixel clock list.
Camera The camera currently in use and the type of camera signal are shown
in the Camera field.
Shading Use the Shading correction group to have rigid image structures
correction corrected during image acquisition - these structures may result from,
eg, an irregular illumination of the image field.
Offset Select the Offset check box to have the offset image ("dark image")
subtracted during image acquisition.
Gain Select the Gain check box to have the image divided by the gain
image during image acquisition.
Info Click on the Info... button to view information on the device in use and
the current temperature of the CCD chip and the camera housing - in
the MegaView II Information dialog box:

14
 MegaView II

The Display tab

Entries for the three sample
channels:
MegaView II Search:
Over exposure:
"Display warning" selected,
Overflow 0.50%;
Automatic gain display:
"Activate" selected, Left and
Right overflow 0.50%;
"Online histogram" selected
Image scaling:
"Adjust to viewport".
MegaView II Focus:
Over exposure:
"Display warning" selected,
Overflow 0.50%;
Automatic gain display:
"Activate" selected, Left and
Right overflow 0.50%;
"Real time FFT" selected;
Image scaling:
"Adjust to viewport".
MegaView II Snapshot:
same entries as for
"MegaView II Search".

Over exposure This is where you determine when, or if an overexposure warning is

to appear - in the Over exposure group. analySIS® is continually
checking image intensity in the live mode and will warn you as soon
as the live image becomes too bright.
Display warning Select the Display warning check box to have an overexposure
warning appear when relevant:

Overflow An image is overexposed when a certain percentage of all pixels have

reached the maximum possible gray value. You enter this percentage
into the Overflow field.
Automatic gain Use the functions of the Automatic gain display group to have the
display image optimally displayed onscreen independent of the actual
illumination conditions. The system will assess the current gray-value
histogram in real time in order to find the optimal onscreen display of
the live image.
The automatic gain display affects monitor display only - it does not
alter actual image information.
Activate Select the Activate check box to turn on the automatic gain display.

15
 MegaView II

Left overflow When the automatic gain display is active, image intensity as far as
Right overflow monitor display is concerned, will be spread between the gray values
Gmin and Gmax. You determine the values of these Gmin and Gmax
gray values in the Left and Right overflow fields: this is where you
enter the percentage of pixels with the lowest gray values which are
to be displayed black, and which percentage of the highest gray
values are to be displayed white.
This is how you can clip gray values that are to be ignored at both the
upper and lower ends of the gray-value histogram. Make use of the
overflow values so that individual pixels or minute image areas that
are too bright or too dark do not influence the automatic gain display.
Fixed scaling Instead of using the Automatic gain display group, you can use the
Fixed scaling group. The group whose Activate check box has been
selected will be the one available - the other will not be available.
The functions of the Fixed scaling group are for manually influencing
onscreen image display. You select definite limits - Gmin and Gmax -
for the gray-value range that you wish to have optimally spread for
onscreen display. Any gray values occurring outside of this defined
gray-value range will appear either black or white.
Application This is how you can, for example, improve the recognizibility of the
specimen structures of interest to you - by having the dark grid bars
of the specimen mount displayed entirely in black.
As is the case with the automatic gain display, this manual definition
of a gray-value range affects monitor display only - actual image
information is not altered.
Activate Select the Activate check box to switch on the manual definition of
the gray-value range to be displayed.
Left limit When this manual option is active, image intensity for monitor display
Right limit will be spread between the gray values Gmin and Gmax. Enter the
Gmin gray-value limit into the Left limit field - the G max gray-value limit
into the Right limit field.
Online Select the Online histogram check box to be able to keep an eye on
histogram the gray-value distribution during image acquisition. During image
acquisition, a window showing the current histogram will appear
automatically. This histogram will be updated continually.
Beneath the histogram, mi-
nimum, mean and maximum
gray values of the image will
be shown. Percentages are
in relation to the maximum
obtainable gray value.
When working with Mega-
View II (with its 12-bit
depth), the maximum ob-
tainable gray value is 4095. This gray value corresponds to 100%. A
mean of 84.4% would thus correspond to a gray value of 3457.

16
 MegaView II

Real time FFT Select the Real time FFT check box to have not only the live image
shown during a live acquisition, but the continually-updated fourier
image shown as well (the absolute value of the fourier transform).
This will simplify the adjustment and focussing of the TEM. This See also
option is described in greater detail in the "Real-time functions" sec- Real-time functions 26
tion of the chapter on "Image Acquisition".
Image scaling Select one of the four selections for displaying an image in a Viewport
in the Image scaling list.
Underscan Select the Underscan entry to have the whole image displayed. The
system will select the largest zoom level - 25%, 50% or 100% - where
the whole image can still be displayed within the Viewport. In certain
cases not all of the Viewport’s available area will be used.
Overscan Select the Overscan entry to ensure that all the Viewport’s available
area will be used. The system will select the smallest zoom level -
25%, 50% or 100% - where the image fills the whole Viewport. In
certain cases parts of the image will not be visible.
Adjust to Select the Adjust to viewport entry to have the image size adjusted
viewport to optimally fit the Viewport. The image will be adjusted to fit the
current size of the Viewport.
Full size (100%) Select the Full size (100%) entry to have the image displayed without
any zooming at all. If the Viewport is smaller than the image, as much
of the image will be shown as fits - starting from the upper-left corner
of the image.

The Format tab

Entries for the three sample
channels:
MegaView II Search:
Image format:
640 x 512 Pixel, Binning 2 x 2;
Binning:
Horizontal and Vertical 2;
Clipping border:
Left, Right, Top and Bottom 0
respectively (unless clipping
is necessary).
MegaView II Focus:
same entries as for
"MegaView II Search".
MegaView II Snapshot:
Image format:
1280 x 1024 Pixel, Binning 1 x 1;
Binning:
Horizontal and Vertical 1;
Clipping border:
Left, Right, Top and Bottom 0
respectively (unless clipping
is necessary).

Image format The Image format list contains a series of predefined standard image
formats:
• 1280 x 1024 pixels, Binning 1 x 1

17
 MegaView II

• 640 x 512 pixels, Binning 1 x 1

• 640 x 512 pixels, Binning 2 x 2
• 320 x 256 pixels, Binning 1 x 1
• 320 x 256 pixels, Binning 2 x 2
• 160 x 128 pixels, Binning 1 x 1
• 160 x 128 pixels, Binning 2 x 2
The very first time you call up this tab the maximum obtainable resolution
of MegaView II is a preset. Images will not be clipped according to this
setting. In addition, this list contains all user-defined image formats. You
can define your own image formats in the Clipping border (pixel) group.
User-defined image formats are denoted by the appearance of the word
"free" along with the entry - eg, "free, 512 x 512 Pixel, Binning 1 x 1".
You may select either a predefined or a user-defined image format from
the list. The format you select will be the one set the next time you call
up this tab.
A graphic display of clipped borders can be seen in the Active area
field. The values are shown in the fields of the Clipping border
(pixel) group.
Binning Binning is where the camera integrates the contents of neighboring
Definition pixels. This will result in a more speedy read-out and greater
sensitivity - image resolution will however decrease.
Application Use Binning for when you, eg, are working with an electron
microscope at low intensities - binning will reduce noise. Binning can
also be useful if a rapid frame rate is of greater priority than a high
image resolution. A rapid frame rate is important for the proposed
input channel called "MegaView II Search", for example.
Horizontal Enter the number of neighboring pixels to be integrated into the Ho-
Vertical rizontal and Vertical fields. Two entries are permissible for Mega-
View II: enter a value of 1 to not make use of the binning option. Enter
a value of 2 to have two neighboring pixels integrated - vertically and
horizontally.
You cannot alter the values in the Horizontal and Vertical fields
independently of one another when using MegaView II. If you alter
one value, the other will automatically be adjusted accordingly.
Clipping border Define the number of lines and columns, and the edges where they
(pixel) are to be clipped in the Clipping border (pixel) group. The corres-
ponding format will be indicated in the Image format field and
displayed (graphically) in the Active area field. The result will be
included in the Image format list.
Application You can define your own image formats by clipping the borders of a
predefined image format. Clipping is advisable if distortion or other
kinds of disturbances due to the microscope and/or camera occur at
image edges. These disturbances can thus be eliminated directly at
image acquisition.
Top/Bottom/ Enter the number of lines and columns to be clipped in the Top,
Left/Right Bottom, Left and Right fields.

18
 MegaView II

Interactive Click on the Interactive button to define borders directly within the
image. This button is only available if the active image buffer contains
an image at the same resolution of the logical input channel. A red
frame will appear within the image which can be defined using the Set
Frame command (in the Image menu). This frame can be enlarged
or reduced by keeping the left mouse button depressed and adjusting
the frame size by moving the mouse accordingly. Position the frame
by moving the mouse (the left button no longer depressed). Confirm
the frame via rightclick. The image area not within the frame will be
the image borders and thus will be clipped.
No Clipping Click on the No Clipping button to reset the number of lines and
columns back to zero ("0") - meaning that no borders will be clipped.
The Image format field will once again show the maximum image
format corresponding to the resolution of the logical input channel.
Active area For estimation purposes, the Active area field will provide you with a
schematic preview of how much area is to be clipped at the image
borders. The blue frame represents the maximum image format
available for this logical input channel. The cross-hatched area
represents the image area. The white areas represent the borders to
be clipped.

Shading-Correction Set-up
Definition The Online shading correction is how analySIS® corrects fixed
image structures (resulting from the imaging system) during
acquisition.
What’s shading • Irregular illumination of the CCD chip can, eg, lead to a dip in
correction for? intensity at image borders. This means that image edges are
darker than the more central image areas, meaning that specimen
structures and image gray values are no longer in clear correlation
with one another. Setting of thresholds is thus impossible and a
successful conducting of many automatic measurement operations
is also not feasible.
• The amplification of individual camera-array pixels can fluctuate
slightly in CCD cameras. Individual pixels - so-called "warm pixels"
- are thus somewhat brighter or darker than neighboring pixels
which were illuminated with the exact same intensity. This pheno-
menon can be particularly disturbing when working with long ex-
posure times.
• Luminescent screen thickness in the reflecting prism can vary
slightly, resulting in fluctuations in light yield.
What does The following image operations are executed during a real-time
shading shading correction:
correction 1) The offset image is subtracted from the original image.
involve? 2) The resulting image will then be divided by the gain image - (the
gain image has already been offset corrected at this point - ie, the
appropriate offset image has already been subtracted from the
gain image).

19
 MegaView II

3) The resulting corrected image will then be multiplied by the mean

gray value of the offset-corrected gain image. Mean image bright-
ness is thus not affected by shading correction.
Shading Prerequisite for shading correction is your having acquired a valid
correction gain image and a valid offset image - using the Acquire Reference
prerequisites Images... command. When you begin image acquisition and
analySIS® cannot find any valid reference images, you’ll be instructed
to acquire reference images.
Definition A camera offset image is acquired without any illumination. An offset
correction is necessary when using a CCD camera in order to
compensate for a non-homogenous dark-current signal. A shading
correction involves subtracting the offset image from the original
image.
To be precise, two offset images with varying exposure times are
acquired and saved. Common exposure times are 50 ms and 1000
ms. A third offset image - when necessary - is then computed via
inter- or extrapolation of the first two offset images for the current
exposure-time setting. If you alter exposure time, and then acquire an
image using real-time shading correction, the requisite offset image
will be automatically recomputed - without your noticing.
Definition The camera gain image is acquired at homogeneous illumination -
without any specimen. The original image is divided by the gain
image during shading correction.
Both reference image will automatically be saved to the computer
hard disk.
Warning If you have to work with more than one TEM acceleration voltage you
will have to acquire reference images for each of these. If analySIS®
can directly read out the voltage set on your TEM via remote it
(analySIS®) will automatically select the correct reference images as
needed. If you do not have remote control you will have to acquire a
new gain image for each input channel used anytime you alter the
voltage level.
You also have to have reference images for each image format
selected for a MegaView II input channel. If you alter the image
format of an input channel you will subsequently have to acquire new
reference images for that channel.
How to... How to set up shading correction for a MegaView II input channel:

Offset image How to acquire an offset image for MegaView II:

1) Switch on your TEM and the MegaView II Controller Box.
2) Make sure that no light can reach the CCD chip of the camera. To do
this, cover up the window of the TEM projection chamber and turn off
the electron beam. Move the camera into the TEM column.
3) Select the logical input you’re acquiring reference images for. Clear
both check boxes of the of the Shading correction group in the Input
tab to have the automatic shading correction turned off during
acquisition of reference images.
4) Select the Acquire Reference Images... command (in the Image
menu) to acquire the offset image for correction of camera dark

20
 MegaView II

current.
→ The Acquire Reference Images dialog box will be opened:

5) Enter a value of 4 into the Averaging cycles field - in the Offset

image group.
→ An averaging cycle of 4 means that 4 images will be read in
successively and the arithmetical mean will be saved as the
reference image. (To be exact, there are in fact eight images being See also
acquired - four images twice - at two different exposure times.) Definition offset image 20
→ The control lamp will indicate whether there is an valid reference
image existent (green) - or there is not (red).
→ The word "missing" will appear beside the control lamp if there is
no valid reference image available, or if there is one, that image’s
creation date.
6) Click on the Acquire button.
→ The warning lamp will now be green - the date will be updated.
→ The offset image acquired ought to look something like the one
shown below:
A typical MegaView
II offset image (this
image (right) has
been contrast en-
hanced).
Any structures
resulting from a
non-homogeneous
dark-current signal
are barely visible,
but these can -
especially where
low-intensity
acquisitions are
concerned - lead to
artifacts, which
impede quantitative
image analysis.
7) Click on the Close button.

21
 MegaView II

Gain image How to acquire a gain image for MegaView II:

8) Make sure that illumination of the luminescent screen (on the topside
of the MegaView II reflecting prism) is even. Move the camera into
the TEM column, turn on the electron beam and remove the speci-
men from the ray path. You can also make use of a hole through the
specimen - instead of removing the specimen - so that the beam can
pass through it unhindered.
9) Select the Configure Input... command (in the Image menu).
10) Select the Online histogram check box in the Display tab.
11) Confirm the setting of the current input via OK.
12) Select the Camera Control... command (in the Image command).
→ The Camera Control dialog box will be opened.
13) Select the Acquire command (in the Image menu) to switch over to
the live mode.
→ A live image will be shown in the active Viewport.
→ The Histogram window will be opened. The diagram shows the
current gray-value distribution of the live image.
14) Make a note of the value shown in the Exposure time field - in the
Camera Control dialog box. Then enter a value of at least 500 ms.
15) You can use the histogram to check whether the gray values are
situated centrally in the diagram. As necessary you can, eg, alter
illumination intensity of the electron microscope, or the Exposure
time of the camera to shift gray values roughly into the middle of the
graph. The mean value shown ought to be between 45 % and 55 %.
To ensure good results, make sure that shading correction has been
switched off in step 3.
16) Select the Acquire Reference Images... command (in the Image
menu) to now acquire the gain image for correction of camera
amplification.
→ The Acquire Reference Images dialog box will be opened.
17) Enter a value of 4 into the Averaging cycles field of the Gain image
group.
18) Click on the Acquire button.
→ The gain image will be saved offset corrected. This is why you can
only acquire a gain image if a valid offset image is available.
→ The control lamp will now be green, and the date will be updated.
→ The gain image acquired ought to look something like the image
shown below:

22
 MegaView II

A typical MegaView
II gain image.
The non-homo-
geneous illumina-
tion of the image
field is readily
visible. The image
appears less
homogeneous than
it in fact is - this is
because image
contrast has been
automatically
enhanced for
monitor display.

19) Click on the Close button.

Switching shading Here’s how you switch on real-time shading correction for the current
correction on input channel:
20) Select the Configure Input... command (in the Image menu).
21) Click on the Input tab.
22) Select the Offset check box of the Shading correction group to
have the camera’s offset image automatically subtracted during
image acquisition.
23) Select the Gain check box to have each image acquired auto-
matically divided by the camera’s gain image.
24) Click on OK.
Checking Be sure to check the reference images acquired afterward:
reference images
25) Set TEM illumination at medium brightness.
→ Generally, after having acquired reference images correctly, you
will not need to adjust current brightness. During the subsequent
image acquisition, the online histogram ought to display a mean
value between 40 % and 60 %.
26) Acquire an image - without specimen - using the Acquire or
Snapshot command (in the Image menu).
→ The corrected acquisition cannot have any visible structures due
to the imaging system. The only thing you should be able to see
is homogeneous noise:

23
 MegaView II

A microscope
image acquired
without specimen
shows the
homogeneous
noise visible when
the shading
correction was
conducted
successfully.
This noise is caused
by the electrons’
quantum noise.
The image (right)
has been contrast
enhanced.

→ If shading correction was not conducted successfully, please

check and see whether the reference images acquired look
essentially the same as the sample images shown here. To do
this, use both Show buttons (in the Acquire Reference Images
dialog box - which is opened using the Acquire Reference
Images... command (in the Image menu)). Then - if necessary -
repeat acquiring reference images for shading correction. Be sure
to follow all instructions as described above.
27) Reset the Exposure time (in the Camera Control dialog box) to the
value you noted in step 14. This is normally the standard value of the
current input channel - that you can look up, eg, in the table providing
you with an overview of channel properties.

24
 MegaView II

Image Acquisition
Before acquiring Turn on the TEM and the MegaView II Controller Box - supplied with
images compressed air, and start analySIS ® up on your computer. Your See also
system is now ready for image acquisition. Turning on the hardware 7
Moving in the The MegaView II camera is moved into the TEM column via com-
camera pressed air. As this happens, the reflecting prism - connected rigidly
to the camera - is positioned into the electron ray path such that the
electrons fall onto the luminescent screen of the reflecting prism.
Generated photons are directed into the camera via an optics system.
MV button bar or There are two ways to move in and retract the camera:
manual • The MegaView II software - integrated into the analySIS® program
switchbox - provides you with a button bar (two buttons):
Click on the "I" button to move the camera into the ray
path. Click on on the "O" button to retract the camera
from the ray path.
If this button bar is located in the header area of the
analySIS ® Graphical User Interface (GUI), directly next to the other
button bars, its own MV header will not be visible.
• The manual switchbox can be used for camera moving/retraction in
a darkened laboratory - for example, without your having to look at
the computer screen.
The manual switchbox has
two easily-found-in-the-dark
buttons - PLUS - they can be
seen in the dark, being
lighted. Only one is lighted
at a time - the one that can
be used next. When the
green button is lit, this
means that the beam is
passing through unhindered,
meaning that the camera is
in a position retracted from
the ray path. When you
press the green button, the camera will be moved into the TEM
column. If the red button is lit, the beam is hindered - ie, the camera
is in the moved-in position and ready for acquisition. When you
press the red button the camera will be retracted from the ray path.
Set Input To set the logical input channel desired select the Set Input...
command (in the Image menu). Instructions on how to do this are See also
found in the chapter entitled "Software Camera Control". How to..., Software
Camera Control 8
Snapshot As when using other image-acquisition devices, for acquiring a single See also
Acquire image you select the Snapshot command (in the Image menu). To Acquire/Snapshot, Image
have a continually-updated live image, select the Acquire command menu, User’s Guide
(in the Image menu). The camera icon (to the lower right in the status
bar channel field) will blink during live acquisition. The live-acquisition
mode will be terminated when you select the Snapshot command.

25
 MegaView II

Camera Control Calling up the Camera Control... command (in the Image menu) will
open a dialog box in which you can interactively influence the quality
of your images by varying Exposure time - while at the same time
keeping an eye on the live acquisition:

- + Exposure time During MegaView II acquisition this dialog box will remain open. To
adjust the value in the Exposure time field click on either the minus
sign ("-") or the plus sign ("+") - or you can adjust the value right in the
Exposure time field. This is how you interactively set the length of
time the CCD chip is to be exposed. Continue adjustment until image
quality meets your satisfaction. When using these buttons ("-" or "+")
the value will be adjusted in pseudo-logarithmical intervals: 1, 2, 5, 10,
20, 50, 100, 200, ...
The following applies to image quality in general: short exposure
times result in a more rapid frame rate and a low signal/noise ratio,
whereas long exposure times result in a slower frame rate and a high
(ie, desirable) signal/noise ratio. As there is a linear correspondence
between light incidence and electron yield with the CCD chip, you
need not set the mean value to half maximum.
When you alter exposure time in the Camera Control dialog box, the
value in the Exposure field (in the Input tab) will be updated
correspondingly. This means: the channel property exposure time will
change for all images acquired afterwards using this channel.
Ref. The color of the Ref. lamp will indicate reference image status:
• if this lamp is grey, online shading correction has been deactivated;
reference images are thus not required.
• if the lamp is green, online shading has been activated and the
requisite reference images are available for the exposure time
currently selected.
• if the lamp is red, online shading correction has been activated but
the offset image for the exposure time currently selected has not yet
been computed. This computation will be executed automatically
as soon as you acquire an image; the color of the lamp will then go
to green. When you then alter exposure time the lamp will turn red
again until you acquire the next image.

Real-time functions
Real-time Several analySIS ® real-time functions ease image acquisition with
functions MegaView II:
1) Real-time shading correction corrects image interference resul-
ting from the imaging system right at acquisition.

26
 MegaView II

To activate shading correction go to the Input tab and select the

check boxes in the Shading correction group.
2) Use the Online histogram to keep an eye on gray-value distribu- See also
tion in the live image during acquisition. This makes setting opti- Online histogram 16
mal imaging paramaters (on both camera and TEM) as well as
screen contrast enhancement alot easier. The histogram is up-
dated continually. Turn it off with the [Esc] key.
Select the Online histogram check box in the Display tab to
activate the online histogram.
3) The Over exposure warning appears if the live image is too See also
bright. Over exposure 15
Activate this warning by selecting the Display warning check box
in the Over exposure group in the Display tab.
4) Automatic gain display ensures that image structures are See also
optimally visible onscreen independent of the actual lighting Automatic gain display 15
conditions.
Activate automatic gain display by selecting the Activate check
box in the Automatic gain display group in the Display tab.
5) The Real-time Fast Fourier Transformation (RTFFT) is a See also
tremendous help when interactively adjusting the electron beam in Real time FFT 17
the TEM and when focussing the image. During live acquisition,
besides having the live image shown, you can have the
continually-updated fourier image (the absolute value of its fourier
transform):
The fourier image
of a 2-D catalase
crystal.
The light spots re-
present the per-
iodic structures in
the microscope
image. The closer
such a spot is
located to the center
of the fourier image
the lesser the local
frequency of the
corresponding
periodic structure -
ie, the greater the
periodic distances
within the micro-
scope image. (The
light ‘cross’ is a
result of margin
effects.)
Adjustment and • When working with thin, amorphous specimens, rings (so-called
focussing using ‘Thon rings’) result in the diffraction patterns of the TEM. The dark
RTFFT rings indicate where the phase-contrast transfer function of the
TEM is either minimal or zero. The fourier image computed will also
contain corresponding ring-shaped structures. If these are elliptical
in shape this indicates that an astigmatism exists. If this astigma-
tism is corrected in the TEM, the ellipses within the fourier image
will become circular in shape.

27
 MegaView II

Two fourier images

of an amorphous
specimen.
The elliptical shape
of the structures in
the left image in-
dicates an astig-
matism.
The astigmatism
has been corrected
in the right image:
the ellipses have
become circular.

• If the live image acquired becomes out of focus, the high local
frequencies - ie, the structures located further away from the center
- will disappear.
• If you’ve switched on a Wobbler on your TEM that rapidly flips the
electron beam back and forth, fringes may appear within the fourier
image. These fringes indicate that the electron beam is not pre-
cisely focussed on the specimen. When you focus the beam the
distance between the fringes will increase and the fringes will then
move out of the image.
Microscope image
and corresponding
fourier image of
latex beads.
Two partial images
overlap one another
in the microscope
image (left)
because the (not-
precisely-focussed)
electron beam is
being flipped back
and forth by the
Wobbler.
This is why a fringe
pattern occurs in the
fourier image (right).

28
 MegaView II

• Make use of the RTFFT Control dialog box to improve onscreen

display of the fourier image:

Use the scroll bars to adjust contrast and brightness. Determine the
size of the (centered) image segment used to compute the fourier
image in the Size group.

The RTFFT function with RTFFT Display and RTFFT Control

dialog boxes is activated by selecting the Real time FFT check box
in the Display tab.

Frequently-used-channels button bar

The Acquisit Use the Acquisit standard button bar - provided by analySIS ® - to
button bar speed up the (likely often necessary) switching back and forth
between various input channels.
How to... If your current analySIS ® configuration does not make the Acquisit
button bar available, you’ll need to carry out the following steps to
have it appear:
1) Open the Special menu.
2) Select the Edit Button Bars... command
→ The Edit Button Bars dialog box will be opened.
3) Select the check box next to the Acquisit entry in the Button bars
list.
4) Close the dialog box via OK.
Hooked up to The array of 2X3 buttons are hooked up to
the first three the first three input channels set up via
channels analySIS®. To check and see which chan-
nels these are, open the Set Input dialog
box using the command of the same name (in the Image menu).
Altering channel If you find more than three channels in this dialog box and you wish
order to alter their order, do the following: select the channel you’d like to
shift by clicking on it with the mouse. Then position the mouse cursor
between two of the other channels, right where you wish to have the
channel selected placed. The mouse cursor will change its shape at
exactly one of these ‘in-between’ positions - into a horizontal line with
arrowheads pointing in opposite directions. Following another
mouseclick the channel selected will be shifted into its new position.
After altering channel order as desired close the dialog box by clicking
on Close.
29
 MegaView II

This is a way, for example, how you ensure that the sample channels
you’ve set up will be in the top three positions in the Set Input dialog
box, and thus will be available to you via the Acquisit button bar.
What the 6 A single mouseclick on any of the three left buttons (having the video
buttons do camera symbol) will activate the corresponding channel and switch
you over to the live mode. This mouseclick thus serves the same
function as when you do so with the Set Input... command and then
calling up the Acquire command (in the Image menu).
A single mouseclick on one of the three right buttons (with the
"action!"-board symbol) activates the corresponding channel and
acquires a single image. This mouseclick thus serves the same
purpose as when you activate this channel using the Set Input...
command and then call up the Snapshot command (in the Image
menu).
Example Let’s say your second input channel - "MegaView II Focus" -
comprises interactive fourier-image computation and you then click
on the button with the video camera # 2 on it, analySIS® will switch
over to this channel and use it for a live image acquisition plus fourier
image display.
Your own button Moreover, analySIS® enables you to define your own button bars - via
bars which you have convenient (just-a-mouseclick-away) access to
frequently-used commands. You’re thus able to, for example, set up
a "MegaView II" button bar to make accessing the commands
necessary for working with this camera much easier. One of these
commands might be, eg, the Set Magnification... command (in the
Image menu). Have a look at the User’s Guide to find out how button
bars are defined.

30
 MegaView II

Common image-acquisition steps

How to... The following instructions indicate how you will generally proceed
when acquiring TEM images using the MegaView II camera. These
instructions have been kept succinct that they be easier to follow. For
more detailed information on any of the following steps have a look at
the other chapters of this documentation or in the User’s Guide.
1) Open the analySIS ® database where you wish to save images
acquired.
2) Retract the camera from the TEM column.
3) Set illumination intensity (at the TEM) to a value for which a photo
plate would have to be lighted about a half second.
4) Move the camera into the TEM column.
5) Set the "MegaView II Search" input channel.
6) Start up the live-acquisition mode. (To do this, select the Acquire
command (in the Image menu) or click on the appropriate button.)
7) Using the live image as a guide, determine the portion of the
specimen you wish to examine.
8) Terminate the live-acquisition mode. (To do so, select the Snapshot
command (in the Image menu) or click on the appropriate button.)
9) Set the "MegaView II Focus" input channel.
10) Restart the live-acquisition mode.
11) Adjust the TEM and focus the electron beam on the specimen. The
fourier image shown will help you to accomplish this.
12) Terminate the live-acquisition mode.
13) Set the "MegaView II Snapshot" input channel.
14) Restart the live-acquisition mode.
15) Select the Camera Control... command (in the Image menu).
16) Check the quality of the live image: is exposure time long enough to
yield a desirable signal/noise ratio? As needed, you can adjust the
exposure time (in the Camera Control dialog box) until image quality
is satisfactory.
17) Terminate the live-acquisition mode. The live image will be "frozen"
within its image buffer.
18) If your TEM does not have remote control you will now have to
indicate to analySIS® which magnification level you chose on your
TEM. To do this select the Set Magnification... command (in the
Image menu).
19) Now save the image in the database. The easiest way to do this is
per drag&drop - dragging it from its analySIS® image buffer onto the
database window.
20) Continue processing the image and conduct measurements and
evaluations as desired.
21) To acquire the next image, return to step 5.

31
Index

MegaView II Digitizer 8
C MV button bar 25
Camera Control 26
Channel properties, overview 11
Configure Input O
Display 15 Offset image 20
Format 17
Input 13
Magnification 12
R
Real-time functions 26
E Remote Interface 9
RTFFT Control 29
Error message, MegaView II Digitizer 8

S
G
Sample input channels 11
Gain image 20 Select Device 9
Set Input 8
Shading correction 19
H Gain image 20
Hardware Components 5 Offset image 20
How to...
Activate module 7
Common image acquisition steps 31
Configure Input 8
Display Acquisit button bar 29
Set up shading correction 20

I
Image acquisition 25, 31
Camera Control 26
RTFFT Control 29
Information 14

M
Manual switchbox 25
MegaView
Hardware Components 5
Image acquisition 25
Information 14
MV button bar 25
Remote Interface 9

32