Bio Optics World Sep 2014

Advances in lasers, optics, and
imaging for the life sciences

Sept/Oct 2014
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Optogenetics
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linked emotion
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SEPT
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OCT 2014
VOLUME 7, NO. 5
F E A T U R E S
OPTI CAL COHERENCE TOMOGRAPHY
16 High-resolution micro-OCT uncovers correlations
MEDI CAL AND AESTHETI C LASERS
21 Semiconductor diode laser advances
enable medical applications
CYTOMETRY/ CELL ANALYSI S
26 Optical cell volume measurement
3D OPTI CAL MOLECULAR I MAGI NG
29 Developing drugs in 3D
SPECTRAL I MAGI NG
31 Imaging spectrometers look at life in two ways
SUPER- RESOLUTI ON MI CROSCOPY
35 New twists on superlenses improve
subwavelength microscopy
www.BioOpticsWorld.com
Advances in lasers,
optics, and imaging
for the life sciences
Depar t ment s
Editors Column 2
Online 4
BioOptics Breakthroughs 6
6 Raman signal boost for cell/tissue analysis
6 Low-cost fluorescence diagnoses Type 1 diabetes early
7 Optical absorptions suppression of interference
promising for biomedical imaging
8 Photoacoustic method images the small intestine
9 Molecular movies promise life sciences discoveries
9 Direct monitoring of singlet oxygen in individual cells
11 Optical amplifier enables external
transmission of bio signals
News & Notes 12
12 Pioneering products for global priorities
12 Regulatory approval takes photonics-
based systems to the clinic
14 Fast microbe ID system aids multiresistance fight
15 CBORT, BiOptix each attract $1.4M
Products 38
End Result 40
29 Cover Feature
A 3D luminescence system
enables bioluminescent
and magnetic resonance
imaging (MRI) from
the same sample.
(Image courtesy of
Aspect Imaging)
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E d i t o r i a l
2 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com
O
n average, a new study on optogenetics is reported every day. Thats been
the trend for the past two years. According to PubMed, 359 research papers
on optogenetics were published in 2013; this year through the end of
August, it was 229.
When you consider that until 2010, only a dozen papers were published on
optogenetics in total, thats impressive.
Optogenetics, which uses light to control neuron activity, is highly intriguing
and a little unnerving. As with plenty of other technological developments, it carries
opportunity for misuse. But in the history of humanity, technology has never been
required for abusive treatment of our fellow humans. And, importantly, optogenetics
potential benefits are absolutely astounding.
Karl Deisseroth, the Stanford professor and psychiatrist who coined the term, has
been heard to comment that optogenetics impact on humans will be the facilitation
of drug development for treatment of psychological disorders. Indeed, the past couple
of months have brought news of studies that seem promising in this regard. Weve seen
reports of optogenetics being used in animal studies to:
1. Learn how memories become linked with emotionsand to show that its possible to
reverse the positive/negative emotional association of specific memories;
1
2. Understand the dynamics of cellular signaling;
2
3. Clarify the operation of the thalamic reticular nucleus (TRN), which is linked to
human brain disorders such as schizophrenia, autism, and post-traumatic stress dis-
order (PTSD);
3
and
4. Control muscle movement to the spinal cords of awake, alert animals.
4
Clearly, each of these studies has important and exciting implications for treatment
of disorder and injury in human subjects. Equally exciting, then, is the translation of
optogenetics technology to the marketplace. As I write this, Strategies in Biophotonics
is about to take place in Boston (Sept. 911), and the Innovators Demo Stage, a late
addition to the conference program, will feature a number of biophotonics-based
technology demonstrations. Among these is Kendall Research Systems (Cambridge, MA)
FireFly, an ultralight, wirelessly powered and controlled headstage system for chronic,
freely behaving optogenetics research. The folks behind this company and technology are
MIT professor Ed Boyden and technology entrepreneur Christian Wentz, who earned his
PhD at MIT and founded both Kendall Research and another company, Cerenova.
Optogenetics is still in its infancy, but its hockey-stick growth trajectory portends an
exciting future that we look forward to reporting.
REFERENCES
1. R. L. Redondo et al., Nature, doi:10.1038/nature13725
(2014).
2. M. Grusch et al., EMBO J., 33, 17131726 (2014);
doi:10.15252/embj.201387695.
3. M. M. Halassa et al., Cell, doi:10.1016/j.cell.2014.06.025
(2014).
4. V. Caggiano, M. Sur, and E. Bizzi, PLoS One, 9, 6, e100865
(2014); doi:10.1371/journal.pone.
The rise of optogenetics
Christine A. Shaw Senior Vice President/
Group Publishing Director
(603) 891-9178; cshaw@pennwell.com
Barbara Goode Editor in Chief
(603) 891-9194; barbarag@pennwell.com
W. Conard Holton Executive Editor in Chief
(603) 891-9161; cholton@pennwell.com
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CONTRIBUTING EDITORS
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Susan M. Reiss
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Near-infrared imaging helps make cancer
glow to improve surgical outcomes
A team of researchers has developed
a new method involving near-infrared
light to help surgeons see an
entire tumor in a patient,
increasing the likelihood of a positive
outcome.
http://bit.ly/1sR1qJA
OCT imaging of the inner ear promising
for new hearing loss therapies
Optical coherence tomography (OCT), which generates
high-resolution 3D images, enables mapping of the
tissues within the cochlea, the portion of the
inner ear responsible for hearing. The work
could lead to new therapies for hearing loss.
http://bit.ly/Z0MLmy
Pen-sized device employs
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Visit our website to catch up on the latest developments in biophotonicsboth in optical technologies and
instrumentationby attending webcasts, watching videos, and following our continually updated news and analysis.
Correction: In the feature article Painting tumors to guide cancer surgery that ran in the May/June 2014 issue, the
acknowledgement should have stated that Tumor Paint is a trademark of Blaze Bioscience, Inc., rather than PerkinElmer.
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B R E A K T H R O U G H S
CELL BI OLOGY/HYPERSPECTRAL RAMAN
Dramatic boost in
Raman signal facilitates
cell/tissue analysis
A new form of spontaneous Raman spectroscopy deliv-
ers signals 10,000 times stronger than those obtained
from spontaneous Raman scattering, and 100 times
stronger than signals generated by comparable coher-
ent Raman instruments.
1
Furthermore, the technique
uses a much larger portion of the vibrational spectrum
of interest to cell biologists. A version of broadband
coherent anti-Stokes Raman scattering (BCARS), the
technique is fast and accurate enough to create high-
resolution images of biological specimens. Such images
contain detailed spatial information on the specific bio-
molecules present at speeds fast enough to observe
changes and movement in living cells.
Most current coherent Raman methods obtain useful
signal only in a spectral region containing approximately
five peaks with information about carbon-hydrogen and
oxygen-hydrogen bonds. The improved method adds to
this high-quality signal from the fingerprint spectral
region, which has approximately 50 peaksmost of the
useful molecular ID information.
Also, conventional coherent Raman instruments must
tune two separate laser frequencies to excite and read dif-
ferent Raman vibration modes in the sample. The new
MOBI L E HE ALTH/ F L UORESCENCE
Portable, low-cost fluorescence
biosensor diagnoses Type 1
diabetes early
A portable, inexpensive, and microchip-based fluorescence biosensor
could improve Type 1 diabetes diagnosis and care, and help research-
ers better understand the disease.
1
Evidence suggests that early detection and aggressive new therapies
may halt Type 1 diabetes autoimmune attack on the pancreas and pre-
serve some insulin-making ability. But distinguishing between Type 1 and
Type 2 diabetes now requires a time-consuming, expensive test thats lim-
ited to sophisticated health-care settings. The traditional test detects the
auto-antibodies using radioactive materials, takes several days, requires
highly trained lab staff, and costs several hundred dollars per patient.
By contrast, the microchip, developed at the Stanford University School
of Medicine (Stanford, CA), uses no radioactivity, produces results in min-
utes, and requires minimal training to use. Each chip, expected to cost
about $20 to produce, can be used for upward of 15 tests. In addition,
instead of requiring a lab-based blood draw, it can be done with blood
from a finger prick.
The device relies on a fluorescence-based method, near-infrared fluo-
rescence-enhanced (NIR-FE) detection, for detecting the antibodies. The
teams innovation is that the glass plates forming the base of each micro-
chip are coated with an array of nanoparticle-sized islands of gold, which
intensify the fluorescent signal to enable reliable antibody detection.
The test was validated with blood samples from people newly diag-
nosed with diabetes and from people without diabetes, and com-
pared with tests on the same samples using the older method.
In addition to people newly diagnosed with diabetes, the test may
benefit people who are at risk of developing Type 1 diabetes, such as
patients close relativesbecause it will allow doctors to quickly and
cheaply track their auto-antibody levels before they show symptoms.
Because it is so inexpensive, the test may also allow the first broad
screening for diabetes auto-antibodies in the population at large.
Stanford University and the researchers have filed for a patent
on the microchip, and the researchers also are working to launch a
startup company to help get the method approved by the FDA and
bring it to market, both in the U.S. and in parts of the world where
the standard test is too expensive and difficult to use.
1. B. Zhang, R. B. Kumar, H. Dai, and B. J. Feldman, Nature Med., 20,
948953 (2014); doi:10.1038/nm.3619.
High-speed BCARS allows detailed mapping of specific components
of tissue samples. A false-color BCARS image of mouse liver tissue
(left) picks out cell nuclei in blue, collagen in orange, and proteins
in green. An image of tumor and normal brain tissue from a mouse
(right) has been colored to show cell nuclei in blue, lipids in red, and
red blood cells in green. Images show an area about 200 m across.
(Image courtesy of Camp/NIST)
1409bow_6 6 9/4/14 3:40 PM
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Optical absorptions suppression of interference
promising for biomedical imaging
A new discovery could improve med-
ical imaging within biological tissue:
Physicists from the University of Twente
(Enschede, The Netherlands) and Yale
University (New Haven, CT) found that
light traveling through a diffusing mate-
rial follows a straighter path if the mate-
rial partially absorbs the light.
1

Photons traveling through a scattering
medium perform a random walk, which
resembles an uncoordinated stagger. The
researchers found that in opaque media
such as biological tissue, light absorp-
tion actually straightens this path, leading
to less diffraction by scattering and thus
improved imaging through the material.
This seems counterintuitive: Light
absorption is usually detrimental for
imaging, as it reduces the intensity of the
visible image. But the researchers dis-
covered that if enough light is absorbed,
interference is suppressed; numerical calculations showed that long, meandering light paths
are suppressed far more than short, straight paths. With increasing absorption, straight (bal-
listic) light paths persist while the number of scattered paths is considerably reduced.
1. S. F. Liew et al., Phys. Rev. B, 89, 25 (2014); doi:10.1103/PhysRevB.89.224202.
instrument uses ultrashort laser pulses to
simultaneously excite all vibrational modes
of interest; this intrapulse excitation pro-
duces its strongest signals in the finger-
print region. Because too much light will
destroy cells, weve engineered a very effi-
cient way of generating our signal with lim-
ited amounts of light. Weve been more
efficient, but also more efficient where
it counts, in the fingerprint region, said
chemist Marcus Cicerone, one of the proj-
ects researchers from the National Institute
of Standards and Technology (NIST; Gaith-
ersburg, MD) working with others from the
Cleveland Clinic in Ohio.
Raman hyperspectral images are built
up by obtaining spectra, one spatial pixel
at a time. The hundred-fold improvement
in signal strength makes it possible to col-
lect individual spectral data much faster
and at much higher quality than beforea
few milliseconds per pixel for a high-quality
spectrum vs. tens of milliseconds for a mar-
ginal-quality spectrum with other coherent
Raman spectroscopies, or even seconds for
a spectrum from more conventional spon-
taneous Raman instruments. Because its
capable of registering many more spec-
tral peaks in the fingerprint region, each
pixel carries a wealth of data about the bio-
molecules present. This translates to high-
resolution imaging within a minute or so,
whereas, notes NIST electrical engineer
Charles Camp, Jr., Its not uncommon to
take 36 hours to get a low-resolution image
in spontaneous Raman spectroscopy.
Camp adds, There are a number of
firsts in this paper. Among other things,
we show detailed images of collagen
and elastinnot normally identified with
coherent Raman techniquesand mul-
tiple peaks attributed to different bonds
and states of nucleotides that show the
presence of DNA or RNA.
1. C. H. Camp et al., Nature Photon., 8,
627634 (2014); doi:10.1038/npho-
ton.2014.145.
Numerical calculations reveal the distribution of light
intensity inside an opaque diffusing medium. Light enters
the material from the left. The top image demonstrates
multiple scattering, which causes the light paths to become
random walks (blue arrows). The light exits in random
directions, which precludes imaging. The bottom image
illustrates an absorbing opaque medium. The transport of
light occurs via straighter paths, which results in a coherent
image on the right-hand side. (Image courtesy of the Dutch
Foundation for Fundamental Research on Matter)
1409bow_7 7 9/4/14 3:40 PM
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Nanoparticles suspended in liquid, ingested by
patients, promises to enable photoacoustic tomog-
raphy (PAT) imaging for a noninvasive, real-time
view of the small intestine.
1
The advancement could
help doctors better identify, understand, and treat
gastrointestinal ailments such as irritable bowel syn-
drome, celiac disease, and Crohns disease.
To assess the small intestine, doctors typi-
cally require patients to drink a thick, chalky liq-
uid called barium. They then image using x-rays,
magnetic resonance imaging (MRI), or ultra-
sound, but these techniques are limitedin
terms of safety, accessibility, and lack of adequate contrast, respec-
tively. Also, none effectively provides real-time imaging of move-
ment such as peristalsis, the contraction of muscles that propels
food through the small intestine. Dysfunction of these movements
may not only be linked to gastrointestinal illnesses, but also be side
effects of thyroid disorders, diabetes, and Parkinsons disease.
Conventional imaging methods show the organ and blockages,
but this method lets you see how the small intestine operates in real
time, says corresponding author Jonathan Lovell, Ph.D., assistant
professor of biomedical engineering at the University of Buffalo (UB),
where researchers are pursuing the work.
The researchers worked with a family of dyes
called naphthalcyaninessmall molecules that
are highly light-absorbent in the near-infrared
spectrum, which is the ideal range for biological
contrast agents. However, the dyes are unsuitable
for the human body because they dont disperse
in liquid and they can be absorbed from the intes-
tine into the bloodstream. To address these prob-
lems, the researchers formed nanoparticles called
nanonaps that contain the colorful dye mole-
cules and added the abilities to disperse in liquid
and move safely through the intestine.
In laboratory experiments with mice, the researchers adminis-
tered the nanojuice orally. They then used PAT, which uses pulsed
laser lights to generate pressure waves that, when measured, pro-
vide a real-time and more nuanced view of the small intestine. The
researchers plan to continue to refine the technique for human tri-
als, and move into other areas of the gastrointestinal tract.
1. Y. Zhang et al., Nature Nanotechnol., 9, 631638 (2014);
doi:10.1038/nnano.2014.130.
PHOTOACOUST I C TOMOGR APHY/ NANOTECHNOL OGY
Photoacoustic method enables noninvasive, dynamic imaging of small intestine
The combination of nanojuice and
photoacoustic tomography (PAT)
illuminates the intestine of a mouse.
(Image courtesy of Jonathan Lovell)
1409bow_8 8 9/4/14 3:40 PM
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Molecular movie technology promises
life sciences discoveries
A powerful new imaging technology involving femtosecond laser pulses and bio-
luminescent proteins is fast enough to observe life processes as they happen at the
molecular level, according to the researchers who devised it.
1
With this technology,
were going to be able to slow down the observation of living processes and under-
stand the exact sequences of biochemical reactions, says Chong Fang, an assistant
professor of chemistry at Oregon State University (OSU; Corvallis, OR) and leader
of the research team, which also involves scientists at the University of Alberta
(Edmonton, AB, Canada). We believe this is the first time ever that you can really
see chemistry in action inside a biosensor, he says.
The new approach offers sufficient speed to allow scientists to see what is
happening at the molecular level and create whatever kind of sensor they want by
rational design. This will improve the study of, for example, cell metabolism to nerve
impulses, how a flu virus infects a person, or how a malignant tumor spreads.
The technology, for instance, can follow the proton transfer associated with the
movement of calcium ionsone of the most basic aspects of almost all living sys-
tems and also one of the fastest. This movement of protons is integral to everything
from respiration to cell metabolism and plant photosynthesis. Scientists will now be
able to identify what is going on, one step at a time, and then use that knowledge
to create customized biosensors for improved imaging of life processes.
Fang explains, Were making molecular movies. And with this, were going to
be able to create sensors that answer some important, new questions in biophysics,
biochemistry, materials science, and biomedical problems.

1. B. G. Oscar et al., Proc. Nat. Acad. Sci., 111, 28, 1019110196 (2014); doi:10.1073/
pnas.1403712111.
CEL L BI OL OGY/ SPEC T ROSCOPY
Microspectroscopy setup enables
direct monitoring of singlet
oxygen in individual cells
Singlet oxygen, the first excited state of molecular oxy-
gen, is a highly reactive species that plays an important
role in a wide range of biological processes, including
cell signaling, immune response, macromolecule degra-
dation, and elimination of neoplastic tissue during pho-
todynamic therapy (PDT). Now, researchers have devel-
oped an experimental setup that enables direct micro-
spectroscopic monitoring of singlet oxygen.
1
The Charles University (Prague, Czech Republic)
scientists used two detection channelsvisible and
near-infrared (NIR)to perform real-time imaging of
the very weak NIR phosphorescence of singlet oxygen
and photosensitizer simultaneously with visible fluo-
rescence of the
The experimental setup for direct
monitoring of singlet oxygen in
cells includes a 2D InGaAs camera
coupled to an imaging spectrograph.
(Image courtesy of Princeton
Instruments)
continued on page 11
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photosensitizer. Their experimental setup
enables acquisition of spectral images
based on singlet oxygen and photosen-
sitizer luminescence from individual cells,
where one dimension of the image is spa-
tial and the other is spectral, covering a
spectral range from 500 to 1700 nm.
To achieve these results, the research-
ers coupled a 2D-array indium gallium
arsenide (InGaAs) camera with NIR sen-
sitivity to an imaging spectrograph. The
2D detection array in the camera dramati-
cally reduced acquisition times and helped
to avoid some of the problems caused
by photobleaching as compared to the
groups previous 1D InGaAs detectors. A
back-illuminated, silicon CCD camera was
used to detect visible light in the setup.
The researchers indicate that the intro-
duction of spectral images for such stud-
ies addresses the issue of a potential spec-
tral overlap of singlet oxygen phosphores-
cence with NIR-extended luminescence of
the photosensitizer and provides a power-
ful tool for distinguishing and separating
them, which can be applied to any photo-
sensitizer manifesting NIR luminescence.
1. M. Scholz, R. Dedic, J. Valenta, T.
Breitenbach, and J. Hla, Photochem.
Photobiol. Sci., 13, 12031212 (2014);
doi:10.1039/C4PP00121D.
MICROSPECTROSCOPY continued from page 9
BI OL OGI C AL SI GNAL I NG
Laser-like optical amplifier portends
external transmission of bio signals
Potential applications in medicine are exciting, says J. Gary Eden, professor of elec-
trical and computer engineering (ECE) at the University of Illinois Urbana-Champaign
(UIUC). We have made optical systems at the microscopic scale that amplify light and
produce ultra-narrowband spectral output, he adds, explaining a new optical amplifier
(or laser) design that paves the way for power-on-a-chip applications. Actuated by light
that penetrates human skin, the amplifiers can transmit signalsproduced by cells and
biomedical sensorsto electrical and optical networks outside the body.
1

The speed of currently available semiconductor electronics is limited to about
10 GHz due to heat generation and interconnect delays. Dielectric-based photon-
ics, though not limited in speed, are limited in size by the laws of diffraction. The
researchers, led by Eden and ECE associate professor Logan Liu, discovered a path
to the best of both worlds: Plasmonicsmetal nanostructurescan serve as a
bridge between photonics and electronics, to combine small size and high speed.
We have demonstrated a novel optoplasmonic system comprising plasmonic
nanoantennas and optical microcavities capable of active nanoscale field modula-
tion, frequency switching, and amplification of signals, states Manas Ranjan Gar-
tia, lead author of an article describing the work.
1
This is an important step for-
ward for monolithically building on-chip light sources inside future chips that can
use much less energy while providing superior speed performance of the chips.
At the heart of the amplifier is a polystyrene or glass microsphere about 10 m in
diameter. When activated by an intense beam of light, the sphere generates inter-
nally a narrowband optical signal that is produced by a process known as Raman
scattering. Molecules tethered to the surface of the sphere by a protein amplify
the Raman signal, and in concert with a nano-structured surface in contact to the
sphere, the amplifier produces visible light having a bandwidth that matches the
internally generated signal.
Precise manipulation of light at the micro- and nano-spatial scales is necessary for
realizing physical analogs of optical processes in biology, and for pursuing applica-
tions in areas such as embedded biomedical sensors.
1. M. R. Gartia et al., Sci. Rep., 4, 6168 (2014); doi:10.1038/srep06168.
1409bow_11 11 9/4/14 4:28 PM
News Notes
Compi l e d by BARBARA GOODE
Biophotonics-based systems earning recent regulatory approval
include a welcome advance in colorectal cancer screening that
gives patients a pass on the usual dietary restrictions and bowel
prep. Also included is a laser-based adjunct to balloon angioplasty
for peripheral artery disease, and a system for tattoo removal and
treatment of pigmented lesions on all skin types.
A comfortable colo-cancer screen
Exact Sciences (NASDAQ:EXAS; Madison, WI) has received FDA
approval for the first at-home, noninvasive test for colorectal can-
cer that analyzes both stool DNA and blood biomarkers using flu-
orescence. The test requires no medication, dietary restrictions,
or bowel preparation, and demonstrated effectiveness in a pro-
spective, 90-site, 10,000-patient pivotal study.
1
The findings have
proven that this noninvasive test is highly sensitive in detecting
both early-stage colorectal cancer and the most advanced pre-
cancerous polyps most likely to develop into cancer, said David
Ahlquist, MD, a Mayo Clinic (Rochester, MN) gastroenterologist
who co-invented the test. Mayo researchers developed the tech-
nology, and licensed it to Exact Sciences.
When a physician orders the Cologuard test, the kit is mailed
directly to the patients home. And once the patient collects a sam-
ple, he or she sends the kit to the Exact Sciences lab. There, Colo-
guards Quantitative Allele-specific Real-time Target and Signal Ampli-
fication (QuARTS) technology looks for biomarkers that are shed from
the colon as part of the digestive process, and blood released in the
stool. Multiplexed QuARTS reactions are processed using a real-time
cycler, with each biomarker (NDRG4, BMP3, KRAS, and ACTB) moni-
tored separately through independent fluorescent detection channels.
The sample is prepared and analyzed for fecal occult blood in a quan-
titative Enzyme-Linked Immunosorbent Assay (ELISA) that determines
the concentration of hemoglobin in the sample.
Patients learn their results from their prescribing physician. The test is
available in the U.S. for $599. The company plans to make Cologuard
available in select countries in Europe pending CE Mark approval.
Laser-based adjunct to balloon angioplasty
Excimer laser maker Spectranetics (Colorado Springs, CO) has
received FDA approval for its laser atherectomy products to treat
in-stent restenosis (ISR; that is, return of
Pioneering products for global priorities
Regulatory approval takes photonics-based systems to the clinic
Dr. Anita Goels ultimate goal, to under-
stand living systems, has led her on a
20-year quest to help decentralize, mobi-
lize, and personalize medicine. Previously
named by MIT Technology Review as one
of the Worlds Top 35 under 35 science
and technology innovators, Goel, an MD
and PhD, is a pioneer in the emerging field
of nanobiophysics, a new convergence of
physics, nanotechnology, and biomedi-
cineand was a featured speaker at Strat-
egies in Biophotonics (Boston, MA; Sept.
9-11, 2014). These days, she carries out her
pioneering efforts as chairman and CEO of
both Nanobiosym Inc. (NBS) and Nanobio-
sym Diagnostics (NBSDx).
NBS is a technology incubator for funda-
mental research funded at various points
of time with grants from agencies such as
DARPA, DoD, and NSF. The institute/incu-
bator has a three-fold mission: 1) To create
new science and disruptive technologies that
emerge at the convergence of physics, bio-
medicine, and nanotechnology; 2) to spin off
new companies and joint ventures that cap-
ture the commercial impact of the promise
of nanotechnology; and 3) to transition these
technologies to solve pressing healthcare,
energy, and environmental challenges.
By contrast, NBSDx is NBSs commer-
cial engine, with a focus on commercializa-
tion of its diagnostic technology platform,
which Goel explains will empower people
worldwide with rapid, accurate, and porta-
ble diagnostic information about their own
health. Called Gene-RADAR, the chip-
based platform, which is the size of an iPad,
can diagnose any disease with a genetic
fingerprintincluding HIV, Ebola, tuber-
culosis, malaria, and drug-resistant muta-
NANOT ECHNOL OGY/ MOBI L E HE ALT H
F L UORESCE NCE / MEDI C AL L ASE RS
continued on page 14
Anita Goel, MD, Ph.D., who was a featured
speaker at Strategies in Biophotonics (Boston;
Sept. 9-11, 2014), pursues global health advances
as chairman and CEO of both Nanobiosym Inc.
(NBS) and Nanobiosym Diagnostics.
1409bow_12 12 9/4/14 3:40 PM
Be brilliant.
Announcing the
ORCA-Flash4.0 LT
The benefits of sCMOS technology at the price of a CCD.

Find out more by visiting
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O
R
C
A
-
F
l
a
s
h
4
.
0

L
T
tions thereof, Goel told BioOptics World. As
ever, early detection means the opportunity
for both better containment of commu-
nicable diseases and better treatment for
those infected. In a dramatic example, Goel
explains how Gene-RADARs ability to mea-
sure viral load in HIV-positive mothers and
their infants will enable real-time custom-
ization of antiretroviral therapy and moni-
toring of treatment.
Flexible and direct
Gene-RADAR uses light to measure
directly from a single drop of blood or
saliva. Unlike approaches that are based
on antigens or antibodies, Gene-RADARs
approach is very simple in that it measures
directly. There is no hybridization involved;
instead, it is based on actual direct detec-
tion, Goel explains. And it requires no lab
infrastructure, no running water or constant
electricity, and no trained healthcare staff,
so it has potentially dramatic humanitar-
ian impact in areas and situations previously
inaccessible to high tech. In 2013, Nano-
biosym was awarded the grand prize at
the Nokia Sensing XCHALLENGE for Gene-
RADARs advanced sensing technology.
Goel credits tools based on optics and
quantum opticsfor instance, optical
tweezers and atomic force microscopes
for enabling study at the single-molecule
level and for facilitating exploration of the
dynamics of enzymes such as polymer-
ases that operate as they read and write
DNA. Such research, she says, has pro-
vided unprecedented insight into these
structures context-dependent function
insight that has become foundational
for NBSDx.
Gene-RADAR is based upon findings
that these enzymes (which Goel says can be
viewed as nanoscale bio-motors able to con-
vert chemical energy stored in nucleotides
into mechanical work) serve to replicate, tran-
scribe, or otherwise process biological infor-
mation.
1
Precision control of these molecular
engines has broad implications for biomed-
icine (including whole-genome sequencing
with ultra-high precision and accuracy, and
molecular manufacturing of biopolymers)
and beyond (for instance, biological compu-
tation and information storage).
2
The global health impact of inexpen-
sive mobile technologies such as Gene-
RADAR was the focus of Goels presenta-
tion at Strategies in Biophotonics. Her talk
followed the opening keynote by lauded
engineer and entrepreneur Robert Langer
of MIT. For more on the event, see www.
strategiesinbiophotonics.com.
ACKNOWLEDGEMENT
Gene-RADAR is a registered trademark of
Nanobiosym Diagnostics.
REFERENCES
1. A. Goel and V. Vogel, Nature Nanotech-
nol., 3, 465475 (2008); doi:10.1038/
nnano.2008.190.
2. A. Goel, Sci. Am. India, 5, 12, 5357
(2010).
1409bow_13 13 9/4/14 3:40 PM
Cleavage
Marker 1 Marker 2 Marker 3
Quencher
DNA amplication and target probe cleavage
FRET cleavage and fuorescent signal generation
5
5
3
3
5
Cleavage
Quencher
Cleavage
HEX Dye FRET Cassette
Quencher
Cleavage
5
5
3
3
5
Cleavage Cleavage
5
5
3
3
5
Fluorescent signal emitted if target is present
Marker-specic probe Marker-specic probe Marker-specic probe
Forward primer/Invasive oligo Forward primer/Invasive oligo Forward primer/Invasive oligo
Reverse primer Reverse primer Reverse primer
Quasar670 Dye FRET Cassette FAM Dye FRET Cassette
News Notes
continued from page 12
blockage following stent placement) for patients with peripheral
artery disease (PAD).
The development prompts a new standard of care in ISR treat-
ment with improved clinical outcomes, and it follows clinical find-
ings of the EXCImer Laser Randomized Controlled Study for Treat-
ment of FemoropopliTEal (the arteries above and behind the
knee) In-Stent Restenosis (EXCITE ISR). The study, reportedly the
first multi-center, randomized prospective trial ever conducted for
ISR treatment, demonstrated highly superior safety and efficacy
of laser atherectomy with adjunctive percutaneous transluminal
angioplasty (PTA, or balloon angioplasty) compared with PTA
alone. The trial shows a 94-percent procedural success rate using
laser atherectomy with PTA vs. 83 percent with PTA alone.
In the study, the average lesion length was approximately 20 cm
compared to various stent IDE studies with average lesion lengths of 4
to 6 cm. Additionally, a high number of complex or advanced disease-
state patients were enrolled in the trial, a fact that indicates success in
treating all types of ISR lesions, including the most complex. Complete
results from the EXCITE
trial have been submit-
ted to a peer-reviewed
medical journal.
While stents deliver
improved overall out-
comes compared to
PTA treatment, reste-
nosis is common and
stent re-obstruction or
ISR remains therapeuti-
cally challenging. Once
ISR develops, there is a 65-percent chance of recurrence after PTA,
which is considered the standard of care. With over 115,000 ISR pro-
cedures performed annually in the U.S., Spectranetics says it is posi-
tioned to capitalize on potential market opportunities of $350 million
domestically and up to $750 million worldwide.
Removing pigments from all skin types
Aesthetic laser system maker Syneron Medical (Irvine, CA) has
received CE Mark approval for its dual-wavelength picosecond laser
to remove tattoos and pigmented lesions on any skin type. Delivering
532 and 1064 nm wavelengths, the laser uses proprietary PicoWay
technology to apply high peak power and ultrashort pulses for strong
photomechanical impact. The company will begin a staged launch of
the laser in the international market during the third quarter of 2014,
and anticipates FDA clearance by the end of 2014.
REFERENCE
1. T. F. Imperiale et al., N. Engl. J. Med., 370, 12871297 (Apr. 3,
2014); doi:10.1056/NEJMoa1311194.
The first at-home,
noninvasive test for
colorectal cancer,
by Exact Sciences,
analyzes DNA and
biomarkers using
fluorescence.
Fast microbe ID system aids multiresistance fight
The China Food and Drug Administration
(CFDA) has given clearance to Bruker Corp.
(Billerica, MA, and Beijing, China; NAS-
DAQ: BRKR) to market and sell its in vitro
diagnostics (IVD) MALDI Biotyper system
as a medical device for identifying micro-
organisms isolated from human specimens.
Starting from a culture, the IVD MALDI
Biotyper allows for microbial identifica-
tion in a few minutes without further
incubation steps. The system features
the companys microflex matrix-assisted
laser desorption ionization time-of-flight
(MALDI-TOF), mass spectrometry-based
identification to enable analysis of species
that are difficult to identify using other
microbiology techniques. An increas-
ing number of case reports describe the
identification of microorganisms causing
human infections, which previously have
been isolated only from environmental
sources. The companys extensive refer-
ence library covers more than 2300 micro-
bial species of gram-negative and gram-
positive bacteria as well as anaerobes and
yeasts, and often enables the identifica-
tions of unexpected microorganisms.
The approval is expected to facilitate
clinical microbiology in China. With the
significant reduction of the time to result
MAL DI - TOF/ MI CROBI OL OGY
1409bow_14 14 9/4/14 3:40 PM
High Performance
Priced Right
Ultra Series Filters
Learn more at
www.alluxa.com/learning-center
1-855-4ALLUXA sales@alluxa.com
Designed specifcally to improve
optical performance in the
most demanding life sciences
applications.
News Notes
for identification of pathogenic bacte-
ria, the patient outcomes will improve
and the cost of care will be reduced
significantly, said Dr. Lisong Shen,
Director of Laboratory Medicine at Xin
Hua Hospital (affiliated with Shanghai
Jiao Tong University School of Medi-
cine) and president of the Shanghai
Society of Laboratory Medicine.
Fast species identification is important
for helping to guide selection of thera-
peutic drug(s) because of the increas-
ing threat by multiresistant bacteria caus-
ing severe infections. The system is avail-
able in most of Europe; in the Ameri-
cas in Canada, Argentina, Colombia,
Ecuador, and Mexico; and in the Asia/
Pacific region in Japan, China, Hong
Kong, Singapore, Taiwan, and Australia.
In November 2013, the MALDI Biotyper
CA System received clearance for a first
claim by the U.S. FDA.
Two entities working on photonics-based
biomedicine have recently attracted funding
in the same amount: $1.4 million.
Massachusetts General Hospital (Bos-
ton, MA) has received a National Institutes of
Health grant totaling $1,449,151 for its Cen-
ter for Biomedical OCT Research and Trans-
lation (CBORT). With CBORT, principal inves-
tigator Brett Bouma (a Strategies in Biopho-
tonics advisory board member) aims to bring
groundbreaking advances in optical coher-
ence tomography (OCT) to biomedicine. The
center seeks to address the deficient acces-
sibility of cutting-edge OCT instrumentation
and technology through innovation of new
technical capabilities that are motivated by
significant biological and clinical challenges,
and through translation, facilitated by direct
collaboration. Since CBORTs 2011 launch,
its members have initiated six driving bio-
medical projects and have identified techni-
cal projects that will have significant biologi-
cal and clinical impact. The projects fall into
three core thematic areas: advanced struc-
tural imaging, functional and compositional
contrast, and hybrid imaging modalities.
Colorado-based optical biodetection com-
pany BiOptix (Boulder) reports that it has
received an additional $1.4 million from
investors, which they will use to expand sales
and marketing and ramp up manufactur-
ing to meet growing customer demand. The
company sees opportunity in the label-free
marketplace, which CEO Rick Whitcomb says
is looking for a reasonably priced high-per-
formance surface plasmon resonance (SPR)
instrument. They have developed and pat-
ented an ultra-sensitive detection platform
known as Enhanced Surface Plasmon Reso-
nance (ESPR), which pairs the high sensitivity
of SPR with the high stability and lower noise
of common-path interferometry.
CBORT, BiOptix each attract $1.4M
OP T I C AL COHE RE NCE TOMOGR APHY/ SURFACE PL ASMON RESONANCE
1409bow_15 15 9/4/14 3:40 PM
d)
MCT
a) b) c)
5
6
7
8
9
4
3 2
1
0
O P T I C A L C O H E R E N C E T O M O G R A P H Y
By Kengyeh K. Chu, Susan E. Bi r ket , Li nbo Li u, St even M. Rowe, and Gui l l er mo J . Tear ney
High-resolution micro-OCT
uncovers correlations
With a resolution of 12 m, OCT is the only noninvasive method able to comprehensively
and simultaneously study both the subcellular structures and functions important for diseases
such as cystic fibrosis (CF), and the correlations between them, in vivo. Thus, OCT promises
to become an important new tool for understanding the mechanisms of such pathologies.
A
new, high-resolution
form of optical coher-
ence tomography
(OCT), micro-OCT
(OCT) images at 12 msuf-
ficient to visualize individual cells
and even subcellular features in
vivo. Developed by the Tearney Lab
at the Massachusetts General Hospi-
tal in collaboration with Dr. Steven
Rowe at the University of Alabama
at Birmingham, OCT was origi-
nally demonstrated for imaging ath-
erosclerosis, where microscopic fea-
tures (e.g., cholesterol crystals, mac-
rophages, platelets, and fibrin) were
revealed in arterial plaques.
1
More recently,
the technique is being applied to investigate
airway surfaces, where tiny, hair-like organ-
elles known as cilia protrude approximately
7 m and beat in a whipping motion 10 to 20
times per second to carry dirt- and bacteria-
trapping mucus away from the lungs.
2
This
process, known as mucociliary clearance, is
sometimes impaired by disease, which leads
to increased incidence of infection, airway
obstruction, and decreased lung function.
Cystic fibrosis (CF) is perhaps the
best-known example of a disease associ-
ated with mucus transport impairment.
3

Affecting 30,000 patients in the United
States and 70,000 globally, CF results from
mutations in the cystic fibrosis transmem-
brane conductance regulator (CFTR)
gene, which acts primarily as a chloride
and bicarbonate ion channel. The cell
membranes of CF patients are abnormally
impermeable to these anions. CF lungs
are characterized by impaired mucocili-
ary transport, highly viscous mucus, and
are prone to frequent and persistent infec-
tion. Even though the genetic defect and
the resulting pulmonary consequences
are understood, the intermediate links in
the chainincluding the mechanism that
causes mucus transport to break down
remain unknown.
Imaging cilia motion
While living cilia have been difficult to
study noninvasively because of their small
KENGYEH KEN CHU, Ph.D., is Research Fellow in Dermatology at Massachusetts General
Hospitals Wellman Center for Photomedicine. SUSAN E. BIRKET, PharmD, Ph.D., is a
postdoctoral trainee in Pulmonary, Allergy, and Critical Care Medicine at the University of Alabama
at Birmingham (UAB). LINBO LIU, Ph.D., is Assistant Professor of Electrical and Electronic
Engineering at the Nanyang Technical University in Singapore. STEVEN M. ROWE, MD, MSPH,
is Associate Professor of Medicine, Pediatric Pulmonary Medicine, and Physiology & Biophysics at
UAB and the Director of the UAB Cystic Fibrosis Transition Clinic. GUILLERMO J. TEARNEY,
MD, Ph.D., is Mike and Sue Hazard Family MGH Research Scholar and Professor of Pathology
at Harvard Medical School and the Wellman Center for Photomedicine. Contact Dr. Tearney at
tearney@partners.org; www.tearneylab.org.
FIGURE 1. Produced by OCT,
images of cilia (false-colored in green)
on human bronchial epithelial cells
show a low path during the recovery
stroke (a), a high path during the
effective stroke (b), and an ellipsoidal
trajectory over a complete cycle
(c), with distinguishable effective
stroke (yellow arrows) and recovery
stroke (orange arrows). Effective and
recovery stroke positions are visible
in a densely ciliated region of the
epithelium (d). Scale bars: 10 m.
(Reprinted from Liu et al.
2
)
1409bow_16 16 9/4/14 3:43 PM
a)
b)
air
mu
pcl
ep
air
mu
air
mu
ep
pcl
ep
DUAL INVERTED SELECTIVE PLANE
ILLUMINATION MICROSCOPY
For More Information
Visit: www.asiimaging.com
Email: info@asiimaging.com
Call: (800) 706-2284 or (541) 461-8181
ASI has developed a new form of light sheet microscopy with our
collaborators. The system utilizes two water emersion objectives
mounted at 45 degree angles above the sample on an inverted
microscope, a third objective also can be used to view the sample
from below. The top objectives are mounted on piezo positioning
devices so precise image stacks can be obtained.
O P T I C A L C O H E R E N C E T O M O G R A P H Y c o n t .
size and high rate of motion, OCT offers
the resolution and speed to image them
directly. The tip of a single cilium can be
imaged as it progresses through its whip-
like motion, which can be divided into
effective and recovery strokes. Figure
1 demonstrates this: Panel A shows the
recovery stroke of the cilium; on OCT,
the cilium tip, colored in green, traces
a path close to the cell surface as it arcs
backwards in preparation for the effective
stroke, shown in Panel B. In this stroke,
the cilium tip travels forward at a greater
height than during the recovery stroke.
Panel C illustrates the full cycle of the cil-
ium through one full period, as the com-
plete arc trajectory of the tip is seen. Panel
D shows a time-averaged region of densely
ciliated swine epithelium in which the
stroke arc extents are also observed.
For CF research, several quantitative
measures are often used to characterize the
functional health of the respiratory epithe-
lium and consequential mucus clearance.
Two such metrics are airway surface liquid
(ASL) and periciliary liquid (PCL) depth,
which together indicate the hydration status of the airway surface.
The ASL depth quantifies the total thickness of mucus overlying
the cells, and the PCL depth measures the thin sheet of liquid gel
that encompasses the cilia; it is primarily within the PCL that the
cilia beat. The ciliary beat frequency (CBF) is a measure of the cilia
stroke rate. The mucociliary transport (MCT) velocity quantifies
the objective of mucociliary clearance: the removal of mucus. MCT
rate is the speed at which mucus is conveyed over airway surface.
These four parameters (ASL depth, PCL depth, CBF, and MCT
velocity) are essential metrics for analyzing CF because diseased
airways often exhibit dehydration (depressed ASL and PCL), lag-
ging ciliary beat (reduced CBF), and of course mucus clearance
difficulty (significantly decreased MCT). Though methods are
available to measure each of these parameters in cultured cells
or tissue, no pre-existing technique is able to quantify all of these
metrics together.
Fortunately, in addition to the already described imaging of
individual cilia, OCT is able to quantify each of these measure-
ments, as demonstrated in Fig. 2, which shows OCT images of
cultured ciliated airway cells derived from a healthy patient.
4

Because OCT is a cross-sectional technique, the thicknesses
of the ASL and PCL can be easily measured from images. The
yellow bar in Fig. 2a is drawn between the bright line that rep-
resents the top of the mucus layer and the top of the epithe-
lium, and corresponds to the thickness of the ASL layer. The
red bar similarly indicates the PCL layer, which can be more
distinctly seen on a time-averaged image (right side). As OCT
can image the up-and-down periodic motion of cilia tips (green
arrows in Fig. 2a), the frequency of the beat (CBF) can also be
FIGURE 2. Single-frame (left) and time-averaged (right) OCT images of normal human bronchial
epithelial cells show depth measurements of the airway surface liquid (ASL, yellow bar) and periciliary
liquid (PCL, red bar). Cilia tips (green arrows) are seen statically in the single-frame image to the left
and as motion-blurred arcs in the time-averaged image to the right. The motion of the mucus induces
a streaking effect of the mucus in the time-averaged image (a). A OCT time-averaged image of
bronchial epithelial cells from a CF donor reveales compacted ASL/PCL (b). Scale bar: 10 m. (Reprinted
from Birket et al.
4
, with permission from the American Thoracic Society)
1409bow_17 17 9/4/14 3:43 PM
ASL (m) PCL (m)
CBF (Hz) MCT (mm/min)
Non-CF CF
Non-CF CF Non-CF CF
Non-CF CF
40
30
20
10
0
8
6
4
2
0
40
30
20
10
0
30
20
10
0
a) b)
c) d)
MCT (mm/min)
MCT (mm/min)
MCT (mm/min)
CFTR (+/+)
CFTR (/)
DNDS
PCL (m)
PCL (m) PCL depth (m)
15
10
5
0
15
5 10 15
10
5
0
6
5 10 15
4
2
0 5 10 15
a) b) c)
directly determined from OCT movies.
Finally, as particles in the mucus are swept
across the OCT field of view by mucocil-
iary clearance, the speed of their motion
defines the rate of mucociliary transport
(MCT). The streaking pattern within the
mucus on the time-averaged right-hand
image of Fig. 2a results from the motion
of the particles seen within the mucus on
the left-hand image.
Figure 2b depicts an image from cells
taken from a CF patient and cultured. This
OCT data shows very different character-
istics compared to the healthy sample. The
mucus layer is very thin and the cilia-con-
taining PCL layer is compressed as well.
The cilia also beat more slowly and mucus
transport is significantly reduced in the CF
case. Figure 3 shows the quantitative differ-
ences observed in the non-CF and CF cells.
Revealing relationships
Thus, OCT is the only method avail-
able to comprehensively and simultane-
ously study airway hydration, ciliary func-
tion, and mucus transport, and OCTs
ability to derive measurements from the
same regions of the same samples at the
same time makes it possible to study cor-
relations between these factors. Because
the cause-and-effect relationship between
parameters (e.g., airway hydration and
transport rate) can be determined only
by studying them together, OCT has the
potential to be an important new tool for
understanding the mechanisms of CF and
other ciliary disorders.
In a recent study, OCT was used to
analyze the relationship between the
periciliary liquid (PCL) and the rate
of mucus transport (MCT), which is pos-
sible only using a method that can cap-
ture both measurements at once.
4
In nor-
mal airways, it is expected that a higher
amount of liquid in the PCL is associ-
ated with higher MCT rates, reflecting
the importance of hydration for mucus
transport. And indeed, OCT imaging
revealed a positive correlation between
PCL thickness and MCT rate in nor-
mal explanted airways.
4
However, OCT
also showed that this relationship was
disrupted and even reversed in CF sam-
ples, indicating that a factor beyond air-
way hydration alone is responsible for
delaying mucus transport in CF, and
the slight negative slope in the correla-
tion even indicates that more PCL hydra-
tion is associated with decreased mucus
FIGURE 3. Cultured non-CF and CF donor bronchial epithelial cells have statistically significant
differences in the measured depths of airway surface liquid (ASL, a) and periciliary liquid (PCL, b),
ciliary beat frequency (CBF, c) and mucociliary transport velocity (MCT, d), as demonstrated in these
OCT images. (Reprinted from Birket et al.
4
FIGURE 4. Correlated OCT image-derived measurements show that in normal swine tracheas, increased hydration of the periciliary liquid (PCL) is
associated with higher mucus transport rates (MCT; a), but in CF-model tracheas, the relationship is inverted (b). The inverted relationship observed in the
CF trachea is duplicated in normal tracheas exposed to DNDS, a bicarbonate transport inhibitor, suggesting a bicarbonate-related defect in CF tracheas (c).
(Reprinted from Birket et al.
4
1409bow_18 18 9/4/14 3:43 PM
transport.
4
These results suggest that
there is an innate defect in the CF mucus
itself such that more of its presence is, in
fact, counterproductive.
One possible explanation for this is
that CF mucus is inherently more vis-
cous. Though elevated viscosity of expec-
torated CF sputum is a well-established
fact, it is not known whether the viscos-
ity increase is inherent or results from
other CF-related phenomena such as
chronic infection or mucociliary clear-
ance failure. One hypothesis suggests
that a mucus viscosity anomaly in CF
can be caused by a decreased concen-
tration of bicarbonate ions (also result-
ing from CFTR dysfunction), disrupting
the ability of mucins (the long molecu-
lar chains that mesh around a watery
medium to form mucus) to unfold and
causing them instead to stick together
to form an abnormally viscous mucus.
5
Using OCT to test this hypothesis, we
analyzed the response of the PCL/MCT
relationship following application of the
bicarbonate transport inhibitor 4,4- dini-
trostilbene-2,2-disulfonic acid (DNDS)
to normal airways. Indeed, OCT mea-
surements showed that the PCL/MCT
relationship is inverted when bicar-
bonate is inhibited, duplicating the CF
observation. The viscosity of mucus was
also found to be elevated in both CF and
bicarbonate-inhibited airways, which
suggests that bicarbonate inhibition
does cause a viscosity increase in situ that
could explain the newly discovered PCL/
MCT inversion.
Another revelation enabled by OCT
was a previously unobserved phenome-
non that occurs when cilia on normal cul-
tured human airways cells are subjected
to an increased workload when encoun-
tering mucus rafts.
6
Imaging with OCT
showed that a small compression (up to
2 m) of the cilia caused by a mucus load
stimulates the cilia to beat faster and to
transport mucus more rapidly. Further
load that compresses the cilia beyond
2 m negates this increase, and the beat
frequency further declines in response
to additional compression. Simultane-
ous fluorescence imaging revealed a cal-
cium spike at the onset of the mucus
load, and the application of BAPTA-AM
(a chemical that binds calcium and thus
reduces its availability to the cell) pre-
vents the ciliary beat increase. These
results indicate that a calcium-signaling-
based feedback mechanism helps airways
to cope with periods of increased load by
increasing ciliary beating and that this
feedback mechanism fails when the load
is too great.
Recent and future discoveries
The ability of OCT to measure multi-
ple facets of airway function and mucus
transport has proven to be very use-
ful, and has already generated new dis-
coveries enabled by simultaneous and
co-localized measurements of mucus
1409bow_19 19 9/4/14 3:43 PM
c)
d)
e)
Baseline
Mucus-invasion
Mucus
7 m
R
0.5
0.6
0.7
0.8
Baseline
Mucus
invasion
0.9
1.0
1.1
1.2
1.3
2.0
1.5
1.0
0.5
0
M-mode image
Post-invasion
E
R
E
Mucus invasion
Mucus invasion
Baseline
Baseline
Time
MCT
OCT beam
Z
Z
2X 2X
t
X
Time-averaged
B-mode image
b)
4
8
6
4
3
2
1
0
10 20 30 40 50
Mucus invasion
CBF
PCL height reduction
Baseline
PCL compression (m)
e) Normalized CBF
a)
f) Normalized cilia tip intensity
CBF (Hz)
o
Time (sec)
PCL height reduction (m)
60 70
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2
Effective Recovery
FIGURE 5. This diagram of a mucus compression experiment represents horizontal (X), vertical (Z), and time axes. A
human bronchial epithelial culture is imaged before, during, and after the application of a mucus load, during which
time the cilia are subject to compression and a change in the time spent in the recovery (R) and effective (E) strokes
(a). A single-column-over-time presentation (M-mode) shows ciliary beat before and during mucus invasion. The
cilia begin to beat more rapidly under pressure (b). A time-averaged, cross-sectional image of cilia shows unloaded
cilia beating in an uncompressed arc (c), while cilia beating under load are flattened (d). PCL compression and CBF
plotted over the same time axis show that the compression and CBF elevation occur simultaneously at the onset of
the mucus load (e). The amount of CBF elevation is dependent on the amount of PCL compression, with response
peaking at 0.8 m compression and decreasing below baseline at about 2 m compression (f). The effective and
recovery stroke distribution is altered under compression, with the mucus causing the cilia to spend a greater ratio of
time in the effective state (g). (Adapted from Liu et al.
6
volume, ciliary beating, and
transport rate. Furthering sci-
entific understanding of the
basic mechanisms of mucus
transport, including charac-
terizing the effects of diseases
such as CF, clarifies suitable
therapeutic targets for investi-
gation. For example, the find-
ing that the absence of bicar-
bonate transport is associated
with failure of mucus clear-
ance, despite adequate air-
way hydration in CF, suggests
a pathway to address the vis-
cosity or adherence of CF
mucus itself.
Furthermore, OCT could
help identify new drugs by
detecting beneficial changes
to cultured airway cell systems
induced by investigational sub-
stances, and quantitatively
characterize the functional
therapeutic effect of drugs
that have shown promise in the
laboratory or in human trials.
OCT can also characterize
the airway function of new CF
animal models to determine
their suitability for represent-
ing human CF airway disease
features, as recently done for
the new CF rat.
7
And now, OCT endoscopic
probes are being developed to
characterize the same bench-
marks of airway function in liv-
ing patients. In vivo imaging
will allow direct characteriza-
tion of the progression of CF
lung disease, mitigating the
need for cell culture and ani-
mal models.
In addition, OCT has potential
as a clinical tool for diagnosing the
severity of lung disease and monitor-
ing responses to treatment. Besides
CF, other common respiratory diseases
that affect the airway epithelium or
the mucociliary transport apparatus
for instance, primary ciliary dyskinesia
(PCD) or chronic obstructive pulmo-
nary disease (COPD)can be investi-
gated by OCT imaging.
ACKNOWLEDGEMENT
Figures 2, 3, and 4 are reprinted and
Figure 5 is adapted with permission
from the American Thoracic Society.
Originally published in references 4
and 6. Copyright 2014 American Tho-
racic Society.
REFERENCES
1. L. Liu et al., Nat Med., 17, 10101014
(2011).
2. L. Liu et al., PLoS One, 8, e54473 (2013).
3. S. M. Rowe, S. Miller, and E. J. Sorscher,
N. Engl. J. Med., 352, 19922001 (2005).
4. S. E. Birket et al., Am. J. Respir. Crit. Care.
Med., 190, 421432 (2014).
5. P. M. Quinton, Am. J. Physiol. Cell Physiol.,
299, C1222-33 (2010).
6. L. Liu et al., An autoregulatory mecha-
nism governing mucociliary transport is
sensitive to mucus load, Am. J. Respir. Cell
Mol. Biol., in press (2014).
7. K. Tuggle et al., PLoS One, 9, e91253
(2014).
1409bow_20 20 9/4/14 3:43 PM
Absorption (log scale)
Wavelength (nm)
Diode
Alexandrite
300 500 700 1000 2000
Ruby
Pigmented lesion
Melanin
Oxyhemoglobin
GaN GaP GaAs InP GaSb
Bulk tissue
Nd: YAG
Water
Vessel Vessel
Vessel
M E D I C A L A N D A E S T H E T I C L A S E R S
By St ewar t W. Wi l son
Semiconductor diode
laser advances enable
medical applications
Advances in semiconductor diode laser sources are facilitating the migration of medical
and aesthetic lasers to consumer markets. Semiconductor diode lasers offer advantages
over other light sources for applications in dermatology, dentistry, and more
and continued advancements promise to make them increasingly compelling.
W
hile medical and aes-
thetic lasers have been
largely limited to pro-
fessional markets, tech-
nological advances in semiconduc-
tor diode sources are facilitating their
migration to consumer markets. The
U.S. Food and Drug Administration
(FDA) approved the first- ever high-
power diode laser product for hair
removal in 1997 for the professional
market. Nine years later, the com-
pany that had won the approval, Palo-
mar Medical Technologies (acquired
in 2013 by Cynosure [Westford, MA]),
received FDA clearance for an over-the-
counter (OTC) product for home use.
In 2009, the company was the first to
receive FDA clearance for a semicon-
ductor laser-based OTC device for wrin-
kle removal, which it released to the
consumer market in 2010. Advances in
diode laser technology made both of
these cases possible.
Selective photothermolysis
and common chromophores
What makes a light source a good choice
for a given application is the ability of its
emitted light to interact with the tissue
so as to achieve the desired effect. This
is known as selective photothermolysis.
Selectivity is accomplished by matching a
specific wavelength of light to a chromo-
phorethat is, the light-absorbent part
of a moleculein the tissue. During the
absorption process, an electron is raised
to its excited state from a ground state. In
biological molecules that serve to capture
light energy, the chromophore is the moi-
ety that causes a conformational change
of the molecule when hit by light. The
energy directed into the target area pro-
duces sufficient heat to damage or alter
FIGURE 1. Each of the major biological chromophores has an absorption spectra. The wavelength of
light needed to activate each of these correlates with a semiconductor laser material. Above are shown
typical solid-state lasers used for these wavelengths. Penetration depth of light is strongly dependent
on wavelength, and must be considered when determining the energy for a particular application.
STEWART W. WILSON is a principal partner of Integrated Engineering Consulting LLC,
Burlington, MA; e-mail: stewart@iecteam.com
1409bow_21 21 9/4/14 3:43 PM
PuIsew!dth < IkI
TRT
d
2
cm
2
sec
K
*

IhermaI reIaxat!on t!me (IkI}

(sec)
where:
0.1
8 K 24 (Geometric factor)
Iyp!caI IkI ranges:
- 0.005 mm melanosome 0.001 ms
- 0.01 mm vessels 0.010 ms
- 0.1 mm vessels 1 ms
- Hair follicle 10 200 ms
- 5 mm of subcutaneous fat 1 min
( ((Thermal diffusivity of tissue)
Penetration depth (mm)
Wavelength (nm)
10
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500
9
8
7
6
5
4
3
2
1
0
Dermis
Subcutaneous fat
M E D I C A L A N D A E S T H E T I C L A S E R S c o n t .
the target while allowing the surround-
ing area to remain relatively untouched.
There are three light-related knobs
to adjust when employing selective
photothermolysis: wavelength, pulse
duration, and energy level. As already
described, the wavelength chosen assists
in selective absorption of light by the tar-
get vs. the surrounding tissue. The pulse
duration should be significantly shorter
than the targets thermal relaxation time
to avoid interaction with the area out-
side of the target. And the light energy
selected should be higher than the dam-
age threshold of the target. Proper selec-
tion of these critical parameters ensures
the desired results.
Common tissue chromophores tar-
geted in dermatology applications are
water (which makes up 70 percent of tis-
sue), hemoglobin (blood), melanin (in
the epidermis, pigmented lesions, and
hair), lipids (subcutaneous fat and seba-
ceous glands), and protein (specifically,
collagen). Each of these has a specific
absorption spectra that corresponds to
a semiconductor laser material able to
produce the matching wavelength (see
Fig. 1). And certain solid-state lasers
used correlate with the materials and
wavelengths. Because penetration depth
depends highly on the wavelength of
light, it must be considered when deter-
mining the energy needed for a particu-
lar application (see Fig. 2).
For effective treatment and to pre-
vent interaction with the surrounding
area, the pulse width should be suffi-
ciently shorter than the thermal relax-
ation time (TRT) of the targeted chro-
mophore. While there is no substitute
for testing and experimentation, Fig-
ure 3, which provides guidance for
determining approximate pulse width,
can serve as a starting point. The single
limiting factor for using semiconduc-
tor lasers is high peak power. Improve-
ments in this area make their use quite
compelling since semiconductor lasers
also offer performance in the other
parameters.
Light source effectiveness
Semiconductor laser light sources
have many advantages over other light
sources for a variety of medical appli-
cations. Emphasis is on conversion effi-
ciency, temperature sensitivity, and
cost rather than beam quality and
brightness, which tend to be the driv-
ing factors for applications outside
biomedicine.
Lamps generate polychromatic diver-
gent light, whereas lasers generate mono-
chromatic light beams. Using f lash
lamps to target chromophores requires
use of optical filters to narrow the spec-
tral widthand reduces conversion
efficiency to 1020 percent. Addition-
ally, a flash lamp-pumped laser has a
conversion efficiency of around 110
percent. Semiconductor-based lasers, on
the other hand, offer electro-optic con-
version efficiency of 4065 percent for
wavelengths between 780 and 2000 nm.
Recent advances have enabled efficien-
cies as high as 72 percent in the 780
1100 nm range, and 80 percent is theo-
retically possible.
Using a more efficient light source
has two major advantages: The power
FIGURE 2. Depth of light penetration into tissue depends on wavelength and is limited by scattering
and absorption.
FIGURE 3. For effective treatment, the pulse duration should be sufficiently shorter than the thermal
relaxation time (TRT) of the targeted chromophore to allow selectivity from surrounding tissue.
Typical optical fluence needed for effective treatment targeting is between 1 and 100 J/cm
2
; the
corresponding spot size ranges from 0.1 to 10 cm
2
. Typical durations for common treatments include
0.001 ms for a melanosome (the organelle responsible for melanin in animal cells) to 1 min for 5 mm of
subcutaneous fat.
1409bow_22 22 9/4/14 3:43 PM
Mpa
Tooth surface after acid etching
Tooth surface after laser processing
d)
a) b)
c)
25
20
15
10
5
0
Self etch
60%
Self etch and superBonding
Shear bond
strength
Reduce the time and cost of expensive prototyping with Zemax OpticStudio 14.2 optical design software. Simulate optical system performance
and predict manufacturing impact using advanced software features engineered for the way you work:
DeLailed uorescence modeling or radiology
Model light scatter in biological tissue
Confocal image modeling for better imagery
World-class CAD support
Test drive it now at www. Zemax.com/demo
Design biomedical imaging systems, reduce cost, and get to market faster.
supply needed to gen-
erate the optical energy
is significantly less, and
the required cooling sys-
tem is smaller because
there is less waste heat.
Both factors allow for
a more compact, cost-
effective design. As an
example, in comparing
a system having a 25-per-
cent vs. 65-percent con-
version efficiency and
producing the same
optical power, the lower
efficient system would
requi re ~2X more
electrical power and
produce ~6X more
waste heat.
Semiconductor lasers
can produce a variety of
monochromatic wave-
lengths and generate
significant optical power
FIGURE 4. A compact multi-wavelength dental handpiece is designed for both soft tissue treatments and hard, including
etching of dental surfaces (a). Acid etching of dental surfaces is prone to failure of dental restoration (b), while laser
etching (c) produces more reliable outcomes (d).
1409bow_23 23 9/4/14 3:43 PM
in a small, compact footprint. By
contrast, sources such as lamps
tend to be not only inefficient, but
also clumsyand unable to focus
on spot sizes smaller than 5 mm,
which practically eliminates their
use for tissue cutting and fiber
delivery. Additionally, lamps are
unable to generate pulses shorter
than 1 s, which makes them
unsuitable for applications such as
tattoo removal.
While gallium arsenide (GaAs)-
and indium phosphide (InP)-
based semiconductor lasers
emit quite well, gaps have tradi-
tionally existed between their wave-
length ranges. In recent years, though,
more wavelengths have become
available, and, for instance, 1200 nm
(in the gap between GaAs and InP)
has been demonstrated. Likewise, a
system using gallium antimonide
(GaSb) to generate type-I lasing (that
is, typical laser operation in which
light emission is generated by transi-
tion from the conduction to the valence
band) in the 20003000 nm range has
been shown to produce optical power
between 0.5 and 2 W from a single
device. These wavelengths are ideal for
applications involving both soft and
hard tissue, such as epidermal ablation,
drug delivery, and dental treatment.
Lastly, with the advent of type II inter-
band and quantum cascade lasers (in
which light is generated by transitions
from a single level within the conduc-
tion band), even longer wavelengths
beyond 3000 nm (that is, mid IR)are
possible. It will be interesting to see
what medical applications emerge for
these longer wavelengths.
Direct and indirect confgurations
Medical applications using semiconduc-
tor lasers as the main light source can be
configured either directly (non-pump-
ing) or indirectly (pumping)the differ-
ence being whether or not light is being
delivered directly to the treatment site.
Indirectly configured semiconduc-
tor lasers are commonly used as optical
pump sources for solid-state crystals
known as diode-pumped solid-state
(DPSS) lasers. Another example of an
indirect configuration is a diode-pumped
fiber laser, which is also a solid-state
laser, but in this case, the pumping gain
medium is optical fiber. Choosing one of
these would depend on the gain medium
used and the wavelength needed.
A compact DPSS laser pen-like hand-
held dental device has been shown to pro-
duce multiple wavelengths simply by chang-
ing the pulse width of the semiconductor
pumping laser (see Fig. 4). It produced five
distinct wavelengths (2.66, 2.71, 2.81, 2.83
and 2.85 m) using only a single compact
semiconductor laser pump module lasing
at one discrete wavelength: 975 nm. This is
of particular interest for soft- and hard-tis-
sue applications that typically involve five
different lasers for use in deep soft-tissue
coagulation (2.66 m, holmium laser), cut-
ting (2.71 m, CO
2
laser), and hard- and
soft-tissue ablation (2.81, 2.83, and 2.85
m; Er:YAG laser) with minimal thermal
damage (see Fig. 5).
An example of a hard-tissue application
would be in the creation of ultra-retentive
dental surfacesthat is, the preparation of
tooth surfaces to improve bonding with den-
tal prostheses or composites. Retentive den-
tal surfaces are conventionally formed using
acid etch, which creates inconsistent surface
structures and frequently results in the fail-
ure of dental restorations, especially those
bonded to dentine. This failure rate can
be as high as 40 percent. Using a laser for
this same application creates a super-reten-
tive bond on dentine and sclerotic dentine,
and produces a 60-percent improvement in
bond strength to dramatically reduce bond-
ing failures, especially for dentine.
Some of the first direct-diode prod-
ucts to enter the consumer market have
been demonstrated by Palomar Med-
ical Technologies (with the PaloVia
skin-renewing laser system in 2010) and
Tria Beauty (then SpectraGenics, Dub-
lin, CA, with the Tria hair removal sys-
tem in 2008). PaloVia is the first home-
use semiconductor laser device that is
FDA-cleared for removing fine lines and
wrinkles. This battery-operated, hand-
held device delivers 1410 nm in small spot
patterns to the skin, creating a fraxel
pattern of damage sites and thus initiat-
ing the bodys natural healing response
FIGURE 5. Soft- and hard-tissue applications typically involve five different lasers.
Medical applications using semiconductor lasers
as the main light source can be confgured either
directly (non-pumping) or indirectly (pumping)
the difference being whether or not light is
being delivered directly to the treatment site.
Wavelength
(m)
Coeffcient of
absorption (cm
-1
)
System
currently used
Clinical
application
2.66 210 Ho laser
Deep soft-tissue
coagulation and cutting
Hard- and soft-tissue
ablation with minimal-
residue thermal damage
2.71 760 C0
2
laser
2.81 7260 Er:YAG
2.83 8480 Er:YAG
2.85 9470 Er:YAG
1409bow_24 24 9/4/14 3:43 PM
PHONE: 415. 883. 0128 | FAX: 415. 883. 0572 | EMAI L: I NFO@SUTTER. COM | WWW. SUTTER. COM
MOM: 2-Photon Movable Objective Microscope
Versatile The Movable Objective Microscope (MOM) is two
independent microscopes in one. Use the wide eld
setup for epiuorescence, or add a Ti- Sapphire laser for
2- photon imaging.
Flexible Its unique 3- dimensional objective movement and
rotation allows the specimen to remain stationary and
the scope to convert from an upright
to an inverted microscope.
Adaptable We constantly work with highly
regarded imaging labs around the
world to customize our open plaform
design to respond to advances in the
needs of researchers.
Contact Sutter Instrument to see how the MOM can open up new worlds of imaging for your research.
to generate more collagen while at
the same time removing the wrinkles.
Although Palomar was the first to gain
FDA clearance for a home-use semicon-
ductor laser for hair removal (2006),
the purchase of Gillette by Procter &
Gamble in 2005 significantly disturbed
relationship and focus, thus enabling
Tria Beauty to become the first and
only company to deliver a semiconduc-
tor laser hair-removal system to the con-
sumer market.
Impact for the future
An example of a direct- diode laser
product that makes evident the signif-
icant improvements in semiconductor
laser technology is the Vectus Laser
designed by Palomar Medical Technol-
ogies in 2012. With Vectus, Palomar
took direct aim at Lumenis (Yokneam,
Israel) LightSheer hair-removal sys-
tem for the professional market (which
Palomar actually designed in 1997
and sold to Coherent in 1999). Both
systems emit at ~808 nm, but the
Vectus uses an advanced design that
requires four times fewer diode laser
bars and produces significantly more
optical energy in a wider treatment
range. In a split-body direct- compari-
son study, the Vectus significantly out-
performed LightSheer. This is not to
trivialize the success of LightSheer at
all; rather, the fact that LightSheer has
been on the market since 1997 con-
firms the importance of using semi-
conductor laser technology for medi-
cal applications.
Semiconductor diode lasers already
offer many advantages over other
light sources for medical applications.
Improvements in electro- optic con-
version efficiency, optical power, and
wavelength options make the technol-
ogy ideal for targeting chromophores.
Continued advancements will make it
increasingly compelling.
REFERENCES
1. M. Inochkin et al., High efficiency
diode pumped Er:YLF laser with
multi-wavelength generation, Proc.
SPIE, 8234, 8234- 4, session 1 (Jan.
22, 2012).
2. S. Wilson et al., Long pulse compact
and high brightness near 1-kW QCW
diode laser stack, Proc. SPIE, 8241,
8241-14, session 3 (Jan. 22, 2012).
3. L. Shterengas et al., High-power 2.3-
um GaSb-based linear laser array,
IEEE Photon. Technol. Lett. (2004).
4. A. Samad-Zadeh et al., The inf lu-
ence of laser-textured dentinal sur-
face on bond strength, American
Dental Association, 2005-2007, IADR
(2009).
5. J. Lowery et al., Comparative study
between the Vectus diode laser sys-
tem and LightSheer Duet for long-
term hair reduction in the axilla,
Palomar Medical Technologies white
paper (2013).
1409bow_25 25 9/4/14 3:43 PM
Cell
_
+
i
V
a) b) c)
Cell
t
Electrolyte
C Y T O M E T R Y / C E L L A N A L Y S I S
By Et han Schonbr un
Optical cell volume measurement
The ability to measure cell volume enables greater understanding of biological processes, including
disease development. A new method based on light absorption enables high throughput, less sample
processing, and a simpler setup. Plus, it allows for multiplexing, which could produce greater insights.
C
ell volume is a
critical parame-
ter in both biol-
ogy and med-
icine: Understanding cell
growth and death, quanti-
fying intracellular concen-
trations of ions and pro-
teins, and diagnosing most
hematological disorders all
require accurate measure-
ment of cell volume.
Optical technology offers
many benefits for cytome-
try, but obtaining an accu-
rate optical measure of cell
volume is challenging.
1
Often, cells have
complex, three-dimensional shapes and
are composed of heterogeneous materi-
als that have various optical properties.
In addition, because there is consider-
able cell-to-cell variability in a popula-
tion, making assumptions based on aver-
age cell properties is of limited value.
A clever solution for finding the vol-
ume of an object with a complex shape
and composed of an unknown material
was proposed by Archimedes more than
2000 years ago. By submerging the object
in a fluid-filled container, the displaced
volume can be easily measured and the
measurement corresponds absolutely to
the object volume (see Fig. 1a). As long
as the object is not porous, the displaced
volume is nearly independent of the phys-
ical properties of the object. The volume
displacement method works effectively
even on objects with extremely complex
three-dimensional shapes that would be
incredibly difficult to quantify with any
other method. These same advantages
make volume displacement appealing for
measuring cell volume.
2
Not coincidently, an electrical ana-
logue to the volume displacement prin-
ciple, called the Coulter counter, has
become the most widely used method
for quantifying cell volume. Cells are
immersed in a conducting electrolyte
and the suspension is passed through a
small aperture (see Fig. 1b). A constant
current is applied across the aperture
and voltage pulses are obtained as cells
traverse the opening. If the cells are
non-permeable to the electrolyte and
assumed to be insulators, the voltage
pulse height is proportional to the cell
volume. In order to retrieve an accurate
absolute volume, however, a shape fac-
tor needs to be used: While the method
is nearly independent of the cells elec-
trical properties, it is not (unlike Archi-
medes displacement) completely inde-
pendent of a cells geometry.
An optical Coulter counter
Scientists at Harvard Universitys Row-
land Institute have developed a volume
displacement method based not on dis-
placed fluid volume or free electrons, but
on light absorption.
Cells are immersed in a buffer that
contains an absorbing dye, and, in direct
analogy to the electrical Coulter counter,
are passed through a microfluidic chan-
nel with a finite height. The presence of
a cell in the field of view displaces a num-
ber of dye molecules that is proportional
to the cell volume and the dye concen-
tration. The concentration of the dye
FIGURE 1. Methods of measuring displacement include Archimedes approach, which yields the object volume
independent of the objects properties or geometry, as in the case of a kings crown composed of unknown materials
(a). An electrical Coulter counter uses displaced free electrons in an electrolyte and a measurement of voltage to
determine cell volume (b). The optical Coulter counter uses the displacement of dye molecules and optical transmission
to measure cell volume (c).
3
ETHAN SCHONBRUN, Ph.D., is a junior fellow and the principal investigator of the Optofluidics
Cytometry Group at Harvard Universitys Rowland Institute; e-mail: schonbrun@rowland.harvard.
edu; http://www2.rowland.harvard.edu/book/ethan-schonbrun.
1409bow_26 26 9/4/14 3:43 PM
Volume (fL)
e)
c) d)
a) b) f)
Volume (fL)
Height
(m)
150
8
6
4
2
0 m
100
50
0
500
(i) (ii)
5 m 5 m
(iii) (iv)
(i) (ii)
(iii) (iv)
10001500 2000 2500 3000 3500
150
100
12
10
8
6
4
2
0
50
0 500 10001500 2000 2500 3000 3500
673 fL
(i)
(ii) 971 fL
(i) 1046 fL
(ii) 1375 fL
N = 552
(iii) 2364 fL
(iv) 3028 fL
N = 648
(iii) 1792 fL
(iv) 2926 fL
C Y T O M E T R Y / C E L L A N A L Y S I S c o n t .
can be measured before beginning the
experiment and is the only parameter
that needs to be known a prioriunlike
the samples shape factor in the Coul-
ter counter.
For an object that does not scat-
ter light, a single-intensity measure-
ment would be enough to reconstruct
an accurate volume estimate from the
measured intensity. Cells do scatter
light, however, and the measured inten-
sity has scattering contributions in addi-
tion to dye exclusion. We have mini-
mized the contribution from scatter-
ing in two ways. First, we added bovine
serum albumin (BSA) to raise the
refractive index of the absorbing buffer
to approximately match that of the cell.
In addition, we image each cell with
a second color that is not absorbed by
the dye and consequently can be used
to subtract out the remaining scatter-
ing component.
In this setup, a color camera mea-
sures at a throughput of approximately
1000 cells per minute. Figure 2 shows
results for optical displacement imag-
ing of leukemia cells and a compar-
ison of volume distributions for two
different strains. Intracellular scatter-
ing is nearly eliminated (see Fig. 4d),
and the height map does not suffer
from speckle, haloing, shade- off, or
any other artifacts commonly found in
quantitative phase microscopes.
No need for sphering
The most common application of cell
volume measurements is in hematology
where complete blood cell counts quan-
tify red blood cell volume and distribu-
tion width. Using these measurements,
hematologists can diagnose whether the
patient has macrocytic (in which cells
are too large) or microcytic (cells are too
small) anemia.
Current clinical hematology ana-
lyzers use optical scattering to quan-
tify both cell volume and hemoglobin
concentration. But because scattering
couples geometry to optical proper-
ties, cells are required to be sphered
(that is, processed to swell from disc-
to sphere-shape) before measurement.
Departure from a spherical shape pro-
duces artifacts in measurement that
lead to inaccurate results.
Instead, we have applied the optical
displacement method to measure cell vol-
ume while cells are in their natural, non-
spherical state. In addition, we have com-
bined volume displacement with a simul-
taneous hemoglobin absorption measure-
ment to quantify single cell hemoglobin
mass.
4
Our results are similar to those
obtained with a clinical hematology ana-
lyzer, Siemens Advia 2120, but we mea-
sure a smaller coefficient of variation of
hemoglobin concentration (see Fig. 3).
Studies have demonstrated that hemoglo-
bin concentration is tightly regulated in
the body
5
and a more narrow distribution
of hemoglobin concentration might more
accurately represent the actual physiolog-
ical state.
Multiplex-able
Another major strength of an opti-
cal technique, compared to an elec-
trical one, is that it is straightforward
to multiplex with other optical tech-
niques. In our tests, we combined this
method with optical absorption to cre-
ate a method for measuring red blood
cell volume and hemoglobin massa
method that we believe is more accu-
rate than the clinical standard. In addi-
tion, cell volume measurements can be
matched with a fluorescence image or
signal to correlate volume and organ-
elle morphology.
Figure 4 shows thickness maps of neu-
trophils that are captured simultane-
ously with fluorescence images of their
nuclei. Neutrophil volume distributions
have been linked to early detection of
FIGURE 2. In optical displacement imaging, color images using blue and green illumination are collected by a color camera (a). The green channel is
used to compensate for intracellular scattering (b), while the blue channel contains contributions from both volume displacement and scattering (c). After
processing, a thickness map can be retrieved where contributions from scattering have been removed (d). Measurements enable display of the thickness
map and volume distribution for 648 HL60 leukemia cells (e) and 552 K562 leukemia cells (f).
3

1409bow_27 27 9/4/14 3:43 PM
Hb mass (pg)
Volume (fL) Volume (fL)
Hb concentration (g/dL)
a) c)
b)
60
50
40
30
20
10
55
45
50
40
35
30
25
20
0
50 100
0
Advia
QAC
50 100 150 150
20
15
10
5
0 fg
40
30
20
10
0 fg
2.0 50
40
30
20
10
0 fg
3
2
1
(i)
(i)
(i)
(ii)
(ii)
(ii)
(iii)
(iii)
(iii)
(iv)
(iv)
(iv)
1.5
1.0
0.5
0m
3.0
2.0
1.0
0m
0m
A1 A2
5m 3m 3m
3m
3m
Advia
QAC
5m
C Y T O M E T R Y / C E L L A N A L Y S I S c o n t .
infection.
6
Nuclear morphology of neu-
trophil cells has also been studied in
the context of infection due to the fact
that nuclei become more segmented
as the cell ages. We have begun a proj-
ect to correlate these two behaviors in
blood samples. A better measurement of
cell age could have major implications in
the early detection of infection when the
body is trying hard to make more white
blood cells.
Simpler and full of potential
By reducing and correcting for scatter-
ing, we have enabled this optical method
to be, like Archimedes approach, nearly
invariant to both the objects shape and
physical properties. Unlike volume mea-
surements based on optical scattering
and interferometry, the optical Coulter
counter does not rely on a priori knowl-
edge or measurement of a cells refractive
index, which results in a significant sim-
plification of the system.
We are also investigating correla-
tions between cell volume and nuclear
morphology in white blood cells in the
hope of building a diagnostic system
for the early detection of infection.
The ability of optics to multiplex mea-
surable quantities will further enable
cell volume to be correlated with a host
of other biochemical and morphologi-
cal cell properties.
REFERENCES
1. A. Tsur, J. K. Moore, P. Jorgensen, H.
M. Shapiro, and M. W. Kirschner, PLoS
One, 6, e16053 (2011).
2. W. H. Grover et al., Proc. Nat. Acad. Sci.,
108, 1099210996 (2011).
3. E. Schonbrun, G. Di Caprio, and
D. Schaak, Opt. Exp., 21, 87938798
(2013).
4. E. Schonbrun, R. Malka, G. Di Caprio,
D. Schaak, and J. M. Higgins, J. Cytome-
try, 85, 332338 (2014).
5. J. M. Higgins and L. Mahadevan,
Proc. Nat. Acad. Sci., 107, 2058720592
(2010).
6. F. Chaves, B. Tierno, and D. Xu, Am. J.
Clin. Pathol., 124, 440444 (2005).
FIGURE 3. Complete blood counts analyze red blood cell volume and hemoglobin mass and are one of the most frequent clinical tests. To measure both these
parameters, the system uses two-color absorption, where red light is used to retrieve cell volume and blue light retrieves hemoglobin mass (a). Color, thickness,
and mass maps represent a discoid and parachute red blood cell (b). A comparison of the optics-based system (red) and a clinical hematology analyzer (blue)
demonstrates that in addition to volume and mass, the optical system captures images of every cell, which enables study of the morphology of outliers (c).
4
FIGURE 4. Images of height maps of neutrophil cells collected by the optical Coulter counter are shown in red. The nuclear morphology is also
simultaneously observed using a nucleic acid fluorescence stain, Syto16, shown in blue.
1409bow_28 28 9/4/14 3:43 PM
3 D O P T I C A L M O L E C U L A R I M A G I N G
By Mi ke May
Developing drugs in 3D
New 3D imaging tools and techniques are enabling drug development with multiple, simultaneous
views; tracking of multiple targets; greater depth and resolution; multimode operation; and in vivo
monitoring. In doing so, they provide promise that tomorrows medicines will be safer and more effective.
T
hree-dimensional (3D) imag-
ing is changing biology
and changing the world of
drug development. This
truth becomes clear in talking with 3D
imaging specialists like Jacob G. Tes-
dorpf, director of high-content instru-
ments and applications for life sciences
and technologies at PerkinElmers facil-
ity in Hamburg, Germany. For Tesdorpf,
biology intertwines with drug discovery,
and he sees 3D imaging used here in two
ways. A scientist can look at a single cell
or something made of many cells, from a
cell culture to a small model organism,
like a zebrafish. If you image in 3D, he
explains, you can tell the spatial rela-
tionship of the components, regardless
of scale. For example, two structures
might appear side by side in two dimen-
sions, but 3D could reveal that they actu-
ally lie far apartat least in terms of cel-
lular space.
Even cells themselves know that 3D is
better. That is, cells live in 3D environ-
ments in the body. As a result, 3D culture
tells scientists more about how cells really
behave. As Tesdorpf says, 3D cell culture
is a better proxy for how cells really func-
tion and how they interact with neigh-
boring cells. To see those 3D effects in
action, scientists need tools that provide
3D images.
Tracking multiple targets
In drug development, a scientist might
want to track more than one targetsay,
a couple of proteinsin 3D, and look
for interactions between those targets.
To get the most from drug-development
experiments, that researcher must quan-
tify the imaging results.
The recent Opera Phenix High
Content Screening System from
PerkinElmer provides a high-through-
put approach to 3D confocal imag-
ing to track fluorescent targets in
microplates. This platform uses a
scientific CMOS (sCMOS) camera
that captures larger structures
three times larger than the plat-
form that it updatesin a single
image (see Fig. 1). A scientist can
even deck out this system with four
cameras to get simultaneous images
from four fluorophores. Moreover, this
system uses objectives from Carl Zeiss
(Oberkochen, Germany), which pro-
vide large apertures and water immer-
sion. With water-immersion theres not
as much change in the refractive index,
says Tesdorpf, so the resolution remains
high as you move deeper into a sample.
How deep, though, really depends on the
sample at hand. Here, deep might only
mean 80 m, but that gets paired with the
ability to quickly explore many samples.
Matching modalities
In addition to tracking multiple targets,
some medical researchers want to image
a sample in more than one way. For exam-
ple, the new InSyTe series from TriFoil
Imaging (Chatsworth, CA) allows drug
developers to combine optical imaging
with other modalities, such as positron
emission tomography (PET) and com-
puted tomography (CT). As TriFoil CEO
Kevin Parnham explains, If you can
image a subject in vivo, you can watch
the progress of a treatment or the effi-
cacy of a drug. To have any idea of the
volume that youre looking at, you need
to image in 3D. With the InSyTe system,
a researcher can label a chemical in the
body and then see where it gets taken up
in a sample.
Some scientists will desire different
combinations of modalities than oth-
ers. To accommodate such a variety
of uses, the InSyTe comes with a single
modality or as many as three. It can also
be upgraded later if needed. The opti-
cal modality provides 3D fluorescent
FIGURE 1. In this 3D InSight tumor microtissue
from InSpheroa supplier of 3D microtissues for
predictive drug testingan image captured with
the Opera Phenix shows expressed GFP (green)
and Hoechst dye (blue) stains the nuclei. (Image
courtesy of InSphero)
1409bow_29 29 9/4/14 3:43 PM
pco.edge gold
from the pioneers
in sCMOS
image sensor technology
deep
cooled
www.pco.de
www.pco-tech.com
37 000 : 1
intrascene
dynamic
on the
cutting
edge
0.8 e
-
read out
noise
3 D O P T I C A L M O L E C U L A R I M A G I N G c o n t .
imaging. The systems laser rotates
around the sample and captures image
information at 49 positions. For depth,
the system samples in 1 mm steps. The
systems software combines the images
to build a 3D representation of the sam-
ple. Moreover, the system accommodates
organisms from neonatal mice to small
rats, up to about 300 g for the latter.
Although the InSyTe provides
advanced imaging modalities, it does not
require a highly controlled environment
for use. Scientists can place the InSyTe
on a benchtop in an ordinary lab. It can
tolerate a wide range of temperatures,
Parnham explains.
Creating combinations
A combination of hardware and software
makes up the LumiQuant from Aspect
Imaging (Toronto, ON, Canada). This
system consists primarily of a miniatur-
ized magnetic resonance imaging (MRI)
platform, which can be used without the
cooling and shielded rooms needed for
larger superconducting MRI systems.
The LumiQuant integrates with leading
luminescence platforms, including the
IVIS technology from PerkinElmer, to
bring in optical capabilities. For example,
the LumiQuant allows MRI plus biolumi-
nescent imaging (see Fig. 2). The com-
bination of hardware and software in the
LumiQuant, says Robert Sandler, senior
VP of marketing, allows us to look at an
optical bioluminescent signal localized
in 3D with exquisite morphological ref-
erence from the compact MRI.
LumiQuant collects an MRI and
optical signal from the same sample
by using a cassette. A mouse, for exam-
ple, can be anesthetized, placed in the
cassette, imaged with compact MRI,
and then transferred to an optical sys-
tem for bioluminescent imaging. We
use the MRI image for 3D morphologi-
cal imaging, and then apply organ-spe-
cific filtersfrom an atlasto the bio-
luminescence to detect elements of a
disease at the molecular level and with
organ- specific attenuation factors,
Sandler explains.
Although the LumiQuant
system provides high-powered
imaging options, users do not
need extensive training to use
it. The optical platform is lit-
erally push-button technology,
Sandler says. The compact MRI
side is a little more complicated,
but our users are usually biolo-
gists and not MRI experts. He
adds, With only marginal train-
ing, biology researchers and stu-
dents can generate and quantify
images on the Aspect Imaging
M3 compact MRI platform.
Turning high-tech imag-
ing capabilities into far more
user-friendly tools will surely
entice more basic researchers
and industrial scientists to go
3D in medical research. By col-
lecting an added dimension in
drug research, scientists will get
a better understanding of dis-
ease mechanisms, find safer and
more effective medicines and,
thereby, create new treatments
that go far beyond todays medi-
cal options.
FIGURE 2. LumiQuant can combine and co-register 3D

images from luminescence and compact MRI. (Image courtesy
of Aspect Imaging)
1409bow_30 30 9/4/14 3:43 PM
S P E C T R A L I M A G I N G
By J ohn Wal l ace
Imaging spectrometers
look at life in two ways
Spectral imaging is finding more and more applications in life sciences, from
noninvasive disease diagnosis to food processing. Various imaging spectrometers
make those applications possibleand promise more for the future.
I
maging spectroscopy combines the
chemical detection capabilities of
spectroscopy with the image-mak-
ing of camera visualization to
reveal both chemical signature and spa-
tial structure. The techniqueknown
variously as chemical imaging, spectral
imaging, multispectral imaging, hyper-
spectral imaging, and Raman imaging
is enabled by a subset of optical spectrom-
eters called imaging spectrometers.
Indeed, while optical spectrometers
can be classified in any number of ways
(for instance, by size, wavelength range,
or optical configuration, or whether they
work via absorption, emission, trans-
mission, or scattering), one of the most
fundamental divisions is between imag-
ing and nonimaging varieties. If youre
analyzing a uniform sample or look-
ing at the spectral properties of a single
light source, then nonimaging is fine.
But if you need to get spectral details
for every point across a scene or a medi-
cal specimen, then you need an imaging
spectrometer.
Imaging spectrometers come in dif-
ferent formsfor example, multi- or
hyperspectral instruments, which often
characterize externally illuminated
objects such as those under a microscope;
and Raman instruments, which use an
internal laser to illuminate the object or
area under test, then analyze the result-
ing Raman-scattered (and wavelength-
shifted) light.
At least some of the technology inside
a particular spectrometer, which includes
the optics, the type of sensor array, the
electronics, and the software, is at least
partly unique to the instruments man-
ufacturer. Many imaging spectrome-
ters are tweakedor even specifically
designedfor a certain application.
Because such technology is often specific
to an instruments maker, this article will
cover the topic of imaging spectrometry
by focusing on a few instrument manufac-
turers one at a time.
Many confgurations for science
In science, particularly the biosciences,
imaging spectroscopy finds use in micro-
spectroscopy, multichannel plasma anal-
ysis, photoluminescence and electro-
luminescence, Raman microscopy and
spectroscopy, time-domain-fluorescence
imaging microscopy, time-resolved
imaging and spectroscopy, fluorescence JOHN WALLACE is senior editor for Laser Focus World; e-mail: johnw@pennwell.com.
EDITORS NOTE: This article is adapted
from Laser Focus World, 50, 1, 7076
(Jan. 2014).
FIGURE 1. The shape of Horibas iHR550 imaging spectrometer is dictated by its requirements.
1409bow_31 31 9/4/14 3:43 PM
S P E C T R A L I M A G I N G c o n t .
spectroscopy, and other applications.
Horiba JobinYvon (Edison, NJ) pro-
duces a range of imaging spectrometers
that are paired with the companys scien-
tific cameras.
Horiba designs and manufactures a
range of imaging spectrometers varying
in focal length from 70 to 1250 mm. As
diffraction gratings are a core technology
of Horiba JobinYvon, we have the ability
to optimize each design to best match
the experimental requirements of wave-
length range, resolution, throughput,
size, and cost, notes Joanne Lowy, Hori-
bas marketing manager.
The necessity for a large choice of
designs is driven by applications, cost,
and size constraints set by the research-
ers, says Lowy. In an example of one
design, Horiba produces Czerny-Turner
imaging spectrometers with fully aber-
ration-corrected reflective optics that
the company says are free of redif-
fracted light (the Czerny-Turner design,
the basis for many imaging-spectrome-
ter optical layouts, has two mirrors that
image an entrance slit onto an exit slit; a
diffraction grating between the mirrors
disperses the light). In another design,
Horiba offers supercorrected, concave,
holographic-grating-based spectrom-
eters in which the concave grating per-
forms the function of collimation, disper-
sion, and focus in one element.
The shape of Horibas iHR550 imaging
spectrometer is dictated by the require-
ments of the spectrometer, explains Lowy
(see Fig. 1). All-oversized reflective optics
eliminate chromatic aberration (their
large size is to provide high throughput);
toroidally corrected optics reduce aberra-
tions; and careful selection of the angle
of incidence eliminates rediffracted
light. The last is of particular impor-
tance, as systems that suffer from redif-
fracted light, such as criss-cross Czerny-
Turner spectrometers, will not produce
pure spectra at their output foci, which
can lead to erroneous spectra or images.
Unlike typical imaging spectro-
graphs, which measure over only a nar-
row spectral band and (due to their
optical design) one optical output, the
iHR550 has multiple entrance ports, mul-
tiple gratings with on-
axis rotation, and
multiple array exit
ports, with wide
focal planes acces-
sible outside the
instruments cast-
ing. With selec-
tion of appropri-
ate gratings, the
instrument has
a spectral range
from the deep-
ultraviolet (UV;
122 nm) to the
far-infrared (IR;
40 m).
Uses for the
iHR550 named
by Lowy include
multitrack plasma analysis (which
requires high resolution and imaging
quality); direct coupling to a microscope
for photon-starved applications such as
Raman spectroscopy; broadband-emis-
sion applications such as photolumines-
cence; and time-resolved applications
such as fluorescence-lifetime imaging
microscopy (FLIM).
Abolishing astigmatism
The Czerny-Turner configuration is not
simply a commonly used imaging-spec-
trometer design; it also serves as a spring-
board for inventive improvements. One
of these is a Czerny-Turner-based config-
uration developed by Princeton Instru-
ments (Acton, MA) that contains the
addition of a Schmidt corrector to its
optics; this is a transmissive plate with an
aspheric surface that corrects aberrations
(added to a telescope, a Schmidt correc-
tor plate is designed to eliminate spheri-
cal aberration).
The companys imaging spectro-
graph, the IsoPlane 320, has zero astig-
matism at all wavelengths across the
entire focal plane (see Fig. 2). When
used in multichannel spectroscopy, in
which a number of fiber-optic probes
(the channels) are placed across the
image field to collect information,
the astigmatism-free design allows up
to 200 channels, according to Jason
McClure, chief scientist at Princeton
Instruments and designer of the optics.
The design, which is patent-pending,
also boosts sensitivity by focusing more
photons onto fewer pixels.
FIGURE 2. Princeton Instruments
IsoPlane 320 has zero astigmatism at all
wavelengths across the entire focal plane.
FIGURE 3. BaySpecs OCI-1000 hyperspectral imager is able to serve as a
noninvasive diagnostic tool.
1409bow_32 32 9/4/14 3:43 PM
The IsoPlane 320 is used, for example,
in tip-enhanced Raman spectroscopy,
studies of carbon nanotubes, plasmonic
structures, and quantum dots. The ability
of the IsoPlane to image dozens of optical
fibers greatly enhances the spatial resolu-
tion available for imaging. In single-mol-
ecule tip-enhanced Raman spectroscopy
(SMTERS), both the sample and signal
are minuscule; the IsoPlane is the only
spectrograph to date that has successfully
been used in this technique, says Debby
Flint-Baum, marketing communications
manager at Princeton Instruments.
Using the IsoPlane 320 for this pur-
pose, the Van Duyne group at Northwest-
ern University has combined SMTERS
and scanning tunneling microscopy
(STM) to obtain unprecedented sensi-
tivity and spatial resolution in obtaining
Raman spectra of single rhodamine 6G
molecules, advancing the understanding
of excited-state dynamics in adsorbate-
substrate interactions.
1
Handheld hyperspectral
Hyperspectral cameras divide the light
spectrum into many small wavelength
bands (compared to traditional cam-
eras, which only have three bandsred,
green, and blue); spectrometers based
on this technology thus can give very
detailed information about the mate-
rial constitution of an imaged object.
Based on its own volume-phase-grating
(VPG) technology, BaySpec (San Jose,
CA) has developed two closely related
battery-powered handheld hyperspec-
tral imaging spectrometers that enable
field-based uses such as agriculture and
food inspection.
Both are VPG-based, which gives them
an efficiency of up to 99 percent, along
with high stability and repeatability, as
there are no moving parts. The instru-
ments can be configured for any spec-
tral range within 4001100 nm, with a
spectral resolution up to 0.01 nm. Cou-
pled with a high-speed camera, the sys-
tem can generate hyperspectral image
cubes with up to 2048 spectral bands in
a single scan.
BaySpecs handheld hyperspectral
imager, the OCI-1000, integrates the
spectral dispersing element (which has
more than 100 bands covering the 600
1000 nm range) on an image sensor at
the level of the chip itself, eliminating the
need for the large, expensive optics that
are used on traditional systems (see Fig.
3). The instrument weighs less than 0.5
lb. and can be installed almost anywhere,
says Lin Chandler, spectroscopy product
manager at BaySpec.
The similar (and, at 0.8 lb., just
slightly heavier) OCI-2000 snapshot
imager goes one step further, simulta-
neously dispersing 2D images into mul-
tiple spectral bands (currently 32 bands
covering the 6001000 nm range), elim-
inating the need for mechanical scan-
ning. Because a single snapshot pro-
duces an entire multispectral image
data cube, true multispectral imaging
can be done at video rates.
Chandler adds that one of the novel
applications in development for these
hyperspectral imagers is as a noninva-
sive diagnostic tool. For instance, such
an instrument can achieve an accurate
hyperspectral representation of the anat-
omy of any living organism without dis-
turbing it.
Photon conservation
By ef f icient l y maki ng use of
photons, an imaging spectrometer not
only shortens data acquisition times
it can also minimize damage to pho-
tosensitive samples. Andor (Belfast,
Ireland) is known for its low-light sci-
entific cameras; the company also
produces imaging spectrometers that
conserve photons.
The companys Czerny-Turner instru-
ments have motorized multiport, multi-
grating platforms with toroidal optics for
astigmatism correction of aberrations
inherent to Czerny-Turner design; these
spectrometers provide good multitrack
spectroscopy capabilities, as well as good
sample image relay at zero order in
microspectroscopy configurations (allow-
ing the sample to be visualized and spec-
tral analysis to be performed through
the same optical path with one detector),
according to Marion Mathieu, the com-
panys digital marketing executive.
Andor also makes instruments with a
transmission-optics configuration and
low-stray-light volume-phase-holographic
(VPH) gratings, resulting in high collec-
tion efficiency, throughput, and multi-
track-spectroscopy capabilities across a
large focal plane. The companys third
type of instrument is hyperspectral,
based on either a grism (combined
grating and prism) transmission configu-
ration or an aberration-corrected, reflec-
tive diffraction-grating configuration.
The instruments can be used in trans-
mission- or reflection-based absorption
spectroscopy; Raman spectroscopy at
wavelengths from the UV to the near-IR;
micro-Raman and microfluorescence;
photon counting; single-molecule spec-
troscopy (all requiring efficient use of
photons); and for plasma studies. Appli-
cations are many, including biomedical
FIGURE 4. Headwall Photonics hyperspectral imaging device (right) reveals spatial and spectroscopic
features of tissues, such as these cancerous kidney cells (left).
1409bow_33 33 9/4/14 3:44 PM
screening and diagnosis and analytical
cell biology.
As in any optical system, combining
high light throughput (low f-numbers)
with high resolution (low aberrations) is
challenging. Andors VPH-based trans-
missive-optics imaging spectrometer,
called the Holospec, is an on-axis, high-
throughput instrument with imaging-cor-
rected optics that allow both high-den-
sity, multi-track spectroscopy (clear sepa-
ration of individual channel images from
densely packed fiber-optic bundles) and
high throughput, says Mathieu. The f/1.8
aperture allows 100-percent light collec-
tion from fiber optics with a numerical
aperture (NA) of 0.22.
Up to 32 100-m-core fiber channels
can be individually resolved over a 4-mm-
high sensor with low crosstalk, despite
the high-density fiber-bundle configura-
tion, notes Mathieu.
Applications include intrinsically pho-
ton-starved experiments such as quan-
tum-dot photoluminescence and micro-
Raman of biosamples. The Holospec is
also used when acquisition time is a con-
straint (gathering enough photons in
short periods of time while maintaining
a meaningful signal-to-noise ratio)for
example, in microspectroscopic chemi-
cal mapping, microfluidics such as spec-
trally resolved flow cytometry, and on-
line process control. In addition, effi-
cient photon use minimizes photodam-
age of photosensitive samples such as
live cells or luminescent biotags, protect-
ing them from photodegradation and
phototoxicity.
Near and far
While spectrometers do perfectly well sit-
ting in labs and eyeing microscopic sam-
ples, they can also see the worldfor
example, from the vantage point of an
unmanned aerial vehicle (UAV). Head-
wall Photonics (Fitchburg, MA) makes
instruments that do either: The company
produces both hyperspectral and Raman
imaging spectrometers, with the hyper-
spectral version the one that gets to take
the airborne route.
The type of hyperspectral technology
we deploy is commonly known as push-
broom, meaning that motion needs to
occur, says David Bannon, Headwalls
CEO. Either the sensor moves about the
field of view (as it would in an aircraft), or
the scene moves beneath a stationary sen-
sor (as it would in an in-line inspection
deployment).
Headwalls hyperspectral instru-
ments have an athermalized design,
making them fully functional in
demanding environments such as those
that UAVs endure, and have almost no
moving partsagain, for reliable per-
formance while aloft. The instruments
have aberration- corrected imaging to
provide high spatial and spectral reso-
lution across a wide field of view. They
have excellent image fidelity off to the
sides, says Bannon.
Headwall makes its own diffraction
gratings that provide this aberration-cor-
rected imaging; this aids in designing
a package that tightly integrates optics,
electro-optics, electronics, and software,
but also aids in customization. The com-
pany also customizes its software when
needed, in some cases for the specific
application (for example, UAV platforms)
and in others for a specific customer (for
example, to identify foreign objects in
food processing).
Medical and biotech, one of four key
areas served by Headwalls imaging spec-
trometers, includes establishing noninva-
sive diagnostic techniques as well as labo-
ratory applications for microscopy. Med-
ical analysis and interpretation includes
identifying and differentiating between
types of skin cancer, for example (see
Fig. 4). Industrial inspection, another key
area, includes poultry inspection: Hyper-
spectral imaging spectrometry can dis-
cern diseased vs. healthy poultry. Bannon
says, Imaging spectroscopy is the next
generation of machine-vision capability.
However, UAVs are whats making
the news these days. Headwalls sensor
design is geared toward airborne deploy-
ment, says Bannon. Users of hyperspec-
tral sensors arent always white-coated
scientists. They need help optimizing
these elements so that they work well
together: 1) the sensor; 2) the hyperspec-
tral data processing unit that collects
the incoming data and manages it, also
instructing the sensor when and where
to operate; the software (Hyperspec III)
needs to not only manage the data and
sensor operation, but do so with an easy-
to-assimilate GUI; 3) GPS and inertial-
navigation system (INS); and 4) cabling/
cooling/overall integration.
As a further step into the UAV arena,
Headwall has begun selling a package
consisting not just of its Micro-Hyper-
spec sensor in the airborne configura-
tionbut also the UAV that flies the sen-
sor system.
From UV to IR
Imaging spectrometers can be designed
for use in just about any spectral region
used in life sciences, from UV, visible
(Vis), near-IR (NIR), or short-wave, mid-
wave, and long-wave IR (SWIR, MWIR,
and LWIR). McPherson (Chelmsford,
MA) makes them all.
McPhersons instruments collect spec-
tra from multiple, spatially distinct chan-
nels. They can be one of several differ-
ent optical designs; each is optimized for
a particular spectral range or particular
type of application, says Erik Schoeffel,
marketing and sales at McPherson.
For example, MWIR and LWIR 2D
spectroscopy applications favor low-dis-
persion prism-based systems. These dis-
perse many microns of spectral range
on an array detector and have high
throughput and no multiple- order
effects, albeit relatively low spectral res-
olution, explains Schoeffel.
In contrast, UV-Vis-NIR spectroscopy
applications gravitate towards diffrac-
tion-grating-based, high-resolution, long-
focal-length instruments. These tend to
use fiber optics. These disperse a cou-
ple of nanometers spectral range on the
array detector, and have high line symme-
try and spectral resolution.
This is just a sampling of the imag-
ing spectrometers now available for life
sciences applications. For more, follow
BioOptics Worlds product reporting by
visiting http://www.bioopticsworld.com/
products.html.
REFERENCE
1. M. D. Sonntag et al., J. Am. Chem.
Soc., 135, 17187 (2013); doi:10.1021/
ja408758j.
1409bow_34 34 9/4/14 3:44 PM
S U P E R - R E S O L U T I O N M I C R O S C O P Y
By J ef f Hecht
New twists on superlenses
improve subwavelength
microscopy
Metamaterials can overcome traditional limits on optical resolution, but they pose other
challenges including high losses, dependence on resonances, and limited depths of field.
S
ubwavelength microscopy
has come a long way from its
early days as an exotic con-
cept. The diffraction limit
still rules for conventional bulk optics,
but resolution can be pushed below
half a wavelength in a couple of ways.
Metamaterials and special instru-
ments can achieve true super-resolu-
tion by directly recording transmit-
ted, reflected, or emitted light. Alter-
natively, special processes using flu-
orescence, nonlinearities, or other
techniques can achieve functional
super-resolution.
1
The latter is more
like signal processing. You acquire an
image and try to improve [it], says
George Eleftheriades of the Univer-
sity of Toronto (ON Canada).
So far, the most practical techniques
are functional ones, such as stimulated
emission depletion (STED) microscopy,
developed by Stefan Hell of the Max
Planck Institute for Biophysical Chem-
istry (Gttingen, Germany).
2
STED
allows subwavelength resolution of a
fluorescent spot by de-exciting fluoro-
phores surrounding it. Its major appli-
cations, like those of other functional
superresolution imaging, are in bio-
medicine. For example, in June 2014,
PicoQuant (Berlin, Germany) added
STED to its time-resolved MicroTime
200 confocal microscope, improving
resolution to well below 100 nm.
Scanning near-field optical micros-
copy (SNOM) offers true super-resolu-
tion by moving a tiny aperture across
the near field, but is too time consum-
ing for many applications. Metamaterial
devices, such as the superlens devised
by Sir John Pendry of Imperial College
(London, England), can observe much
larger areas at one time.
3
Thanks to that
advantage, and the flexibility of true
super-resolution, superlenses are com-
ing on fast, although they still face major
challenges, including limited bandwidth
and resolution depth and special mate-
rial requirements.
Superlenses and evanescent waves
The superlens concept is based on meta-
materials in which both electric permit-
tivity and magnetic permeability are neg-
ative at certain wavelengths. That gives
them a negative refractive index, so they
can capture evanescent waves that con-
tain information needed to produce sub-
wavelength images, but are not captured
by conventional optics. As shown in Fig. 1,
a superlens bends light entering it back-
ward, forming an image plane inside it
and another on the opposite side.
The most familiar evanescent waves are
those that leak through a surface where
total internal reflection occurs. They can
be detected very close to the surface, but
decay exponentially with distance from the
surface, so they cant carry energy away.
Pendrys superlens can achieve subwave-
length resolution by focusing the waves
near the surface. Negative-index materials
can actually amplify evanescent waves and
thus restore high-resolution details which
are inaccessible by classical imaging sys-
tems, writes Eleftheriades.
4
A slab of negative-index metama-
terial can serve as a superlens, which
FIGURE 1. A superlens with a refractive index of
-1 bends light entering it backwards, producing an
image plane inside the metamaterial and another
on the opposite side of the material, which can have
super-resolution. (Adapted from Wikipedia)
Object
(in near
feld)
Image
(near
feld)
Negative
refraction
at surface
Air
Metamaterial
Internal
focus
JEFF HECHT is a contributing editor for Laser Focus World; e-mail: jeff@jeffhecht.com.
1409bow_35 35 9/4/14 3:44 PM
Cr
Optical
microscope
Conventional lens
Object
plane
Hyperlens
image plane
Far feld
image plane
Quartz
Hyperlens
h = 365 nm
E
Ag/Al
2
O
3
multilayers
S U P E R - R E S O L U T I O N M I C R O S C O P Y c o n t .
essentially produces a time-reversed
wave with a negative frequency relative
to the original, creating a super-resolu-
tion image, says Stephane Larouche of
Duke University (Durham, NC). Nega-
tive refraction also reverses Snells law
and focuses rather than disperses eva-
nescent waves. The metamaterial has
subwavelength resolution over a large
area, unlike SNOM, which can address
only one point at a time.
Superlenses have their drawbacks.
Attenuation tends to be high, particularly
for metal films that have negative permit-
tivity at visible wavelengths. Metamaterial
properties depend on resonances, so the
negative index crucial for super-resolu-
tion imaging only exists across a limited
band of wavelengths. Superlenses also are
limited to the near field and cannot focus
in three dimensions.
Broadband subwavelength imaging
In 2012, Thomas Taubner of RWTH
Aachen University (Aachen, Germany)
and his colleagues proposed making a
tunable broadband superlens from gra-
phene. Graphene layers can both support
plasmons and guide them, making them
suitable for use in metamaterials. Varying
gate voltage, electric field, or chemical
doping applied to the graphene changes
its conductivity, allowing continuous tun-
ing of graphene properties at infrared
and terahertz frequencies.
5
Imaging in graphene is weaker than
true superlensing because it is not
strongly resonant, but Taubners study
showed that graphene sheets would
enhance evanescent waves for subwave-
length imaging. The RWTH team pre-
dicted that two graphene layers could
achieve resolution of about one-seventh
wave (h/7), and multiple layers could
reach about h/10. Most important, the
team predicted that a graphene lens
could focus to below the diffraction
limit from the mid-infrared to the tera-
hertz band. Tauber and his colleagues
also say their concept could be general-
ized to a two-dimensional conducting
sheet lens of semiconductor hetero-
structures. Results of experiments with
their graphene multifrequency super-
lenses have yet to be published.
Hyperlenses and metalenses
Evanescent waves can be
focused into the far f ield
with a hyperlens, based on
an anisotropic layered meta-
material. The simplest type is
a stack of alternating metal/
dielectric layers much thin-
ner than the optical wave-
length. The original proposal
called for hyperbolic disper-
sion in the metamaterial, but
eccentric elliptical dispersion
is another alternative, write
Dylan Lu and Zhaowei Liu of
the University of California at
San Diego (La Jolla, CA) in a
review paper.
6
Far-field imaging was a sur-
prise to many, but several types
have been demonstrated. Fig-
ure 2 shows how a hyperlens
with hyperbolic dispersion can
focus evanescent waves radially,
effectively converting them into
propagating waves that carry
subwavelength information in the eva-
nescent waves into the far field. The flat
layers are bent so light travels radially
through them and magnifies an object
placed at the inner radial surface so it
can be viewed on the outer surface with
subwavelength resolution. The ratio of
the radii at the inner and outer curved
layers gives the magnification at the
outer surface.
A hyperlens cannot do such standard
optical tasks as focusing plane waves or
performing Fourier transforms. That
requires a metalens, a dense array of
identical resonators fabricated in the
plane of a metamaterial to provide the
phase compensation missing in a hyper-
lens. Metalenses built from such ele-
ments as meta-atoms, plasmonic metal-
insulator-metal waveguide couplers, or
graded index patterns can provide an
exceptional combination of super-resolu-
tion and desirable functions of conven-
tional lenses, write Lu and Liu.
6
Many metalens designs are under
study. Liu proposed using arrays
of bidirectional planar plasmonic
waveguide couplers, as shown in Fig-
ure 3, but has yet to demonstrate it.
Nikolay Zheludev at the University of
Southampton (England) used meta-
atoms that form a metalens to focus
800 nm light onto an array of sub-
wavelength 160 nm hot spots beyond
the near field of the lens.
7
Interfer-
ence among light waves propagating
through the meta-atoms caused an
effect called superoscillation to gen-
erate the spots.
Vladimir Shalaevs group at Purdue
University (West Lafayette, IN) developed
a different planar nanostructure; a 4 m
lens with concentric rings of nanoholes
machined in a 30 nm gold film. The lens
focused 676 nm light only a spot 2.5 m
away; 476 nm light was focused at 7 to 10
m from the lens. Their proposed design
can be machined onto the end of an opti-
cal fiber.
8
Camille Jouvaud and colleagues at
the Langevin Institute (Paris, France)
made magnetic metalenses from arrays
of split-ring oscillators with different res-
onant frequencies. Microwave experi-
ments produced localized modes instead
of extended ones, and they predict their
approach would allow subwavelength
imaging in the visible and infrared.
9
FIGURE 2. Curved stack of layers in a hyperlens captures
evanescent waves from object (inside curvature at top), then
transfers them radially to the outside curvature at bottom
before they are focused with subwavelength resolution.
(Courtesy of Zhaowei Liu)
1409bow_36 36 9/4/14 3:44 PM
a) b) c)
Glass lens
Zone plate
Metal
Air
Air
Air
Air Air
Air Air
Metamaterial
k
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S U P E R - R E S O L U T I O N M I C R O S C O P Y c o n t .
New directions for
subwavelength microscopy
Super- resolution imaging is still
young, and new ideas continue to
emerge. One that particularly intrigues
Larouche is assembling optical meta-
materials by a self-organizing bottom-
up process rather than using the top-
down approach of photolithography.
His lab recently made a near-infrared
metamaterial with 30 nm elements.
We used electron-beam lithography,
which is a great approach for research,
he said, but not for mass production.
That particular device took weeks
to fabricate.
He was intrigued by a recent proposal
by Zsolt Szabo of the Budapest Univer-
sity of Technology (Hungary) and col-
leagues to embed silver nanospheres in
silica to make a composite metamate-
rial for subwavelength imaging. They
calculated that with self- organizing
unit cells smaller than 20 nm, a single-
or multi-layer metal-dielectric compos-
ite metamaterial could achieve 100 nm
resolution.
10
Subwavelength imaging is still
young, and development is so
diverse that there is no room to cover
it all, particularly the many variations
on functional super-resolution. Big
challenges remain, but real progress
is being made in seeing the previously
unseeable.
ACKNOWLEDGEMENT
This article originally appeared in Laser
Focus World, 50, 8, 3337 (2014).
REFERENCES
1. A. Neice, Methods and limitations of
subwavelength imaging, in Advances
in Imaging and Electron Physics, pp.
117140 (2010).
2. S. Hell and J. Wichmann, Break-
ing the diffraction resolution limit
by stimulated emission: Stimulated-
emission- depletion f luorescence
microscopy, Opt. Lett., 19, 780 (1994);
doi:10.1364/OL.19.000780.
3. J. Pendry, Negative refraction makes
a perfect lens, Phys. Rev. Lett., 85,
3966 (Oct. 2000).
4. A.M.H. Wong and G. V. Eleftheriades,
Advances in imaging beyond the dif-
fraction limit, IEEE Photon. J., 4, 586
(Apr. 2012).
5. P. Li and T. Taubner, Broadband
subwavelength imaging using a tun-
able graphene lens, ACS Nano,
6, 11 10107 (2012); doi: 10.1021/
nn303845a.
6. D. Lu and Z. Liu, Hyperlenses and
metalenses for far-field super-resolu-
tion imaging, Nat. Commun., 3, 1205
(2012); doi: 10.1038/ncomms2176.
7. T. Ray, E.T.F. Rogers, and N. I. Zhe-
ludev, Sub- wavelength focusing
meta-lens, Opt. Express, 21, 7577
(Mar. 2013).
8. X. Ni, S. Ishii, A. V. Kildishev, and V.
M. Shalaev, Ultra-thin planar Babi-
net-inverted plasmonic nanolenses,
Light: Sci. Appl., 2, e72 (2013); doi:
10.1038/lsa.2013.28.
9. C. Jourvad, A. Ourir, and J. de
Rosny, Far-field imaging with a
multifrequency metalens, Appl.
Phys. Lett., 104, 243507 (2014); doi:
10.1063/1.4882277.
10. Z. Szabo, Y. Kiasat, and E. P. Li.,
Subwavelength imaging with com-
posite metamaterials, J. Opt. Soc. Am.
B, 31, 1298 (June 2014); doi:10.1364/
JOSAB.31.001298.
FIGURE 3. Proposed metalens design uses an array of waveguidesvertical in the metal layer
to provide phase compensation so it can focus plane waves. (Courtesy of Zhaowei Liu)
1409bow_37 37 9/4/14 3:44 PM
PRODUCTS
Components Systems
Compi l e d By LEE DUBAY
http://www.bioopticsworld.com/products.html
BioOpticsWorld online
FOR CONTINUALLY UPDATED PRODUCT NEWS, GO TO
Pl ease send your new pr oduct announcement to
bowproducts@pennwel l . com
Fiber collimator
The Fiber Focuser fiber collimator can generate micron spot sizes at long working
distances in applications such as flow cytometry and genetic sequencing. They
work with singlemode-type fiber in the 3502300 nm wavelength range. The
focusers optical materials eliminate or minimize any
fluorescence generated by some optics. There
is no epoxy in the optical path, and an all-
stainless-steel housing construction minimizes
temperature effects.
Micro Laser Systems, Garden Grove, CA,
http://bit.ly/1AsvqAq
Microscope camera
A 13 Mpixel microscope camera is based on Sony Exmor CMOS
technology, and can be mounted on a microscopes ocular or
C-mount. The camera, which is equipped with a distortion-free, 5.3
mm (35 mm equivalent) auto-focus lens, allows capture of what is
seen through the ocular rather than just a region of interest. A USB
3.0 interface enables a full HD (1920 1080) preview at 30 frames/s
on the host PC.
The Imaging Source, Bremen, Germany, http://bit.ly/1r4a8qT
Tunable ultrafast laser
The InSight DS+ tunable ultrafast laser system delivers
ultrashort pulses over a 6801300 nm tuning range
from a single beam, while a second beam provides
simultaneous, dual-wavelength output at 1041 nm for
multimodal imaging. Applications include in vivo deep
tissue imaging for neuroscience, cell biology, and other life science research. The laser
offers integrated dispersion pre-compensation for imaging penetration depths beyond
1 mm, which has been demonstrated in live tissue.
Spectra-Physics, a Newport company, Santa Clara, CA, http://bit.ly/VUjdWc
Illumination
system
The Lumen 300-LED white
light excitation illumination
system delivers broad-
spectrum LED illumination
in fluorescence applications.
The system, which fits directly
to most microscopes, offers
instant on/off operation via
transistor-transistor logic (TTL),
manual 0- to 100-percent
intensity control in 1-percent
increments, and on/off control
for each LED. An optional
liquid light guide is available
when needing to keep the
illumination source remote
from the microscope.
Prior Scientific, Rockland,
MA, http://bit.ly/1pFAaPE
1409bow_38 38 9/4/14 3:44 PM
Confocal module
The Revolution DSD2 laser-free confocal
module fits to most fluorescence
microscopes for routine confocal
microscopy. It features the companys
scientific CMOS (sCMOS) camera
technology to deliver a large field of
view and high dynamic range, and a
broadband white light source for imaging
of any fluor by selection of filters. Three
confocal settings enable use across the full
magnification range for developmental
biology, neuroscience, embryology, and
plant biology. The module handles fixed,
live-cell, and embryo specimens.
Andor Technology, Belfast, Northern
Ireland, http://bit.ly/XvyL3J
Laser platforms
ZFSM Series fiber-coupled laser
platforms for diagnostic and intra-
operative medical applications integrate
into standard and intra-operative
diagnostic devices, as well as in surgical
and therapeutical equipment. A
standard 780 nm version can provide
a defect map of the visualized area,
while an RGB version has a miniaturized
optical head for intra-operative
diagnoses and for highly targeted area
illumination applications.
Z-LASER, Freiburg, Germany, http://
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Linear positioning stage
The MPS75SLE linear positioning stage
has use in research applications, including
microscopy. A precision ground, preloaded
ball-screw, and linear encoder provide
accuracy, repeatability, and a positioning
resolution capability to 25 nm. Anti-creep
crossed-roller bearings provide smooth
travel and high-load capacity. A low thermal
expansion (3.3 ppm/C) precision glass
scale assures high accuracy and repeatable
positioning over long periods of time.
Aerotech, Pittsburgh, PA, http://bit.
ly/1reFIjh
Development kit for
spectral sensing
The STS Developer Kit for spectral sensing
includes the companys STS spectrometer,
Raspberry Pi microcomputer, customizable
software, and wireless capabilities. Using
ultraviolet-visible (UV-Vis) absorption
spectroscopy across 2001100 nm, the
kit is programmable for various DNA,
RNA, or protein
measurements,
as well as blood
oxygenation. The kit
can be used as a base
for building more
compact instruments,
including handheld
point-of-care devices,
and offers a choice
between three STS spectrometers: STS-UV
(190650 nm), STS-Vis (350800 nm), or
STS-NIR (650100 nm).
Ocean Optics, Dunedin, FL,
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1409bow_39 39 9/4/14 3:44 PM
By LEE DUBAY
E
n
d
Result
U
niversity of Cincinnati (Ohio)
philosophy and psychology
graduate assistant Luis Favela studies
how people perceive their environment,
and how those perceptions inform
their judgments. Inspired by the CDC
prediction that more than 6 million
Americans aged 40 and older will be
affected by blindness or low vision by
2030, Favela wondered whether an
infrared (IR) light-based handheld device,
developed by University of Reading
(Reading, Berkshire,
England) cybernetics
students Tom Froese and
Adam Spiers
1
, could help
such people.
The device, called the
Enactive Torch (ET), uses
two nonlinear-response
IR rangefinders (one is
a Sharp GP2D12, which
covers a range of 880
cm, and the other is a
Sharp GP2Y0A02YK0F,
which covers 20150
cm) located at the
front to detect
distance information.
The information is
then transferred to
a vibrational motor
that is attached to the
wearers wrist and emits
a vibration similar to a cellphone alert.
This enables the user to feel whether
something is in front of the device and
then how near or far that something is
based on the intensity of the vibrations,
Favela told BioOptics World. The gentle
buzz increases in intensity as the torch
nears the object, letting the user make
judgments about where to move based
on a virtual touch.
Comparative test
Favela conducted
an experiment
to determine the
value of such a
tool. He asked
27 participants
with normal or
corrected-to-
normal vision to
make perceptual
judgments about
their ability to
pass through an
opening a few
feet in front of
them without
needing to shift their normal posture. He
tested the judgments they made in three
modes: using only their vision, using a
cane while blindfolded, and using the
ET while blindfolded. The idea was to
compare judgments made with vision
against those made by touch.
Favela thought that vision-based
judgments would be the most accurate
because vision tends to be most
peoples dominant perceptual modality.
But the data revealed that all three
modalities were equally accurate.
People can carry out actions just about
to the same degree whether theyre
using their vision or their sense of
touch. I was really surprised, he says.
Favela plans on additional
experiments with the ET that require
more complicated judgments, such as
the ability to step over an obstacle or to
climb stairs. He, Froese, and Spiers have
also discussed the possibility of smaller
versions of the ET, considering devices
with other forms of feedback such as
skin stretch feedback, he says.
Favela presented his research
Augmenting the Sensory Judgment
Abilities of the Visually Impaired at the
American Psychological Associations
(APA) annual convention, held Aug.
7-10, 2014, in Washington, DC.
REFERENCE
1. T. Froese, M. McGann, W. Bigge, A.
Spiers, and A. Seth, IEEE Trans. Haptics, 5,
365375 (2012); doi:10.1109/ToH.2011.57.
Handheld IR device could be game-
changer for the visually impaired
The Enactive Torch uses infrared sensors
to detect objects in front of it and, upon
detection, send a vibrational warning to
the wearer. (Both photos courtesy of Colleen
Kelley, University of Cincinnati)
Luis Favela observes Amon as she uses the Enactive Torch during a
demonstration of Favelas experiment.
1409bow_40 40 9/4/14 3:44 PM
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SEPTEMBER 9-11, 2014
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Advances in lasers, optics, and

imaging for the life sciences

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FIGURE 2. LumiQuant can combine and co-register 3D

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