Biophotonics201109 DL

September 2011
Worldwide Coverage: Optics, Lasers, Imaging, Fiber Optics, Electro-Optics, Photonics Component Manufacturing
Photoacoustic Microscopy Fluorescence Imaging FRET
Photoacoustic
Microscopy
Unlocks Secrets of Optical Absorption
Intrinsic Fluorescence
Lights Up Cellular Components
QD-Based FRET Enables
Multiplexed Sensing
Sept 11
www.Photonics.com
Volume 18 Issue 7
t
NEWS 10 BIOSCAN
TABLE OF CONTENTS
www.photonics.com
BioPhotonics editors curate the most significant

headlines of the month for photonics in the life
sciences and take you deeper inside the news.
Featured stories include:
Cornell dots characterized, ready for human trials
Semiconductor nanowire laser tech could
kill viruses, purify water
MicroOCT advances understanding of atherosclerosis
22 BUSINESSSCAN
OCT researchers garner grants for glaucoma detection
FEATURES 24 PHOTOACOUSTIC MICROSCOPY UNLOCKS SECRETS

OF OPTICAL ABSORPTION
by Chi Zhang, Konstantin Maslov and Lihong V. Wang,
Washington University, St. Louis
Unlike mainstream optical microscopy technologies, subwavelength
photoacoustic microscopy sensitively measures optical absorption,
providing physiological information.
28 INTRINSIC FLUORESCENCE LIGHTS UP CELLULAR COMPONENTS

by Angela Goodacre and Dennis Donley, Olympus America Inc.
In conjunction with multiphoton microscopy systems, researchers can detect
intrinsic fluorescence with methods and wavelengths that do not perturb the cell.
31 FLUORESCENCE IMAGING PROGRESSING FROM CELLS TO TISSUE

38
by Marie Freebody, Contributing Editor

Advances in microscopy, computerized software and digital image acquisition,
along with spectral libraries of fluorophores, are helping meet current challenges.
35 MULTIPLEXED SENSING WITH QD-BASED FRET

by Gary Boas, News Editor
Investigators are developing high-performance methods and technologies to exploit
the advantages of quantum dots for multiplexed diagnostics and other applications.
38 PHOTON BY PHOTON: THE EVOLUTION OF THE GEIGER-MODE

SILICON AVALANCHE PHOTODIODE FOR SINGLE-PHOTON COUNTING
by Bernicy Fong, Excelitas Technologies Corp.
A short history of single-photon counting from the 1930s on, along with a summary
of what upgrades future biomedical and life sciences applications will require.
DEPARTMENTS
8 EDITORIAL
42 BREAKTHROUGHPRODUCTS
48 APPOINTMENTS
Upcoming Courses and Shows
49 ADVERTISER INDEX
50 POST SCRIPTS
by Caren B. Les
Seeing the fluorescence for the trees
THE COVER
The cover art is from this
issues feature on photoacoustic
microscopy by a team from Washington University, St. Louis; the
article begins on page 24. Design by
Art Director Suzanne L. Schmidt.
PHOTONICS
The technology of generating and harnessing light and other forms of radiant energy whose
quantum unit is the photon. The range of applications of photonics extends from energy generation
to detection to communications and information processing.
BIOPHOTONICS
The application of photonic products and techniques to solve problems for researchers,
product developers, clinical users, physicians and others in the fields of medicine,
biology and biotechnology.
BioPhotonics September 2011
www.photonics.com
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EDITORIAL
Good news for biophotonics
hether you follow the political or economic stories

or both affecting life and business (if you have the
stomach for it), you know that there hasnt been a lot
of good news lately. And questions abound regarding the impact
of recent moves and markets on science and technology research.
As the two worlds move toward steadier ground, researchers
everywhere continue to push new diagnostic methods closer to
clinical application.
One such arena is Frster resonance energy transfer (FRET)
spectroscopy. For more than a decade, investigators have explored its potential for use with quantum dots in multiplexed
diagnostics, noting the unique photophysical properties of the
particles. In his feature, Multiplexed Sensing with QD-based
FRET, news editor Gary Boas shares some of the latest research
into QD-based FRET from around the world. The article begins
on page 35.
pus America Inc., we learn that windows of opportunity for research into dynamic cellular events and processes are opening,
thanks to a multiphoton combo of intrinsic fluorescence and other
label-free imaging modalities such as second-harmonic generation and coherent anti-Stokes Raman scattering. Read Intrinsic
Fluorescence Lights Up Cellular Components, beginning on
page 28.
Since its invention in the mid-1930s, the photomultiplier tube
has been the principal detector for experiments involving a small
number of photons. However, some disadvantages led researchers
and systems designers to seek alternatives for single-photon
detection. One result is the Geiger-mode silicon avalanche
photodiode (G-SAPD), which enables single-photon detection
with silicon APDs. Bernicy Fong of Excelitas Technologies Corp.
explains it all in Photon by Photon: The Evolution of the GeigerMode Silicon Avalanche Photodiode for Single-Photon Counting, beginning on page 38.
Also in this issue:

Fluorescence Imaging Progressing from Cells to Tissue, by
contributing editor Marie Freebody, explains that, although tissues and even whole animals are not as easily captured with
fluorescence imaging as cells are, recent research and technological developments could change all that. The story starts on
page 31.
In an article by Angela Goodacre and Dennis Donley of Olym-
And, finally, Chi Zhang, Konstantin Maslov and Lihong V.

Wang of Washington University in St. Louis describe how in
vivo, label-free subwavelength-resolution photoacoustic microscopy enables more precise measurement of optical absorption
and, therefore, provides more information. Read about it in
Photoacoustic Microscopy Unlocks Secrets of Optical Absorption, beginning on page 24.
I hope you enjoy the issue.
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BIOSCAN
MicroOCT advances understanding of atherosclerosis
BOSTON Coronary atherosclerosis
could someday be more easily diagnosed
and treated, thanks to a new version of the
intravascular imaging technology optical
coherence tomography that provides a
resolution 10 times greater than that of
standard OCT.
The new version, developed at the Wellman Center for Photomedicine at Massachusetts General Hospital, is called microOCT, and it provides the contrast and
resolution required to show individual arterial and inflammatory cells within coro-
nary artery samples, including subcellular

features that may identify vulnerable
plaques.
OCT, a catheter-based technology, uses
reflected near-infrared light to create detailed images of a blood vessels internal
surfaces. Standard OCT is used to identify
arterial plaques that are likely to rupture,
but it can only clearly image structures
larger than 10 m. Now, using new types
of lenses and advanced imaging components, microOCT can image structures as
small as 1 m. The technique reveals an
These are images of a coronary artery plaque (Ca in image c) produced by (a) standard OCT,
(b) microOCT and (c) tissue histology. Courtesy of Nature Medicine and the Wellman Center
for Photomedicine at Massachusetts General Hospital.
intact tissues detailed information in three

dimensions and does so much more
quickly than with the prepared tissue
slides of traditional pathology.
Using microOCT, researchers were able
to image human and animal coronary artery tissue, revealing inflammatory cells
that contribute to the formation of coronary plaques, endothelial cells that line
coronary arteries, smooth muscle cells
that produce collagen in response to
inflammation, and fibrin proteins and
platelets that are involved in the formation of blood clots.
The technology also can distinguish
between bare-metal stents and drugeluting stents placed in coronary arteries.
During the study, defects in the drugeluting coating were identifiable using
the technology.
The researchers anticipate that observation of cells with microOCT could be performed in humans in roughly three to five
years. They are hopeful that, with the ability to follow and track cells in 3-D, they
can prove or disprove many theories of
coronary artery disease, as well as better
understand how clots form on a microscopic level.
In addition, improved definitions of
high-risk plaques could lead to greater
accuracy in identifying those that could
rupture or block coronary arteries. The
researchers also said that being able to
monitor the healing of implanted stents
could reduce the number of patients who
must be on anticlotting medications,
which have major side effects and are
very costly.
Findings were described in the July 10
online edition of Nature Medicine (doi:
10.1038/nm.2409).
Semiconductor nanowire laser tech could kill viruses, purify water

RIVERSIDE, Calif. A semiconductor
nanowire laser technology that can kill
viruses, purify drinking water and more
has been discovered by scientists at the
Bourns College of Engineering at the
University of California, Riverside.
Although widely used for biology, data
processing and information storage, ultraviolet semiconductor diode laser applications have been limited because of size,
10
cost and power. This breakthrough in zinc

oxide nanowire waveguide lasers offers
smaller sizes, lower costs, higher powers
and shorter wavelengths. Findings appeared online July 3 in Nature Nanotechnology (doi: 10.1038/nnano.2011.97).
Until recently, zinc oxide nanowires
could not be used in real-world lightemission applications because of the lack
of p-type material needed by all semicon-
ductors. Now, the researchers have doped

the zinc oxide nanowires with antimony,
a metalloid element, to create the p-type
material. Connecting the p-type zinc oxide
nanowires with n-type zinc oxide material,
the scientists formed a p-n junction diode.
When powered by a battery, the nanowires
emit highly directional laser light from
their ends.
The discovery could have a lot of im-
pact. For biology and medical therapeutics, living cells could be penetrated with
the laser, or the nanowire laser could excite or change a cells function from bad
to good.
While the scientists have proved that
p-type doping of zinc oxide and electrically powered nanowire waveguide lasing
works, they said that more work must be
done with the p-type material to make it
more reliable and stable.
From left to right, scientists Guoping Wang, a

graduate student, Jianlin Liu, a professor of
electrical engineering, and Sheng Chu, a graduate
student, developed a nanowire laser that can kill
viruses or alter cells. Courtesy of University of
California, Riverside.
Cornell dots characterized, ready for human trials

ITHACA, N.Y. The bright
nanoparticles known as Cornell
dots could light up cancer cells
in PET optical imaging, and the
technology, recently approved
as an investigational new
drug by the FDA, is headed
toward clinical trials in humans.
For the first time, scientists
have reported an advanced
comprehensive characterization
of Cornell dots. The report,
published in the June 13 issue
of the Journal of Clinical Investigation, was a collaborative
effort between Memorial Sloan-
Kettering Cancer Center,

Cornell University and Hybrid
Silica Technologies, a Cornell
business startup.
Cornell dots are silica
spheres less than 8 nm in diameter that enclose several dye
molecules. The silica shell is
chemically inert and small
enough to pass through the
body. For clinical applications,
the dots are coated with polyethylene glycol (PEG) so that
the body does not recognize
them as foreign objects.
To make the dots stick to
tumor cells, organic molecules

that bind to tumor surfaces or
even specific locations within
the tumors can be attached to
the PEG shell. When exposed
to near-infrared light, the dots
fluoresce much brighter than
the dye, serving as a beacon to
identify the target cells. The researchers said that, during surgical treatment, the technology
enables visualization of invasive or metastatic spread to
lymph nodes and distant organs,
and can show the extent of
treatment response.
Nodal mapping is now being

pursued under the award of
a BioAccelerate NYC Prize
from the Partnership for New
York City and the New York
City Economic Development
Corp., which is expected to
lead to another clinical trial
in humans.
The collaboration is in the
process of forming a new commercial entity in New York
that will help transition the
research into commercial products that will benefit cancer
patient care.
GFP helps turn single cell into living laser

BOSTON For the first time, a single living cell has been genetically engineered
to produce nanosecond pulses of laser light.
The living laser, developed by two researchers at the Wellman Center for
Photomedicine at Massachusetts General Hospital, is a single cell genetically
engineered to express green fluorescent protein originally found in a species
of jellyfish that amplifies photons into a visible laser beam.
Lasers have used synthetic gain materials such as gases, crystals and dyes to
amplify photon pulses. Now the scientists are using GFP as the gain material.
To determine GFPs potential for generating light, the team assembled a device
consisting of a 1-in.-long cylinder, with mirrors at each end, filled with a solution
of GFP in water. The researchers estimated the concentration of GFP required to
produce the laser effect after confirming that the solution could amplify input
energy into brief pulses of laser light. Using the information, they developed a
line of mammalian cells that express GFP at the required levels.
The cellular laser was assembled by placing the single GFP-expressing cell, just
Microscope image of a single-cell living laser in action. The

irregular internal structure of the GFP-expressing cell causes
the apparently random pattern of laser light emission.
Courtesy of Nature Photonics and Malte Gather, Wellman
Center for Photomedicine, Massachusetts General Hospital.
11
BIOSCAN
Schematic of a living laser. When a single biological cell genetically programmed to produce GFP is
placed inside an optical resonator consisting of two
parallel mirrors separated by 20 m, the cell can
generate green laser light.
15 to 20 m in size, into a microcavity

that consisted of two highly reflective mirrors spaced 20 m apart. The scientists observed not only that the cell-based device
produced pulses of laser light but also that
its spherical shape acted as a lens, refocusing the light and inducing emissions of
laser light at lower energy levels than are
required for the solution-based device. The
cells used in the lasing process survived
and continued producing hundreds of laser
Microscope gains
a fourth dimension
PASADENA, Calif. Extending the technology of the threedimensional microscope, scientists have added a dimension
that promises sweeping applications in biological research,
medicine and the development of new electronic devices.
The four-dimensional scanning ultrafast electron microscope was developed by chemistry Nobel laureate Ahmed H.
Zewail and his colleagues at California Institute of Technology. They discovered a way to integrate time into traditional
electron microscopy observations, which resulted in the creation of high-resolution images of vanishingly small nanoscale
objects in four dimensions rather than three.
Their laser-driven technology enabled researchers to visualize 3-D structures such as a ring-shaped carbon nanotube
as it wiggled in response to heating, over a femtosecond
timescale. Although they obtained 3-D information from
the approach, it was limited in that it showed the object as
stationary rather than undergoing its natural movements.
The scientists overcame the limitations using their 4-D
scanning ultrafast electron microscopy technique, which
allowed deeper insights into the innermost structure of materials. Their work, which appeared in two papers from the
Journal of the American Chemical Society (doi: 10.1021/
ja203821y and doi: 10.1021/ja2031322), describes how the
technique could be used to investigate atomic-scale dynamics
on metal surfaces, and to watch the vibrations of a single silver nanowire and a gold nanoparticle.
The scientists said the new techniques hold promise for
a variety of applications, including single-particle biological
imaging and materials science.
Funding for the work came from the National Science
Foundation, the US Air Force Office of Scientific Research,
the Gordon & Betty Moore Physical Biology Center at
Caltech, and the Arab Fund for Economic and Social
Development.
12
light pulses. The findings were reported in

the June 30 issue of Nature Photonics
(doi: 10.1038/nphoton.2011.122).
Offering a new way to analyze the properties of large numbers of cells almost instantaneously, the technique could be useful for photodynamic therapies or novel
forms of imaging, the scientists noted.
They are hopeful that their findings will
bring optical communications and computing, currently done with inanimate electronic devices, into the realm of biotechnology. Future work will include proving
the technique useful for interfacing electronics with biological organisms, as well
as implanting a structure equivalent to the
mirrored chamber right into a cell.
Birds-eye view used to craft

autopilot technology
CAMBRIDGE, Mass.
Once used as wartime
messengers, pigeons
now have a new calling:
acting as informants for
navigation technology
design.
Harvard University
research involved training messenger pigeons
to fly through an artificial forest with a tiny
camera mounted to their
heads, literally offering
a birds-eye view and
enabling the scientists
Pigeons were fitted with a tiny head-camera
to reconstruct both what
for their flight through an artificial forest.
the birds saw and how
Images courtesy of Talia Moore.
they moved.
The navigation methods that enable a pigeon to fly quickly
and accurately through difficult environments could be used as a
model for autopilot technology and even lead to new developments in robotics. With >300 panoramic vision, pigeons are
well suited for the task because their wraparound vision makes
it possible to assess obstacles in their periphery. In addition, they
can stabilize their vision and switch rapidly between views by
using head saccade, a small, rapid head movement.
The pigeons also offer other skills important for autopilot
machines. For instance, they tend to choose the straightest,
most direct route, reaching the other side of the forest quickly
and energy-efficiently.
The scientists observed also that pigeons tend to exit the forest
in the exact same direction as they entered, even with all the
twists and turns they make while navigating.
These findings, presented in July at the Society for Experimental Biologys annual conference, could prove crucial when sending a robot or unmanned aircraft to a specified destination.
BIOSCAN
Scientists reach for

the stars to find new
radiation treatments
COLUMBUS, Ohio Many have looked
to the stars for inspiration, but could a
new cancer therapy be up there as well?
Ohio State University astronomers think
so. After studying how different chemical
elements absorb and emit radiation in stars
and around black holes, they found that
heavy metals such as iron emit low-energy
electrons when exposed to x-rays at specific energies. With the help of medical
physicists and radiation oncologists, the
team has developed a radiation treatment
intended to be tougher on tumors but gentler on nearby healthy tissue.
The concept, called resonant nanoplasma theranostics (RNPT), uses x-rays
to fire electrons at malignant cells to destroy them. The methodology could revolutionize both x-ray imaging and cancer
therapy, the astronomers said.
Electrons orbit the nuclei of atoms at
various distances, and when an electron
closer to the nucleus is lost, a more distant
one may drop in to take its place, releasing
energy in a phenomenon known as the
Auger effect.
The astronomers believe that K-alpha
x-ray frequencies kick the close-in electrons out of heavy-metal atoms like platinum, causing many far-out electrons to
fall in while many more are kicked out.
The free Auger electrons are low in energy
but great in number and could easily bombard malignant cells, shattering their DNA.
Typical therapeutic x-ray machines
such as CT scanners generate full-spectrum x-rays, but with the new methodology, hospitals could employ RNPT using
only K-alpha x-rays, greatly reducing a
patients radiation exposure.
The scientists first experiment using
the Auger effect demonstrated that it is
possible to deliver specific frequencies of
x-ray radiation to heavy-metal nanoparticles embedded in diseased tissue. The
team is hopeful that, by targeting heavymetal nanoparticles to certain sites in the
body, x-ray imaging and therapy could
become more precise and powerful, while
radiation exposure would be reduced.
Research was funded by a Large Interdisciplinary Grant award from Ohio State,
and computational resources were provided by the Ohio Supercomputer Center.
13
BIOSCAN
Unnatural amino acids probe mysteries of neural stem cells

LA JOLLA, Calif. Before stem-cell-based regenerative medicine can advance, scientists must understand how stem cells work
and what kinds of cells they produce. Salk Institute research
recently uncovered a way to study neural stem cells by tagging
them with genetically incorporated unnatural amino acids, such
as those that emit green fluorescence. The neural stem cells differentiated into brain neurons with the incandescent tag intact,
allowing scientists to follow the process.
Although stem cells hold great promise for disease treatment, it is difficult to study how they renew themselves and
produce all of the bodys cells, said Dr. Lei Wang, assistant
professor and Frederick B. Rentschler Developmental Chair
in the institutes Chemical Biology and Proteomics Laboratory.
The ability to genetically incorporate unnatural amino acids
in stem cell proteins will accelerate our understanding of the
signaling networks that control these stem cells, Wang said.
Although they had been used in bacteria in 2001 and in mammalian cells in 2007, this marks the first time that unnatural
amino acids (Uaas) have been used in stem cells. The first step
of the study was to see whether Uaas could be incorporated into
neural stem cells without disrupting their process of differentiation and, if that were possible, whether the fluorescent tag the
team had inserted would be carried into neuronal cells created
by the stem cells.
Because added genes are often lost before a stem cell has a
chance to finish differentiation, current methodology for Uaa
incorporation is not appropriate for stem cells. To address this
issue, the scientists developed a lentiviral-based gene delivery
method to incorporate the Uaas into proteins expressed in

neural stem cells.
They used the virus to deliver different components needed
in the Uaas technology, such as synthetic transfer RNA and
enzymatic synthetase. They could then load it with the third
engineered molecule: an unnatural amino acid. Chemically
distinct from the 20 naturally occurring amino acids in the
body, these unnatural amino acids can be engineered for various
desirable properties, such as the ability to fluoresce.
Uaas were successfully incorporated into neural stem cells, and
the incorporation lasted through differentiation; these cells could
produce neurons carrying the fluorescent amino acid. Further
studies demonstrated that these Uaas could be used to help solve
a biological question: How do voltage-sensitive ion channels
work in neurons?
We are trying to understand how the electric field of cell
membranes can turn on or turn off protein activities like a
switch in a house turns on or off lights, Wang said.
To find the answer, the scientists embedded a fluorescent Uaa
into a domain that ion channels and other proteins use to sense
the electric field in neural stem cells. This produced neurons with
the same embedded Uaa, allowing the scientists to detect realtime changes in fluorescence intensity due to changes in electrical
current across the neuron.
The experiment, designed to demonstrate the power of Uaas in
brain cells, could be adapted for studying membrane proteins in
other cells, no matter where they exist in the body, Wang concluded. The new technique was detailed in the June 16 online
issue of Stem Cells (doi: 10.1002/stem.679).
Reward-seeking behavior regulated with optogenetics
Nerve cells in the nucleus accumbens (red) receive

input from amygdala fibers (green). Optogenetic
stimulation of these nerve fibers produces a
rewarding effect in mice. Courtesy of Stuber
Lab, UNC-Chapel Hill.
14
CHAPEL HILL, N.C. Scientists have

used optogenetics to control rewardseeking behaviors such as drug addiction
in rodents, demonstrating the roles of
specific connections in the brain that
control behavior.
Through combining genetic engineering
and laser technology, scientists at the University of North Carolina tweaked the microcircuitry of the brain so that they could
assess how those changes affect behavior.
Their findings, which appeared in the June
29 issue of Nature (doi: 10.1038/nature
10194), suggest that therapeutics that
target the path between two critical brain
regions the amygdala and the nucleus
accumbens, the regions that are associated
with reward could act as treatments for
neuropsychiatric diseases and addiction.
These regions are important for assessing clinical disorders, but until now there
were no proper tools to directly study the
connections between them.
Using the new technique, scientists

transferred light-sensitive proteins called
opsins derived from algae or bacteria
that need light to grow into mammalian
brain cells they wished to study. Shining a
laser beam directly onto the genetically
manipulated brain cells, they could either
excite or block the activity with millisecond precision.
Initial experiments targeted the nerve
cells that connect the amygdala and the
nucleus accumbens. Using light to activate the connection between the regions,
the scientists rewarded the mice with
laser stimulations for performing mundane tasks, such as poking their nose into
a hole in their cage. The mice treated
with opsins quickly learned how to obtain
stimulation of the neural pathway, while
the genetically untouched mice in their
experiment never caught on to the task.
To study whether this brain wiring
plays a role in more natural behavioral
BIOSCAN
processes, the scientists trained the mice

to associate a cue in this case, a lightbulb turning on in the cage with a reward of sugar water. This time, the opsin
that was transferred into the brains of the
mice was one that shut down the activity
of neural connections in response to
light. While delivering the cue to the
control mice, they blocked the neuronal
activity in the genetically altered mice,
and they observed that the control mice
responded quickly to the cue by licking
the sugar-producing vessel, while the

treated mice did not exhibit the same response.
The researchers are exploring how
changes to this segment of brain wiring
could render animals either sensitized or
oblivious to rewards. Proving a useful
tool for studying brain function, the technique could one day provide an alternative to electrical stimulation or pharmacotherapy for neuropsychiatric illnesses
like Parkinsons disease.
Glowing gland could reduce endocrine surgery risk

NASHVILLE, Tenn. Parathyroid glands
four small organs the size of grains of
rice located at the back of the throat are
very difficult to identify with the naked
eye, and they can be damaged during surgery to remove a diseased thyroid. Now,
scientists have discovered that the glands
glow with a natural fluorescence in the
near-infrared region of the spectrum, and
this unique fluorescent signature could
allow endocrine surgeons to detect and
positively identify them to avoid injury.
Not only are parathyroid glands difficult
to see, but they can be found in different
locations from person to person. It takes a
microscope to reliably tell the difference
between the parathyroid tissue and the
thyroid and lymph tissue that surround it.
Even with magnification, it is difficult to
distinguish the parathyroid.
Biomedical engineers and endocrine
surgeons at Vanderbilt University have
discovered that the parathyroid tissue
glows naturally with fluorescence that
is so strong that detecting the tissue
does not require expensive or sophisticated instruments, potentially reducing
the risk of parathyroid damage during
neck surgery.
The team assembled a detector from
off-the-shelf hardware consisting of a lowpower infrared laser connected to an optical fiber probe. As the fiber illuminates
the tissue with invisible near-infrared
light, a detector, connected to other fibers
in the probe, measures the strength of the
fluorescence that the laser excites. The
university has applied for an international
patent to cover the application.
Although accurate, the first generation
of the device was difficult to use because
surgeons had to dim the lights for it to
work. This will not be the case for the
Faster Dynamics
for Fluorescence
Higher Power
Faster Results
Parathyroid tissue in the upper right emits a natural

fluorescence in the infrared spectrum that is twice
as strong as that of thyroid tissue, in the lower left.
The difference is strong enough that a simple optical
system can distinguish between the two during
endocrine surgeries. Courtesy of Mahadevan-Jansen
Laboratory.
Our new higher power solid-state

lasers are ideal for live cell,
spinning disk and TIRF imaging,
and cytometry as well as other
uorescence-based applications
that rely on fast dynamics of
uorophores for results. Diode
laser based technologies, including
488 nm, have direct modulation
capability for reduced system cost
and footprint. All lasers operate
with common control commands
for easy integration.
Lgkf.,dNXk,-(ed
Lgkf,'dNXk+//ed
Lgkf0'dNXk+',ed
newer version, which will include a filter

that will block out visible light. According
to the surgeons, the system will be more
user-friendly with the addition of a camera
that displays the fluorescence of all the
tissues in the throat on a single display.
The cause of fluorescence in parathyroid tissues remains a mystery, but the
scientists asserted that the uncertainty will
not get in the way of using their method to
improve the effectiveness of parathyroid
surgeries and to reduce the damage done
to the glands during other endocrine surgeries.
Details of their work were described in
the June 8 issue of the Journal of Biomedical Optics (doi: 10.1117/1.3583571).
Lgkf.,dNXk-+'--'ed
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BIOSCAN
New gold nanoparticles serve as biomedical test bed
A single sulfur atom at the root of each multiply branched dendron anchors it
to the gold nanoparticle at the center. Researchers at NIST and NCI/NCL are
studying the tiny constructs as a test bed for possible biomedical applications.
Courtesy of Cho, NIST.
16
GAITHERSBURG, Md. Gold nanoparticles could be used as a

test bed to explore how the tiny particles behave in biological
systems, according to a new report. It also suggests a new model
for the characterization of nanoparticle formulations to determine
just what materials scientists are working with. The report was
produced by the National Institute of Standards and Technology
(NIST) and the National Cancer Institutes Nanotechnology
Characterization Laboratory (NCL).
Nontoxic gold can be fashioned into particles in a range of
shapes and sizes. Alone, gold has no biological implications, but
it becomes functionalized when it is attached to protein-based
drugs along with targeting molecules that cluster preferentially
around cancer cells. The nanoparticles generally are coated to
prevent clumping and also to avoid rapid clearance by the bodys
immune system.
The density, stability and coating composition have an impact
on the nanomaterials safety, the efficacy of the delivery system,
and biocompatibility how well the nanoparticles distribute in the
body. Scientists believe that thorough characterization of these
parameters will enable them to develop better nanomaterials.
The team created a nanoparticle test bed to facilitate its studies.
The test bed was a uniform, controllable core-shell nanoparticle
that could be made to order with precise shape and size, and to
which could be attached nearly any potentially useful functional-
BIOSCAN
ity. With this information, the researchers could study how

controlled variations fared in a biological system.
The trial system was based on dendrons, regularly shaped
branching molecules that were suitable for the study because
individual dendrons are always the same size and can readily
be modified to carry payload molecules. In addition, the tip
of the structure is designed to bond easily to the surface of a
gold nanoparticle.
The experiment resulted in an exhaustive set of measurements,
which the team used to thoroughly describe its custom-made
dendron-coated nanoparticles. The scientists established a basic
series of measurement protocols that could be applied to any
gold-based nanoparticles.
The paper, which appeared online April 26 in Chemistry of
Materials (doi: 10.1021/cm200591h), outlines the commencement of a catalog of analysis techniques for gathering a detailed
description on nanoparticles. The techniques include dynamic
light scattering; matrix-assisted laser desorption/ionization mass
spectrometry; and ultraviolet/visible, nuclear magnetic resonance
and x-ray photoelectron spectroscopy.
The dendron-coated nanoparticles were tested also for stability
under biologically relevant conditions of acidity, temperature
and some recognized forms of chemical attacks that could occur
within the bloodstream. In vitro biological tests are pending.
Possible applications include high-precision drug-delivery
systems and diagnostic image enhancers, and chemists are hopeful that gold nanoparticles will be the new gold standard for
medical use.
Forgotten bioluminescent mushroom comes out of hiding

SAN FRANCISCO A glowin-the-dark mushroom not seen
since 1840 has been rediscovered in a Brazilian rain forest
and reclassified.
The bioluminescent fungus
was discovered by San Francisco State University re-
searcher Dennis Desjardin and

his team in 2009. They have
now collected new specimens
of the forgotten mushroom,
which was named Agaricus
gardneri, and reclassified it as
Neonothopanus gardneri. Their
findings were published online
June 23 in Mycologia (doi:

10.3852/11-097).
Neonothopanus gardneri was
last seen in 1840 when George
Gardner, a British botanist,
spotted boys playing with a
glowing object they called flor
de coco.
To catch the green glow of the

bioluminescent fungi, Desjardin
and his longtime Brazilian research partner, Dr. Cassius Stevani, stumbled around the forest
on new moon nights searching
for the ghostly glow. Digital
cameras allowed them to photo-
17
BIOSCAN
graph mushrooms they suspected might be

bioluminescent in darkened rooms. They then
analyzed the photos for a glow sometimes
invisible to the human eye within a few minutes, rather than the 30 to 40 minutes required
of regular film exposure.
Capturing the imaginations of cultures
around the world, bioluminescent fungi have
existed for centuries, from the bright orange
and poisonous jack-o-lantern mushrooms
to the phenomenon called foxfire, where
the nutrient-sipping threads of the honey
mushroom give off a faint, eerie glow in
rotten logs.
But the scientists wanted to know how a
fungus glows, and why. Although they believe
that fungi make light in the same way fireflies
do through a chemical mix of a luciferin
compound and a luciferase they have not
been able to identify either of those in fungi.
As long as water and oxygen are available,
Desjardin said, the fungi glow 24 hours a day,
unlike bioluminescent animals who produce
light only in spurts. This tells us that the
chemical that is acted upon by the enzyme in
mushrooms has to be readily available and
abundant, he said.
The why behind the glow also remains
a mystery. Some scientists think that mushrooms with spore-bearing glowing parts might
attract insects that can help disperse the spores
to grow new mushrooms. However, in the
case of the foxfire, the threadlike mycelium,
which seeks out nutrients for the fungi, is
what glows. Insects drawn to the mycelium
might do more harm than good to the fungi
if they ate the attractively lit structures.
Neonothopanus gardneri. Images courtesy of

Cassius V. Stevani, IQ-USP, Brazil.
Ovarian cancers first stages imaged

CAMBRIDGE, Mass. A new model of time-lapse microscopy
has been created to provide better visualization of metastatic
processes in their early stages.
Researchers at Harvard University have created a laboratory model using time-lapse video microscopic technology that
enables doctors to observe the early stages of ovarian cancer
metastasis. The technique makes it possible to observe key
molecular mechanisms that are necessary for the force-dependent processes associated with metastasis.
Ovarian metastatic tumors are some of the most morbid because they can grow large enough to interfere with organ functions in the peritoneum. Ovarian cancer cells spread throughout
the peritoneum by attaching to the outer cell layer of organs in
this area, clearing away the layer of cells there and embedding
18
themselves onto the organ. Once embedded, these cells proliferate and expand.
The scientists visualized the detailed sequence of the cells by
using the time-lapse video microscopic technique. They observed
the insertion of tumor cells into peritoneal monolayers in cell culture and then determined that the mechanism involves the tumor
cells use of force via 5 integrin, talin I and muscle myosin II.
By targeting those molecules, the scientists believe that it
would be theoretically possible to prevent new metastatic tumors
from forming.
The study, which was funded by the Dr. Miriam and Sheldon
G. Adelson Medical Research Foundation and the National Institutes of Health, was published June 14 in Cancer Discovery (doi:
10.1158/2159-8274.CD-11-0010).
BIOSCAN
Measuring macromolecules in 3-D

BERKELEY, Calif. Three-dimensional
plasmon rulers that can measure nanometer-scale spatial changes in macromolecular systems could provide scientists
with unprecedented details on critical
dynamic events in biology including
the interaction of DNA with enzymes, the
motion of peptides, the folding of proteins
or the vibrations of cell membranes.
The new measurement technique, developed by researchers at Lawrence Berkeley
National Laboratory in collaboration with
the University of Stuttgart, is based on
coupled plasmonic oligomers in combination with high-resolution plasmon spectroscopy. The 3-D plasmon ruler enables
scientists to retrieve the complete spatial
figuring of complex macromolecular and
biological processes, and to track the

dynamic evolution of these processes, said
Paul Alivisatos, Berkeley Lab director and
head of the research.
As human machines and devices shrink
to the size of biomolecules, scientists have
found a need to develop tools that can pre-
cisely measure minute structural changes

and distances on the nanometer scale. To
meet their needs, researchers have developed linear rulers based on plasmons.
Compared to other types of molecular
rulers based on chemical dyes and
Frster resonance energy transfer
The spatial freedom afforded the 3-D plasmon

rulers five nanorods enables it to measure the
direction as well as the magnitude of structural
changes in a macromolecule sample. Images
courtesy of Paul Alivisatos research group,
Berkeley, Calif.
A 3-D plasmon ruler is constructed from five

gold nanorods, one of which (red) is placed
perpendicularly between two pairs of parallel
nanorods (yellow and green).
19
BIOSCAN
plasmon rulers neither photobleach nor

blink, and they offer exceptional brightness and photostability. Until recently,
plasmon rulers could be used to measure
distances only along one dimension, making it difficult to understand the biological
and other soft-matter processes that take
place in three dimensions.
To combat this problem, the scientists
used dipolar and quadrupolar modes to
create sharp spectral features in plasmoncoupled nanostructures. Typical dipolar
plasmon resonances are broad because of
radiative damping. As a result, the simple
coupling between multiple particles produces indistinct spectra that are not readily
converted into distances. The scientists
overcame this problem with a 3-D ruler
constructed from five gold nanorods of individually controlled length and orientation.
The strong coupling of the nanorods
suppressed radiative damping, allowing
the excitation of two sharp quadrupolar
resonances that enabled high-resolution
plasmon spectroscopy to occur.
To prove their concept, the scientists
fabricated a series of samples using highprecision electron beam lithography and
layer-by-layer stacking nanotechniques.

They then embedded these with the 3-D
plasmon rulers in a dielectric medium
on a glass substrate. The end result agreed
with the calculated spectra.
The collaborators say that the 3-D plasmon rulers could someday be attached
through biochemical linkers to samples
of macromolecules such as strands of
DNA or RNA, or at different positions
along a protein or peptide, and the samples optical responses could be measured
via dark-field microspectroscopy upon
exposure to light.
Lab-on-chip combines laser, electric field

WEST LAFAYETTE, Ind. A new technology called hybrid optoelectronic manipulation in microfluidics combines a
laser with electric fields and promises a
new lab-on-a-chip designed to manipulate
DNA, bacteria and viruses for a variety
of fundamental science applications.
Purdue University researchers integrated electric fields and laser light to
manipulate not only large objects like
droplets, but also tiny DNA molecules
within those droplets. They said this
discovery has enhanced the efficiency
of lab-on-a-chip sensors.
The technology works by first using a
red laser to position a droplet on a platform created at Purdue. Then, a highly
focused infrared laser heats the droplet,
and electric fields cause the heated liquid
to circulate in a microfluidic vortex, used
to isolate specific types of particles in the
circulating liquid. Particle concentrations
replicate the size, shape and location of
20
A new technology combines a laser and electric fields to manipulate fluids and tiny particles such as bacteria,
DNA and viruses for a range of potential applications, including drug manufacturing and food safety.
Courtesy of Stuart J. Williams, University of Louisville.
the infrared laser pattern. The process

takes less than a second.
The combined technologies are now
ready for some applications, including
medical diagnostics and environmental
samples, said Stuart J. Williams of the
University of Louisville. There are two
main thrusts in applications, he said.

The first is micro- and nanomanufacturing, and the second is lab-on-a-chip sensors. The latter has demonstrated biologically relevant applications in the past
couple of years, and its expansion in this
field is immediate and ongoing.
BIOSCAN
Systems that use the hybrid optoelectronic approach can be designed to precisely manipulate, detect and screen certain types of bacteria, including particular
strains that render heavy metals less toxic.
The new method also is a potential tool

for applications including medical diagnostics, crime scene forensics, pharmaceutical manufacturing, and testing food and
water. The scientists also said it may be
used someday as a tool for nanomanufacturing because it shows promise of suspended colloids.
The findings appeared online May 20 in
Lab on a Chip (doi: 10.1039/C1LC20208A).
Tiny lens-free camera takes pixel-perfect pictures

ITHACA, N.Y. A microscopic
device that fits on the head of a
pin, contains no lenses or moving parts, and costs just pennies
to make could someday revolutionize surgery, robotics and
other science applications.
The camera, invented and developed by a group led by Cornell University postdoc Patrick
Gill in the lab of Alyosha Molnar, is one-hundredth millimeter
thick and one-half millimeter
on each side. It resolves images
about 20 pixels across. The
quality does not compare to that
of portraits done in a studio, but
it is sufficient to shed light on

things previously hard to see.
Gill, whose other research
interests include making sense
of how the brains neurons fire
under certain stimuli, began this
invention as a side project related to developing lensless implantable systems for imaging
brain activity. This type of system could be used as part of
an implantable probe for imaging neurons that have been
modified to glow when they are
active, he said.
His camera is composed of a
flat piece of doped silicon that
looks like a tiny CD and contains no parts that require offchip manufacturing. As a result,
the lightweight camera costs
mere cents, as opposed to the
$1 or more for a conventional
small camera on a chip requiring bulky focusing optics.
Dubbed planar Fourier
capture array (PFCA), the camera uses the principles of the
Fourier transform, a mathematical tool that allows multiple
ways of capturing the same
information. The sensitivity of
each pixel in the PFCA to a
unique blend of incident angles
allows it to report one component of the Fourier transform

of the image detected.
The scientists said they will
continue to work on improving
the cameras resolution and efficiency. They believe the concept could be used as a component in cheap electronic systems, such as a device that detects the angle of the sun, or a
microrobot that uses a simple
visual system for navigation.
The work is detailed online
in the July 6 issue of Optics
Letters (doi: 10.1364/OL.36.
002949).
Compiled by BioPhotonics staff
21
BUSINESSSCAN
OCT researchers garner grants for glaucoma detection
CLARKSBURG, Md. OCT could become even more useful in glaucoma detection, diagnosis and research, thanks to new
grants from the American Health Assistance Foundation, which funds research
on age-related diseases. The organization
recently awarded 22 grants
totaling nearly $2.2 million
to scientists worldwide for work
on glaucoma and macular degeneration, the two leading causes of
vision loss and blindness in the
US. Two of the grants are for research on OCT for glaucoma.
Right now, researchers in the US
and around the world are getting tantalizingly close to measuring changes in
the brain and eye that were previously
difficult to spot, said Dr. Guy Eakin,
AHAFs vice president for scientific affairs.
Improved testing will lead to earlier and
more effective treatments to prevent blindness.
Dr. Julie Albon, a lecturer in the Optometry and Vision Sciences department at
Biomagnetics Diagnostics Corp., based in

Orangevale, Calif., has accepted delivery of a
biosensor that is proposed for use in diagnosing
cholera, tuberculosis and malaria. The sensor
could be in clinical trials by 2012. Developed
in conjunction with scientists and engineers
at Los Alamos National Laboratory in New
Mexico, the device could allow relatively untrained medical personnel to detect a variety of
diseases rapidly. It uses planar waveguides to
bring protein-receptor pairs into proximity with
one another, thus triggering detectable fluorescence. The company plans to commercialize the
sensor, which was created through a cooperative research and development agreement with
Los Alamos National Security LLC.
Navitar Life Sciences Inc., an affiliated
entity of optics manufacturer Navitar Inc. of
Rochester, N.Y., has agreed to acquire the
assets of Modulation Optics, including its
Hoffman Modulation Contrast (HMC) imaging
technology. The Glen Cove, N.Y., company
makes HMC microscope components and systems for use in live-cell imaging applications
such as stem cell imaging, cancer studies, and
embryo and sperm monitoring during in vitro
fertilization procedures. The technology is used
to view colorless and transparent biological
specimens. The acquisition enables Navitar to
expand its product offering beyond its mainstay ZFL fluorescence imaging optical systems.
HMC will now be used in combination with
Navitars other illumination techniques, such
22
Cardiff University in the UK, and four coinvestigators in Wales, Vienna and London
received one of the grants for their work
using OCT to study changes in the optic
as differential interference contrast, dark field,

bright field and fluorescence to achieve highquality images. HMC can be applied to most
objective lenses.
The OSA Foundation (OSAF) in Washington
has announced a fiscal sponsorship agreement
with the International Commission for Optics
(ICO) that will allow charitable donations made
to OSAF to be earmarked for ICO outreach
initiatives. Donations designated to OSAFs
International Programming Fund will support
international education, professional development and recognition programs currently
conducted through a grant to ICO. OSAF will
support travel grants for students and scientists
from underdeveloped countries to attend conferences and meetings such as Optics Within
the Life Sciences, Information Photonics and
ICO Bureau meetings. It also will support traveling lecturer grants to senior scientists and
engineers. OSAF operates on behalf of the
Optical Society of America.
Dental laser manufacturer Biolase Technology
Inc. of Irvine, Calif., has received two more
patents from the US Patent and Trademark
Office for its laser technologies that treat various eye conditions, including presbyopia the
decline of near vision in midlife. The patents are
the seventh and eighth to be issued under the
Methods for Treating Eye Conditions patent
family. US Patent number 7,967,017 includes
29 claims and introduces a strategy for identify-
nerve head that could enable speedier diagnosis of glaucoma.

The second OCT-related grant went to
Dr. Michael Julien Alexandre Girard at
Imperial College London and Dr. Nick
Strouthidis at Londons Moorfields Eye
Hospital. They will use an OCT scanner to explore stiffness of the cornea,
and whether its presence at the front
of the eye predicts mechanical damage and, therefore, vision loss at
the back of the eye. The correlation
has not yet been established through
scientific testing. Optometrists and
ophthalmologists may someday be
able to use such measurements to determine a patients risk of developing
glaucoma.
Other topics of study that attracted the
new AHAF research grants include the
brains control of eye and brain pressure
changes, adult stem cells for improved
treatments and gene tracking in various
populations.
Laura S. Marshall
laura.marshall@photonics.com
ing optimal treatment zones for the reversal of

presbyopia. US Patent No. 7,909,040 includes
10 claims and relates to the shape of the treatment laser beam and specific ablative patterns
for treating the condition.
Carl Zeiss and Digital Surf have signed an
agreement that enables Zeiss to provide Digital
Surfs ConfoMap surface imaging and analysis
software for use with its confocal microscopes.
The software offers numerous analytical studies,
including geometric and functional. It can be
extended by adding modules for advanced
analysis of surface texture, dimensions, grains,
particles, 3-D Fourier and surface evolution. It
can be used with Zeiss Axio CSM 700 confocal
microscope, its LSM 700 confocal laser scanning
microscope, and with other compound microscopes for topographical research.
Daylight Solutions Inc. of San Diego has
closed a $15 million Series C equity financing
round with Northrop Grumman Corp., the
lead investor, and several of the companys
existing investors. The financing will allow
the company to develop a family of quantum
cascade laser-based products based on a
modular, open source, core technology platform, meeting the requirements of demanding
environments in defense as well as commercial markets. The companys products address
molecular detection and imaging applications
that benefit from mid- and long-wave infrared
illumination.
BUSINESSSCAN
for Escherichia coli and Enterococcus faecalis,

the system will be evaluated by the US Environmental Protection Agency and the US
Army Corps of Engineers.
Safety, health and sensor technology group

Halma of Amersham, UK, has acquired Avo
Photonics Inc. from Blaine Hobson, Whitecap Partners, and other shareholders and
employees for an initial cash consideration of
$9 million. The Pennsylvania-based Avo designs
and manufactures miniaturized photonic components and subsystems for original equipment
manufacturer customers in the medical and
communications markets. Avo joins Halmas
photonics businesses (Fiberguide Industries,
Labsphere, Ocean Optics and Ocean Thin
Films) in the health and analysis sector. The
acquisition, immediately accretive to earnings,
was funded from Halmas existing cash and
debt facilities.
Tyndall National Institute UCC has launched

InfiniLED Ltd., a high-tech spinout that will
commercialize a new generation of LED technology that extends the battery life of portable
devices such as cameras, mobile phones, laptops, and medical and analytical instrumentation. The Micro LED technology produces more
usable light and uses less energy. It was invented by researchers at Tyndall National Institute and supported and funded by Enterprise
Ireland.
Lumex of Palatine, Ill., and element14 have

formed a strategic partnership that allows
electronic design engineers in the Asia-Pacific
region to acquire more than 8000 Lumex
optoelectronic components from element14s
inventory of 130,000 products. Lumexs LEDs
and LCDs are suitable for a variety of applications, including medical devices. The companys standard and customized products
encompass the UV, VIS and IR wavelengths.
A collaborative community, research portal
and e-commerce website, element14 supports
engineers and purchasing professionals worldwide. It has distribution hubs in Shanghai,
Singapore and Sydney, Australia.
In the UK, the University of Warwicks physics

department has been awarded a five-year grant
of 1.7 million (about $2.7 million) for its initiative Creating Silicon Based Platforms for New
Technologies. The project will focus on energy
harvesting, cooltronics and zero-power electronics and could be key in combating global
climate change. The grant from the Engineering and Physical Sciences Research Council
will help the departments Nano-Silicon Group
continue its research. Due to start in October,
the grant will help develop epitaxy techniques,
whereby novel materials are created by depositing one atomic layer at a time. Such materials
could have applications in health monitoring.
Zecotek Photonics Inc. has been granted

Patent No. 7,956,331 B2 by the US Patent
and Trademark Office for a depth of
interaction technology that was developed
to provide improvements in the precision
and performance of PET scanning and other
medical detection systems. Zecotek and the
University of Washington developed the
technology under a joint PET-MRI development
alliance. For the new scanners, the partnership
used Zecoteks patented scintillation crystals
and solid-state multipixel avalanche photodiode photodetector, with the universitys data
acquisition methodologies and image reconstruction algorithms. The project is managed
by Zecotek Imaging Systems (Singapore)
Pte Ltd., a wholly owned subsidiary of
Zecotek.
The Optical Systems Div. of Zygo Corp. has
been awarded a $3 million continuation order
from Commissariat lnergie atomique
for its meter class laser fusion amplifiers. The
order is part of an ongoing contract begun
in 2007 in support of the Laser Mgajoule
Program for the French government. It is
anticipated to run through 2014. Zygo sup
plies optical metrology instruments, precision
optics, and electro-optical design and manufacturing services for the semiconductor
capital equipment, biomedical, scientific
and industrial markets.
Mad City Labs Inc. of Madison, Wis., a manufacturer of flexure-based nanopositioning

systems, has appointed Elliot Scientific Ltd.
of Harpenden, UK, as its exclusive distributor
within the UK and Ireland. The addition of the
product range to Elliot Scientifics portfolio of
micro- and nanopositioning systems strengthens
the British companys presence as a supplier to
the UKs nanotechnology, biophysics, and life
sciences communities in academia and industry.
Its expanded product line covers the spectrum of
nanopositioning in single- and multiaxis stages,
some including rotation/tilt mechanisms.
Medical device company Michelson Diagnostics plans to expand its new US subsidiary,
Michelson Diagnostics Inc., to increase the
availability of its skin cancer imaging products.
The company has recruited Karen Miller Gillis
as vice president of sales and marketing and
general manager of the Boston-based US subsidiary. Michelson anticipates that its VivoSight
OCT scanner, which uses four laser beams to
improve image resolution, will provide dermatologists with new information about what
happens below the surface of the skin of
patients who receive treatment for nonmelanoma skin cancer.
Sporian Microsystems, a developer of sensors and sensor systems based in Lafayette,
Colo., has shipped first article units for a new
in-line biological sensor based on its patented
technology. The system, which could work in
combination with water purification and testing
systems, runs autonomously with LEDs to indicate pathogen concentration. It includes a USB
interface and attaches to a personal computer
or netbook for logging sensor data for trend
analysis. Initially to include sensing capabilities
23
Photoacoustic
Microscopy
Unlocks Secrets of
Optical Absorption
In vivo, label-free
subwavelengthresolution
photoacoustic
microscopy enables
more precise
measurement
of optical absorption
and, therefore,
offers more
information.
BY CHI ZHANG, KONSTANTIN MASLOV
AND LIHONG V. WANG,
WASHINGTON UNIVERSITY, ST. LOUIS
ainstream optical microscopy

technologies cannot sensitively
measure the optical absorption
that provides essential physiological information such as the concentration and oxygen saturation of hemoglobin.
In our research, we have implemented
subwavelength-resolution photoacoustic
microscopy (SW-PAM) to provide highoptical-absorption contrast by using a
water-immersion optical objective with a
1.23 NA. SW-PAM can detect nonfluorescent endogenous pigments in vivo with
100 percent sensitivity to optical absorption.
We found that, by imaging gold nanospheres, SW-PAM approached the ultimate
diffraction-limited optical resolution: 220
nm at a 532-nm wavelength. We imaged
ex vivo cells, including melanoma, red
blood and onion epidermal, using SWPAM with subcellular organelles resolved.
We also imaged in vivo vasculature and
early-stage melanoma with 12:1 and 17:1
contrasts, respectively, without labeling.
For all of these applications, SW-PAM
showed contrasts orders of magnitude
higher than those achieved with wide-field
Figure 1. Schematic diagram of subwavelength-resolution photoacoustic microscopy (SW-PAM).

Reproduced with permission from Reference 15.
Figure 2. Measuring the transverse spatial resolution of SW-PAM. (a) PAM image of gold nanospheres.
(b) System point spread function calculated from the gold nanospheres. Circle: the averaged pixel value.
Line: the theoretical Bessel-form function. Reproduced with permission from Reference 15.
24
Figure 3. Ex vivo images of cells. (a) Melanoma cells imaged by PAM, optical microscopy and PAM combined with an
optical microscopy image of the blue-stained nuclei. CN: cell nucleus. (b) PAM and optical microscopy images of red blood cells.
(c) PAM and optical microscopy images of onion epidermal cells. Reproduced with permission from Reference 15.
optical microscopy, ensuring the technique

as a welcome complement to existing microscopy technologies.
Photoacoustic tomography (PAT)1 is
an emerging biomedical imaging technology that ultrasonically detects optical absorption in biological tissues. Because
of the photoacoustic effect,2 absorbed light
is converted to heat and then induces
acoustic waves. PAT can work in both the
optical ballistic regime (<1 mm deep) and
in the diffusive regime (up to several

centimeters deep), with the same optical
absorption contrast yet scalable resolution
and depth.3 So far, PAT has been successfully applied to in vivo structural, functional and molecular imaging of biological
tissues.4-9
When light is focused into biological
tissue and the resulting excited photoacoustic signals are received by a focused
ultrasonic transducer, the localized optical
absorption within the optical or acoustic

focal spot whichever is smaller is
acquired. A high-resolution image of
the tissue then can be obtained after
point-by-point scanning of the tissue.
This approach is called photoacoustic
microscopy (PAM).
Although mainstream optical microscopy detects scattering (including confocal
microscopy and optical coherence tomography) or fluorescence (including confocal
25
Photoacoustic Microscopy
and two-photon microscopy), PAM is sensitive to endogenous optical absorption

with a relative sensitivity of 100 percent,10,11 providing essential biological
functional information. Because nonfluorescent hemoglobin and melanin are major
sources of biological endogenous absorption in the visible and near-infrared spectral ranges, PAM finds broad application in
imaging tumor angiogenesis, blood oxygen
saturation and early-stage melanoma. Recently, by exciting DNA and RNA with ultraviolet light, PAM also imaged in vivo
cell nuclei without labeling.12
In optical-resolution PAM, the image
resolution is limited by the numerical
aperture (NA) of the optical objective at
the given wavelength. The first opticalresolution PAM system reached a resolution of 5 m.13 In the past few years, the
resolution has been improved for imaging
of subcellular structures. Here we describe
our subwavelength-resolution PAM, which
has reached the finest diffraction-limited
optical resolution.
A system diagram of SW-PAM is
shown in Figure 1.14,15 We used a 532-nm
Nd:YVO4 laser as the irradiation source.
A 1.5-ns laser pulse, focused by the 1.23NA objective, irradiated the sample. An
ultrasonic transducer with a central fre-
quency of 40 MHz and an NA of 0.5

received the photoacoustic waves in transmission mode. The objective and the
transducer mechanically scanned in raster
mode in the X-Y plane to obtain a 3-D
image, which will be shown as a two-dimensional maximum-amplitude projection
image along the Z-axis.
We demonstrated the subwavelength
resolution of SW-PAM by imaging the
gold nanospheres (Figure 2a). The nanospheres had a diameter of 15 nm and,
therefore, were ideal point absorbers to
measure the point spread function of
SW-PAM. The gray values of the typical spheres in the image were fitted by
the theoretical Bessel-form function (Figure 2b). We measured the system transversal resolution as 220 20 nm, given
by the full width half maximum of the
Bessel-form point spread function. The
theoretical resolution was calculated as
0.51 /NA 221 nm, in good agreement
with the measured value.
In Figure 3, the subcellular structures
are shown and the contrast of SW-PAM is
compared with that of wide-field optical
microscopy. In general, the PAM images
have a dark background, while the optical
microscopy images have a bright background. Melanosomes organelles con-
Figure 4. Melanin distribution in a black mouse ear at different depths.

(a) ~10 m deep (the close-up image indicates melanosomes). (b) ~30 m deep.
SG: sebaceous gland. Reproduced with permission from Reference 14.
26
taining melanin appear as bright dots

(Figure 3a, left) in the PAM image, but as
dark dots in the optical microscopy image
(Figure 3a, middle).
The melanosomes in the PAM image
have a contrast-to-background ratio of
54.5 0.4:1 and a contrast-to-noise ratio
(CNR) of 49 dB, while the optical microscopy image has a much lower contrast
of 0.79 0.04:1 and a lower CNR of
25 dB. This difference occurs because
PAM is sensitive to absorption only, but
optical microscopy shows both absorption
and scattering (the latter is relatively close
between melanosomes and other areas).
In the PAM image, the dark holes inside the cells with few melanosomes are
nuclei, confirmed by staining them with
fluorescent dye (Figure 3a, right). In comparison, the nuclei in the optical microscopy image are less visible because of
the low contrast. The hemoglobin contrast
disparity also can be observed by imaging
red blood cells, as shown in Figure 3b (the
two images show different areas of the
cell sample). The characteristic doughnut
shape of a red blood cell is clearly shown.
Moreover, rich contrasts are observed by
PAM in the purple onion epidermal cells
(Figure 3c).
We also imaged the melanin distribution in a black mouse ear (Harlan Laboratories Inc., Indianapolis, C57BL/6NHsd)
in vivo with SW-PAM. We acquired two
images, focusing at ~10 and ~30 m
deep, respectively, as shown in Figure 4.
Most melanocytes reside in the basal
layer of the epidermis, whose thickness is
~10 m in this case.16 Therefore, single
melanosomes can be clearly identified
in the shallower layer (Figure 4a). In the
deeper layer (Figure 4b), more skin structures, such as the sebaceous glands, are
shown, but melanosomes are now out
of focus.
It is worth noting that the signals from
the deeper layer are weaker, but Figure 4b
appears to have higher contrast because it
is scaled to the full color range. The optical microscopy image of the ear, although
not shown here, has extremely low contrast. These results suggest potential applications of PAM in quantifying melanin
distribution in vivo, which is important for
detecting melanoma as well as determining individualized radiant exposure in
dermatological laser therapies.17
The ear of a nude mouse (Harlan Laboratories, Hsd:Athymic Nude-Foxn1nu) also
was imaged in vivo. Here, we used an
Photoacoustic Microscopy
objective with a 0.60 NA (providing 400nm resolution) to extend the focal zone for
the thick layer of vessels. As shown in
Figure 5a, we imaged all the blood vessels, including capillaries, with high contrast and with few background signals. In
some areas, motionless red blood cells can
be identified with the characteristic doughnut shape (Figure 5b). Next, we injected
10 l of suspension containing 1 million
B16 melanoma cells into the ear to induce
a melanoma tumor.
We imaged the same ear again, as
shown in Figure 5c, four days later. The
melanoma generated stronger photoacoustic signals than the vessels and was
easily identified. The vasculature and
melanoma had contrasts of 12 1:1 (33-dB
CNR) and 17 1:1 (36-dB CNR), respectively. The contrast difference between the
vasculature and the melanoma can be further increased if the laser wavelength is
changed to, for example, 650 nm. Under
wide-field optical microscopy (Figure 5d),
the melanoma is obscure, with a contrast
of 0.27 0.02:1 (21-dB CNR).
As shown by Figures 2 to 5, SW-PAM
can resolve structures as small as subcellular organelles, both ex vivo and in
vivo. Melanoma and vasculature can be
visualized with high contrast. Therefore,
SW-PAM has superior potential to detect
melanoma in the early stage, to count
red blood cells as an in vivo flow cytometer and to measure blood flow velocity in
capillaries.
Moreover, although the current transmission-mode configuration of SW-PAM
limits the thickness of tissues that can be
imaged, we are extending SW-PAM to reflection mode for applications in more
anatomical sites. As a result, combining
it with scaled-up macroscopy deeppenetrating photoacoustic tomography 4-18
may bridge microscopic research and
clinical practice.
Meet the authors
Chi Zhang is a graduate student, Konstantin
Maslov a research associate professor and Lihong V. Wang the Gene K. Beare Distinguished
Professor at the Optical Imaging Laboratory in
the Department of Biomedical Engineering at
Washington University in St. Louis; e-mail:
lhwang@biomed.wustl.edu. This work was
sponsored in part by National Institutes of
Health grants R01 EB000712, R01 EB008085,
R01 CA113453901, U54 CA136398 and 5P60
DK02057933. Lihong Wang has a financial
interest in Microphotoacoustics Inc. and Endra
Inc. Neither supported this work.
Figure 5. Monitoring melanoma growing on a nude mouse ear. (a) PAM image of the ear before
injecting melanoma cells. (b) PAM image, where in vivo red blood cells (RBCs) can be identified
(indicated in the close-up image). (c) PAM image of the ear four days after injecting melanoma cells.
MT: melanoma tumor. (d) Optical Microscopy image, in which the melanoma is obscure.
Reproduced with permission from Reference 14.
References
1. C. Li and L.V. Wang (2009). Photoacoustic
tomography and sensing in biomedicine.
Phys Med Biol, Vol. 54, pp. R59-R97.
2. A.G. Bell (1880). On the production of sound
by light. Am J Sci, Vol. 20, p. 305.
3. L.V. Wang (2009). Multiscale photoacoustic
microscopy and computed tomography. Nat
Phot, Vol. 3, pp. 503-509.
4. X. Wang et al (2003). Noninvasive laserinduced photoacoustic tomography for structural and functional in vivo imaging of the
brain. Nat Biotech, Vol. 21, pp. 803-806.
5. H.F. Zhang et al (2006). Functional photoacoustic microscopy for high-resolution and
noninvasive in vivo imaging. Nat Biotech,
Vol. 24, pp. 848-851.
6. L. Li et al (2007). Photoacoustic imaging of
lacZ gene expression in vivo. J Biomed Opt,
Vol. 12, 020504.
7. M.-L. Li et al (2008). Simultaneous molecular and hypoxia imaging of brain tumors in
vivo using spectroscopic photoacoustic tomography. Proc IEEE, Vol. 96, pp. 481-489.
8. J. Laufer et al (2009). Three-dimensional
noninvasive imaging of the vasculature in the
mouse brain using a high resolution photoacoustic scanner. Appl Opt, Vol. 48, pp. 299306.
9. Y. Wang et al (2011). In vivo integrated photoacoustic and confocal microscopy of hemoglobin oxygen saturation and oxygen partial
pressure. Opt Lett, Vol. 36, pp. 1029-1031.
10. L.V. Wang and H. Wu (2007). Biomedical
Optics: Principles and Imaging. John Wiley
& Sons Inc., Hoboken, N.J., pp.3-8.

11. L.V. Wang (2008). Tutorial on photoacoustic microscopy and computed tomography. IEEE J Sel Topics Quantum Electron,
Vol. 5, pp. 171-179.
12. D.-K. Yao et al (2010). In vivo label-free
photoacoustic microscopy of cell nuclei by
excitation of DNA and RNA. Opt Lett, Vol.
35, pp. 4139-4141.
13. K. Maslov et al (2008). Optical-resolution
photoacoustic microscopy for in vivo imaging of single capillaries. Opt Lett, Vol. 33,
pp. 929-931.
14. C. Zhang et al (2010). Subwavelength-resolution label-free photoacoustic microscopy of
optical absorption in vivo. Opt Lett, Vol. 35,
pp. 3195-3197.
15. C. Zhang et al (2011). Subwavelength-resolution photoacoustic microscopy for labelfree detection of optical absorption in vivo.
Proc SPIE 7899, p. 78990L.
16. K. Kietzmann et al (1990). The mouse epidermis as a model in skin pharmacology:
influence of age and sex on epidermal metabolic reactions and their circadian rhythms.
Lab Animal, Vol. 24, pp. 321-327.
17. W. Verkruysse et al (2009). Remittance at a
single wavelength of 390 nm to quantify epidermal melanin concentration. J Biomed Opt,
Vol. 14, p. 014005.
18. M.P. Fronheiser et al (2010). Real-time
optoacoustic monitoring and three-dimensional mapping of a human arm vasculature.
J Biomed Opt, Vol. 15, p. 021305.
27
Intrinsic Fluorescence Lights Up

Cellular Components
BY ANGELA GOODACRE AND DENNIS DONLEY, OLYMPUS AMERICA INC.
Visualization of microregional hypoxia in the mouse cortex. (a) Exposed mouse cortex as seen under bright-field microscopy.
(b) Homogeneous distribution of two-photon excited NADH fluorescence in the cortex layer under baseline conditions.
(c) Heterogeneous distribution of NADH fluorescence after induction of moderate cortical hypoxia (PaO2 48 mmHg).
(d) Localization of microregional tissue hypoxia. The hypoxia-induced percent increases in NADH fluorescence (see legend on right)
exhibit a distinct geometrical relationship to the cortical microvasculature (arterioles labeled red, venules labeled blue), revealing
the tissue boundaries of oxygen diffusion from the vasculature. Scale bars represent 100 m. (b-d) Images in these panels
were captured via intrinsic fluorescence of the specimen using the Olympus FV1000MPE multiphoton system.
Courtesy of Dr. Karl Kasischke, University of Rochester Medical Center, New York.
28
Multiphoton microscopy is
especially useful when
intrinsic fluorescence imaging
is combined with other
label-free imaging modalities,
such as second-harmonic
generation or coherent
anti-Stokes Raman scattering,
which may open new
windows of opportunity for
research into dynamic cellular
events and processes.
he intrinsic fluorescence of cells,

their organelles and other biological
elements can be valuable for uncovering new information about tissue function
and cellular processes. In comparison to
labeling cells with exogenous dyes that
may alter native physiology or morphology, intrinsic fluorescence, or autofluorescence, offers unique advantages in studying living cells and tissue. Using intrinsic
fluorescence to image cellular processes in
real time allows quantitative evaluation
without perturbing the cell.
The power of imaging intrinsic fluorescence has only recently begun to be explored as advances in optics and lasers
have made multiphoton microscopy systems more readily available to biological
researchers.
Most intrinsically fluorescing constituents absorb light in the ultraviolet wavelength range and emit in the violet and
blue. Because of phototoxicity, photobleaching and other problems associated
with ultraviolet transmission through optical components, scientists often image intrinsic fluorescence with a multiphoton excitation system with a pulsed near-infrared
laser. In such systems, two or more longwavelength photons combine their energies to function as a photon of half the
wavelength. Thus, the fluorescence-excitation effects of two photons of 710 nm
(longer wavelength, which is less damaging to living tissue) are equivalent to a
photon of 355 nm (shorter wavelength,
which is usually biologically averse).
In general, the signal from intrinsic
fluorescence is much weaker than from
traditional, exogenously induced fluores-
cence. The two-photon absorption cross

section of nicotinamide adenine dinucleotide (NADH), for instance, is 1 to 0.1
percent the magnitude of conventional fluorophores.1 This means that the multiphoton laser-scanning microscope in autofluorescence experiments must be optimized
for sensitivity. The system should have
transmission characteristics optimized for
the infrared wavelengths of the pulsed
laser and dispersion compensation
matched to the exact optical components
of the light path.
In highly scattering material such as
brain tissue, a tenfold increase in signal
can be attained by maximizing the collection of scattered photons via external or
nondescanned detectors, together with an
objective lens that offers relatively low
magnification combined with a high numerical aperture. To ensure the highest
possible excitation efficiency deep within
tissue, a correction collar allows the user
to minimize the focal volume.
Two-photon excited intrinsic fluorescence has been reported at the following
wavelengths:
Reduced forms of nicotinamide adenine dinucleotide including NADH
and NADPH, with emission peaks
around 450 nm (blue-violet)
Elastin, retinol and folic acid, with
emission peaks around 500 nm
(bluish green)
Riboflavin, with an emission peak at
around 540 nm (green)
Oxidized forms of flavin adenosine
dinucleotide, which emit at around
560 to 590 nm (in the yellow range)
Lignin, a major structural polymer in
plant cell walls, which emits a broad
spectrum of fluorescence that peaks
between 450 and 550 nm.
Multiphoton microscope systems not
only facilitate excitation of ultravioletabsorbing molecules but also alleviate
many of the problems that arise during
excitation and signal collection for autofluorescing specimens because multiphoton imaging systems, in comparison to
their confocal counterparts, rely on much
longer wavelengths of light to induce
fluorescence, which are subject to far
less scatter as they pass through tissue.
Multiphoton excitation is limited to a
small volume corresponding to a single
point in the image, and photons are deliv-
ered in short bursts, or pulses, lasting for

approximately 100 fs. Statistically, the opportunity for two or more photons to interact with the same fluorophore molecule
occurs only at the plane of focus, where
there is a high density of photons, so fluorescence excitation is effectively limited
to the plane of focus, eliminating photobleaching and phototoxicity in the areas
above and below the focal plane.
Redox potential and more
Researchers are using autofluorescence
imaging to investigate oxidative stress,
which has been implicated in a wide variety of diseases, including diabetes, atherosclerosis, neurodegeneration, age-related
macular degeneration and cancer cell
pathology. In particular, researchers have
found that the reduced forms of NADH
and NADPH exhibit intrinsic blue fluorescence. NADH is a major by-product of
respiration, in contrast to NADPH, which
is involved with biosynthesis of lipids and
nucleic acids.
Imaging the fluorescence intensity of
NADH and NADPH as a ratio with the
yellow fluorescence of oxidized flavin
derivatives can provide a sensitive means
to assess oxidative pathways. The ratio between the two serves as a measure of the
reduction-oxidation capacity of the cell
and is known as the cells redox potential,
which can affect a range of cellular
processes including gene expression,
apoptosis, cancer pathology and enzyme
kinetics, allowing the investigation of tissue alterations due to disease or changes
in metabolism.
Dr. Karl Kasischke at the University of
Rochester Medical Center in New York
studies the metabolic activity of brain tissue. Most studies of brain metabolism use
cell extracts, so information about the spatial organization and behavior of different
cell types is lost. Kasischke uses the
Olympus FV1000MPE multiphoton microscope system and intrinsic fluorescence
to image the pattern of metabolic activity
in relation to cell types in specific regions
of the brain and has correlated specific
neural activity with local metabolism.
High-resolution microscopy of the mouse
cortex in situ allows his group to map the
spatial relationship between blood vessels
and local NADH levels to elucidate mechanisms by which the brain regulates blood
flow to maintain critical oxygen supply.2
Stimulating neural activity causes rapid
29
Intrinsic Fluorescence
consumption of glucose, resulting in

increased concentration of tissue NADH.
Conversely, the intensity of NADH fluorescence increases with lower oxygen content in tissue. These findings suggest
NADH imaging as a method to measure
the effects of local oxygen tension during
neuronal stimulation and other brain activity. Because the brain contains 86 percent
NADH and only 14 percent NADPH,
most of the fluorescence measured in the
420- to 500-nm range is due to NADH.
Kasischkes study reveals defined regions
of low NADH concentration, indicating
higher oxygen levels surrounding the termini of these blood vessels (Figure 1). The
geometry of these regions suggests that
they represent the limits of oxygen diffusion from blood in arterioles through tissue to supply neural cells.
Because autofluorescent microscopic
imaging of NADH can be used to localize
metabolic activity within subcellular organelles, investigators are also using it to
study mitochondrial anomalies associated
with neurodegenerative diseases, cancer,
diabetes and aging in living cells without destroying the cells. This is in sharp
contrast to traditional investigations of
mitochondrial function, which generally
must be performed on extracts and thus
lose all information regarding changes in
the number, distribution and morphology
of the mitochondria.
Some plant species also exhibit autofluorescence, and this characteristic has
helped enable studies in areas such as biofuel production, where the technique is
used to monitor and improve the process
of extracting lignin from plant material.
Lignin, a key component of plant cell
walls, can make it difficult to procure usable biomass from readily available plants
such as grasses because the lignin blocks
the availability of cellulose, the raw material of biofuels.
By extracting lignin, engineers can
make more cellulose available for fermentation into the sugars that provide energy,
but such extraction is notoriously difficult
to achieve. Researchers are now using
lignins natural autofluorescence at approximately 530 nm to help measure the
lignin content of various plant sources to
help evaluate their potential suitability as
biomass for biofuels.
In vivo imaging, as with all imaging
of living tissue, presents enormous challenges. Label-free methodologies are particularly attractive for reasons beyond the
desire to circumvent artifacts caused by

reagents. Labeling with a fluorophore can
be prohibitively expensive when the dye
must be administered throughout an intact
animal; tissue barriers such as the bloodbrain barrier can prevent the label from
reaching its target. Further, the foreign
nature of fluorophores sometimes results
in their being delivered to the liver or kidneys for excretion, further complicating
experiments.
Meet the authors
and acknowledgment
Angela Goodacre is field marketing
specialist at Olympus America Inc.;
e-mail: angela.goodacre@olympus.com.
Dennis Donley is marketing business
manager, Scientific Equipment Group,
Olympus America Inc.; e-mail: dennis.
donley@olympus.com.
The authors wish to acknowledge Dr.
Karl A. Kasischke, assistant professor of
neurology at the University of Rochester
Medical Center, for his support and assistance in the preparation of this article.
References
1. K.A. Kasischke et al (July 2004). Neural activity triggers neuronal oxidative metabolism
followed by astrocytic glycolysis. Science,
pp. 99-103.
2. K.A. Kasischke et al (January 2011). Twophoton NADH imaging exposes boundaries
of oxygen diffusion in cortical vascular supply regions. J Cereb Blood Flow Metab, pp.
68-81.
Further reading
1. D. Li et al (January 2009). Two-photon autofluorescence microscopy of multicolor excitation. Opt Lett, pp. 202-204.
2. M. Monici (2005). Cell and tissue autofluorescence research and diagnostic applications. Biotechnol Ann Rev, Vol. 11, pp. 227256.
3. W.R. Zipfel et al (June 10, 2003). Live tissue
intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. PNAS, pp. 70757080.
4. M.J. Levene et al (April 2004). In vivo multiphoton microscopy of deep brain tissue.
J Neurophysiol, pp. 1908-1912.
5. A.C. Kwan et al (March 2009). Optical visualization of Alzheimers pathology via multiphoton-excited intrinsic fluorescence and
second harmonic generation. Opt Express,
pp. 3679-3689.
6. S. Singh et al (September 2009). Visualization of biomass solubilization and cellulose
regeneration during ionic liquid pretreatment
of switchgrass. Biotechnol Bioeng, pp. 68-75.
Fluorescence Imaging
Progressing from Cells to Tissue
Tissues and even whole animals are not as
easily captured with uorescence imaging as
are cells, but recent research and technological
developments could change that.
BY MARIE FREEBODY, CONTRIBUTING EDITOR
luorescence imaging has become one

of the most powerful tools for studying cellular processes. Today, with
an ever-expanding array of available fluorochromes to tag cells of interest, scientists can identify cells and submicroscopic
cellular components with high specificity
amid nonfluorescing material.
Imaging tissue is another story, however. A great challenge faces imaging
specialists, but progress is being made,
and more is predicted.
The application of an array of fluorochromes (also known as fluorophores) has
made it possible for fluorescence microscopes to reveal the presence of a single
molecule. And the use of multiple fluorescence labeling makes it possible for various probes to identify several target molecules simultaneously.
Although the fluorescence microscope
cannot provide spatial resolution below
the diffraction limit of specific specimen
features, detection of fluorescing molecules below such limits is possible.
Modern fluorescence microscopes
combine the power of high-performance
optical components with computerized
control of the instrument and digital image
acquisition.
At present, optical image formation is
only the first step toward data analysis,
said Allison Forlenza, assistant product
and marketing manager at Nikon Instruments Inc. in Tokyo. Microscopy now
depends heavily on electronic imaging to
rapidly acquire information at low-light
levels or at visually undetectable wavelengths.
Computerized control of focus, stage
position, optical components, shutters,
filters and detectors is in widespread use.

The increasing application of electrooptics in fluorescence microscopy has
led to the development of optical tweezers
that can manipulate subcellular structures
or particles, to the imaging of single mole-
cules and to a wide range of sophisticated

spectroscopic applications.
There are a much wider range of
confocal microscopes available, many
of which are now spectral confocal, and
the price range of them has dropped con-
Whole-body image of GFP and RFP (red fluorescent protein) human tumors implanted in the brain of a mouse,
imaged with the Indec Systems FluorVivo imaging system. Courtesy of AntiCancer Inc.
The OV100 Olympus Small Animal Imaging

System offers variable magnification for imaging
mice from macro to subcellular regimes.
Courtesy of AntiCancer Inc.
31
This nude mouse appears normal

under bright-field light (a), but it
glows under fluorescence excitation
with 470-nm blue light (b). Courtesy
of AntiCancer Inc.
The whole skeleton of this mouse appears normal under bright-field light (a), but it glows under fluorescence excitation with blue light (b). Courtesy of AntiCancer Inc.
siderably, with some in the $99,000

range, said James R. Mansfield, director
of tissue analysis applications at Caliper
Life Sciences Inc. in Hopkinton, Mass.
In addition, live-cell imaging methods,
environmental chambers and the ability
to track the growth of individual cells in
three dimensions in embryos and cell
cultures are all growing rapidly.
Naturally fluorescent proteins
A simple description of fluorescence
imaging can be divided into three parts:
excitation, emission and imaging. Fluorochromes are molecules that, when excited
by a photon, will emit a photon of longer
wavelength (toward the red end of the
spectrum) than that which it absorbed.
This process of absorption and re-emission
is known as fluorescence.
Fluorochromes attach themselves to
visible or subvisible structures and are
often highly specific in their attachment
targeting. Such molecules usually have a
significant quantum yield (ratio of photon
absorption to emission).
The discovery and use of naturally
fluorescent proteins, which can be used
to label specific cells, has revolutionized
biology by enabling the formerly invisible to be seen clearly.
One example is taking place at AntiCancer Inc. in San Diego. Robert Hoffman, president and CEO, is using green
fluorescent protein and red fluorescent
32
protein to image cancer cells in real time

in live mice. Such proteins enable scientists to visualize important aspects of cancer in living animals, including tumor cell
mobility, invasion, metastasis and angiogenesis.
AntiCancer is the pioneer of in vivo
imaging with GFP, Hoffman said. This
breakthrough has revolutionized imaging
in small animals. Due to our research,
single cells can be followed in real time
in mice, even at the subcellular level.
These multicolored proteins have
allowed the color-coding of cancer cells
growing in vivo and enabled the distinction of host from tumor with single-cell
resolution. Hoffman and his colleagues
also have used the technique to image
stem cells and bacteria in real time in
live mice.
The company currently is developing
fluorescence-guided surgery with cancers
that have been labeled with GFP and

is looking to determine the efficacy of
cancer therapeutics.
We are also developing tumor-targeting bacteria to cure the tumors, Hoffman
said. We have developed special mutants
of salmonella to selectively target tumors.
We have labeled the bacteria with GFP to
follow them as they attack tumors.
Tissue imaging remains a challenge
Although fluorescence imaging of cells
has progressed by leaps and bounds, when
it comes to tissue imaging, the technique
is not as effective. Fluorescence imaging
of tissues presents a number of challenges
that are not present in live- or fixed-cell
samples and that require a different
approach to obtain optimal results.
In cell-imaging applications, there is
little to no interfering autofluorescence
signal, so highly sensitive cameras and
This whole-body
image of a nude
mouse shows a
highly metastatic
human prostate
cancer expressing
GFP. Courtesy of
AntiCancer Inc.
These images were taken using Nuance, a

multispectral imaging system that enables
imaging of multiple molecular markers in tissue
sections for fluorescence and bright field. Images
courtesy of Pavol Cekan, Rockefeller University.
PMT [photomultiplier tube] detectors can

be used to great effect to obtain excellent
signal-to-noise ratios, Calipers Mansfield said. For tissue imaging, however,
the interfering autofluorescence signal is
what limits detection of immunofluorescence or FISH [fluorescence in situ hybridization] signals, and that needs to be
removed by unmixing of the signals using
multispectral imaging methods.
One critical aspect for obtaining correct
unmixing results is in the generation of the
spectral libraries (or spectral signatures)
of the fluorophores used in the experiment
and of the tissue itself.
Caliper has developed a range of methods designed to generate the correct spectral signatures for its imaging systems,
which Mansfield said has proved critical
in developing tissue-imaging methods.
These methods, known as Compute
Pure Spectrum algorithms, enable the isolation of a pure spectral signature from an
immunofluorescence fluorophore in a
control sample, even when it is mixed
with tissue autofluorescence, he said.
In addition, extracting quantitative data
from tissue samples is more challenging
than extracting similar data from samples
of live or fixed cells. Not only is there
autofluorescence, which must be removed
to obtain accurate intensity quantitation,
but also the determination of which cells
and which compartments of cells need
analyzing.
A tissue section contains many types
of cells, only some of which are generally
of interest to the researcher. For example,
many cancer sections contain cancer cells
and a variety of less important cells (such
as stromal). When pathologists score a
cancer section, they look only at the
cancer cells.
Caliper has developed a software package [inform], which aids in the assessment
and quantitation of tissue section fluores-
cence by limiting the analysis only to cells

of interest, Mansfield said.
The two main areas in which fluorescence imaging of tissue sections is lagging
behind that of cell imaging are in the development of easy-to-use and validated
staining kits and in the development of
clinically useful methods. But Mansfield
expects that both areas will see significant
growth in the near future.
Several companies have begun the
development of validated two-step staining kits for a range of tissue fluorescence
work, he said. In addition, work has
begun on developing fluorescence-based
clinical imaging methods in areas such as
organ transplantation and tumor characterization for personalized medicine.
Imaging advances
Multispectral-photon imaging has become commercially available over the past
few years and, although not inexpensive,
it has enabled high-resolution 3-D imaging
of tissues down to a depth of 400 to
800 m or more. These spectacular and
quantitative images are limited to shallow
depths, however, so only superficial
tissues can be imaged.
On the other hand, some common
species for multiphoton analysis can be
imaged, including embryos and zebra fish,
which are only 1 mm in total thickness.
Nikon is one of at least seven companies that produce multiphoton imaging
33
equipment. Such systems use very high

power, short-pulse infrared lasers to
enable deep imaging.
Nikons goal in designing a multiphoton imaging instrument is to capture the
largest field of view, image the deepest
and at the highest speed, said Ned Jastromb, senior product manager of advanced biosystems at Nikon. We wish
to capture 3-D data over time courses
34
in living organisms. Since most biological

events happen quickly, advances like
resonant scanners and very high numerical
aperture but at the same time long
working distance objectives make this
possible.
When it comes to regular microscopy,
a recent advance in tissue imaging that
promises many important benefits is the
combination of multispectral imaging for
quantitative autofluorescence removal and

fluorophore separation with automated
morphologic analysis, and for cellular
and subcellular segmentation.
This allows extraction of quantitative
per-cell analyte measures from fluorescently labeled tissue sections. This per
cell data can be plotted in a scatter plot
similar to those seen for multicolor flow
cytometry (hence the name tissue cytometry) or can be used to score each of the
markers.
These kinds of analysis methods are
commonly used for cell imaging, such
as for high-content screening systems,
Mansfield said, but have so far been
unavailable for tissue sections without
significant time spent manually drawing
regions of interest around the tissues to
be analyzed.
A possible application of this type of
analysis is for monitoring the viability of
organ transplants, such as the kidney and
heart, and in the future, including lungs
and other organs. Vessels from biopsies
from transplants can be marked using one
label, and automated morphology-based
image analysis can be used to obtain an
objective assessment of rejection.
Another use is in the field of cancer
monitoring in an approach known as automated assessment of tumor-infiltrating
lymphocytes, or TiL counting. The degree
of lymphocytic infiltration is a key determinant of outcome for a variety of malignancies.
Current methods of assessing TiL infiltration are tedious and prone to error.
Caliper is developing a universal, automated multiplexed fluorescence imaging
method for determining TiL infiltration
rates.
Nikon is designing instruments to take
advantage of new probe development and
labeling chemistries in far-red emission
spectra, so researchers can use more
probes simultaneously, Jastromb said.
Furthermore, the emission spectra
of the far-red probes offers unsurpassed
signal-to-noise because these are generally
outside of the spectrum of autofluorescence and do not overlap with conventional blue- or green-emitting probes, he
added. New light sources have become
available to offer excitation wavelengths
for these far-red probes, and our new optical systems, lenses and detectors are continuing to allow us to image further into
the infrared.
marie.freebody@photonics.com
Multiplexed Sensing with QD-Based FRET

For more than a decade, investigators have explored the potential
of quantum dot (QD)-based Frster resonance energy transfer (FRET)
for multiplexed diagnostics, noting the unique photophysical
properties of QDs. This technique has not yet emerged into the
realm of clinical application, but thanks to the efforts of
several groups, it is moving closer to that goal.
BY GARY BOAS, NEWS EDITOR
uantum dot-based FRET has several advantages for multiplexed

diagnostics, including the sizetunability of QDs as well as their broad
absorption spectra and narrow emission
bands, high brightness, high quantum
yields and photostability.
But there are disadvantages, too. Some
are unavoidable, as University of Paris
researcher Niko Hildebrandt noted in a
recent ACS Nano perspective paper, citing
as an example QDs relatively large size,
which could affect the behavior of small
biomolecules. Others, including instability
in biological media, could be addressed
through careful investigation. Still others,
such as blinking effects, might be turned
into advantages.
Investigators are working to develop
high-performance methods and technologies to exploit the advantages of quantum
dots for multiplexed diagnostics and other
applications. Hildebrandt and colleagues,
for example, have reported the use of QDs
as FRET acceptors, rather than as donors,
the more conventional use of QDs in
FRET.
In their recent work, they have paired
QD acceptors with luminescent terbium
complexes serving as donors. This offers
several advantages, Hildebrandt said, including large Frster distances, the almost
complete suppression of background (resulting from the long luminescence lifetimes of the terbium donors), leading to
very high sensitivity and the use of only
one terbium donor for several QD acceptors. This work was initially reported in
a pair of papers in 2010, in Angewandte
Chemie International Edition.
Developing the approach has required a
significant investment of time, but otherwise the researchers have not encountered
any major unanticipated obstacles. It took
us quite a while until we had the first
proof of FRET to QD acceptors, Hilde-
Researchers are working to develop QD-based FRET for multiplexed diagnostics and other applications.
In Paris, Niko Hildebrandt and colleagues have reported the use of quantum dots as FRET acceptors
rather than as donors, as is typically done and have shown that this can offer advantages over
more conventional approaches. Courtesy of Angewandte Chemie International Edition.
brandt said, because you really need the

long lifetimes of the donors to get an efficient FRET. This in addition to the usual
trial-and-error and optimization steps that
you need for most new approaches that
no one else has done before. Once the
system was properly optimized, however,
there were no major challenges beyond
those typically associated with QD-based
FRET.
The next step is to develop the approach
for clinical application. The researchers
have demonstrated a proof-of-principle
bioassay with biotin and streptavidin.

To perform real-world diagnostics, however, they must label the quantum dots
and terbium complexes with antibodies,
aptamers, DNA/RNA or other biological
recognition molecules. They are working
to achieve this.
They also are exploring other possible
applications of QD-based FRET. Alongside several partners in a European project
called Nanognostics, Hildebrandts group
is seeking means for early detection of
Alzheimers disease. At the same time,
35
Multiplexed Diagnostics
they are looking into the potential of FRET biosensing for cellular investigations detecting rare cells such as circulating tumor
cells, for example, or investigating biological processes inside
and outside the cells.
Sensing inside the cell
Among other promising uses, QD-based FRET could be
applied to intracellular sensing. In a 2009 review in Physical
Chemistry Chemical Physics, Igor L. Medintz and Hedi Mattoussi, both scientists at the US Naval Research Laboratory
(NRL) in Washington, noted that the technique could shed light
on a number of biological processes occurring inside live cells,
including protein interactions, enzymatic activity and ion fluxes
in response to external stimuli. In addition, they said, multiplexed
FRET inside cells could offer insight into how cellular processes
are correlated.
Several research groups, including Hildebrandts at the University of Paris, are working to develop QD-based FRET for such
applications. Although some progress has been made toward
intracellular sensing with QD-based FRET, were not quite there
yet, Medintz said. The FRET part itself is very efficient; the
hard part still remains the chemistry needed for assembling the
QD FRET constructs and reliably delivering them where needed
inside a cell.
In an ACS Nano paper published July 26, eBiosciences of
San Diego, in conjunction with the NRL group, reported two new
orthogonal nanocrystal bioconjugation chemistries that overcome
many of the issues that plague currently used methods. The
chemistries target, respectively, the ever-present amines found
on proteins and thiols present in antibody hinge regions or introduced recombinantly into other proteins to aid in site-specific
labeling. They demonstrated the multiplexing potential of these
new QD chemistries in a variety of applications, including threecolor immunoassays and five-color immunolabeling in cellular
and tissues samples, among others.
Medintz and colleagues at NRL also have published a series
of papers exploring delivery of quantum dots into cells. In 2010,
they demonstrated an important step toward engineering QD
FRET constructs intracellularly. They showed that specifically
targeted proteins could be bioconjugated to QDs inside live cells.
This opens the door to having cells presynthesize the protein portion of the sensor, which would provide the recognition or catalytic activity and await final assembly on the QD intracellularly.
To support their work with intracellular sensing, the scientists also have been seeking to understand how QDs and other
nanoparticles engage in different forms of energy transfer. In a
Nature Materials paper published last year, for example, they
showed that QD-dopamine bioconjugates can function as pHbased sensors based not on FRET but rather on electron transfer. This suggests the intriguing possibility of having several QD
sensors functioning simultaneously inside the same cell while signaling by different processes such as FRET and electron transfer.
We are interested in applying the QDs as either FRET or electron transfer donors and/or acceptors, Medintz said. This, of
course, is predicated on a full understanding of the underlying
energy transfer processes, and so requires a lot of basic research
in making the QD assemblies and then looking at their photophysical and energy transfer interactions before we even get to
the biosensing aspect.
gary.boas@photonics.com
Multiplexed Diagnostics
Igor L. Medintz and colleagues at the US Naval Research Laboratory have made strides toward intracellular sensing with QD-based FRET. Shown are
images of COS-1 cells microinjected with 550-nm-emitting QDs labeled with a Texas Red dye acceptor on a peptide and engaged in FRET.
In these images, QDs are being excited and are sensitizing the proximal Texas Red acceptor. The three images are a differential interference
contrast image of the cells (left), the isolated QD emission (center) and the isolated QD-sensitized Texas Red acceptor emission (right).
Courtesy of US Naval Research Laboratory.
Medintz and colleagues also are exploring the use of quantum dots in electron-transfer (as opposed to FRET)-based sensing.
Shown are differential interference contrast and fluorescent micrographs of COS-1 cells microinjected with 550-nm-emitting QD-dopamine
conjugates and FLX nanospheres in PBS at pH 6.5, with merged images shown in the bottom row. Courtesy of Nature Materials.
37
Photon by Photon
The Evolution of the Geiger-Mode Silicon
Avalanche Photodiode for Single-Photon Counting
BY BERNICY FONG
EXCELITAS TECHNOLOGIES CORP.
ince the photomultiplier tube (PMT)

was invented in the mid-1930s, it
has remained the principal detector
for experiments involving a small number
of photons. However, its low photon detection efficiency and high sensitivity to
magnetic fields forced researchers and
systems designers worldwide to find alternatives for single-photon detection.
The foundation for the development of
the Geiger-mode silicon avalanche photodiode (G-SAPD) was laid in 1961, when
Dr. Robert J. McIntyre of RCA ElectroOptics Canada presented his theory of
microplasma instability in silicon. McIntyres team then researched operating the
avalanche photodiode (APD) above the
breakdown voltage the so-called Geiger
mode and enabling single-photon detection with silicon APDs.
The first G-SAPD single-photon-counting detector, generally referred to as the
single-photon avalanche diode, was developed in 1986 using the SLIK structure (su-
Figure 2. (a) Four- and (b) 16-channel

single-photon-counting modules.
Courtesy of Excelitas Technologies Corp.
38
Figure 1. The first commercial Geiger-mode silicon avalanche photodiode single-photon-counting module by
RCA, the SPCM-100, was introduced in 1987. Courtesy of Excelitas Technologies Corp.
perlow K-factor, or superlow excess noise

factor structure) by Excelitas Technologies
Canada, the former Electro-Optics division of RCA Canada. In the wake of
innovations by Rockwell International,
G-SAPD photon-counting detector
structures were introduced by Radiation
Monitoring Devices Inc. and Hamamatsu
Photonics.
In the past 50 years, tremendous progress has been made in the area of singlephoton counting based on G-SAPD.
Applications such as adaptive optics,
single-molecule analysis, positron emission tomography and time-domain fluorescence spectroscopy, as well as market
demand for cost-effectiveness, have been
instrumental in driving its development.
These advances range from a singleactive-area device to multicell silicon
photomultipliers (SiPMs); from the simple
analog-mode avalanche diode to a digital
photon-counting module; and from multiphoton detection to the analysis of their
spatial resolution.
To facilitate the use of G-SAPDs, the
first commercialized single-photon-counting module was introduced in 1987
(Figure 1). It was a self-contained, userfriendly device with built-in temperature
control, a stabilized high-voltage supply
and a Geiger-mode avalanche photodiode
passive quenching circuit. With its low
dark count, low timing jitter, low afterpulse
and a photon detection efficiency (PDE) of
more than 50 percent, this first-generation
module enabled single-photon studies to
move deeper into the red and near-infrared
regions of the electromagnetic spectrum.
In the 1990s, this single-photon-counting module enabled the start and development of fields such as single-molecule
detection, fluorescence sensing and quantum cryptography, which by now are critical in research and commercial fields such
as biology and astronomy. The ability to
detect single photons made it possible to
work with nanoliter or picoliter samples
without amplification. Biomedical studies
of vaccines, bioreagents and pharmaceuticals were some of the first beneficiaries of
this compact solid-state photon-counting
module. Applications such as near-field
scanning microscopy and laser-induced
fluorescence in single-molecule detection
became easier and more effective with
the high PDE. More choices opened up as
well within the laser-induced fluorescence
sector because it was now possible to see
and analyze weak fluorescence in the
green and red.
But a passive quenched solid-state
photon-counting module is quite slow; the
device is inadequate for time-correlated
fluorescence spectroscopy and Frster resonance energy transfer, which need faster
rise times and higher count rates. The
next-generation module evolved to
integrate active quenching circuitry, which
today is the norm for all such devices.
Photon-counting detectors with good
timing resolution or time jitter can measure the molecular dynamics of nanometerscale structures at picosecond-long fluorescence lifetime changes. New applications in time-resolved fluorescence studies
have led to the development of low-timing-resolution G-SAPD single-photon-
Figure 3. (a, b) A silicon photomultiplier by Moscow State Engineering Physics Institute.
counting modules by various companies

within the past decade. These optimized
modules have superior low timing jitter
performance of less than 100 ps, although
most have active detection areas of 50 m
or smaller and lower PDE in the green and
red regions.
In the early to mid-1990s, high-throughput fluorescence detection for drug discovery and genome DNA-sequencing projects
led the trend toward larger-number screening applications in biotechnology using
small amounts of reagents and samples.
At the same time, single-photon counting
in high-resolution imaging was being
explored for time-resolved fluorescence
microscopy, x-ray imaging and PET with
CT imaging. These movements fueled an
increase in demand for multichannel
G-SAPD photon-counting arrays for

increasing throughput in screening experiments and various types of ultrahighresolution imaging.
Such an array would provide researchers the means to study several aspects
of photons simultaneously over many
channels. It would mean that original
equipment manufacturers such as pharmaceutical companies could increase the
speed of multiple drug analysis. Timeresolved acquisition of fluorescence lifetimes in DNA microarray imaging could
be achieved with high resolution in the
red. A PET/CT combination to provide
whole-body anatomical (CT) and functional (PET) diagnostics, including localization of cancer, could significantly reduce the time and cost associated with
disease investigation and treatment.

Various advances were made in building
multichannel G-SAPD single-photoncounting arrays in the 1990s through early
2000s. Formats included monolithic
G-SAPD chip arrays and individual
single-photon-counting modules with
output signals from each detector while
sharing the high-voltage power supply
and quenching electronics (Figure 2).
In the late 1990s came one of the most
promising single-photon-counting arrays
to evolve. It was the complementary metal
oxide semiconductor (CMOS) processbased multipixel Geiger-mode avalanche
photodiode, often referred to as the SiPM,
the solid-state photomultiplier or the multipixel photon counter. This silicon device
is a matrix of small Geiger-mode-operated
Figure 4. (a, b) These graphs demonstrate recently published results from Excelitas Technologies on its 1-mm2 silicon photomultipliers.
Presented in July 2011 in Lyon, France, at the International Conference on New Developments in Photodetection.
39
Single-Photon Counting
APD cells connected in parallel to a

quenching resistor in series. The basis for
this design originated in Russia, and by
and large it owes its existence to the metal
resistor semiconductor APD, a p-n structured silicon APD with a thin layer of
titanium and a layer of high-resistivity
SiC or SixOy to limit its Geiger-mode
breakdown (Figure 3).
The SiPM has some of the most desirable attributes of a solid-state singlephoton-counting device: high gain (105 to
107), high PDE in the blue, low operating
voltage (40 to 100 V), low single-photon
timing resolution (average 100 ps), magnetic field insensitivity, compact size but
larger active areas (1 to 10 mm2), and the
ability to differentiate between single and
multiple photons. The most attractive feature of these microcell G-SAPDs is that
they can be manufactured using the conventional CMOS process. They could be
easily producible in high volume and at
fairly low cost; at the wafer level, lowpower CMOS electronics, such as analogto-digital conversion circuitry, could be
integrated into the SiPM.
40
For applications such as x-ray and medical imaging where many detectors are
needed, the SiPM could significantly reduce the overall system cost and footprint.
For point-of-care diagnostics, where small,
rugged, portable and inexpensive instruments are sought after, the SiPM would
make that possible. Currently, single
SiPMs and SiPM arrays in either diode or
module form are available commercially
from several companies. However, the
weaknesses of the SiPM such as high
dark count, high optical crosstalk, high
afterpulse and low PDE in the green
through red have hindered its adaptation
in many popular biomedical singlephoton-counting domains.
Through ongoing research, many investigators have already found promising
ways to improve the design and performance of the SiPM. Recently announced
results from Excelitas Technologies show
dark counts of their 1-mm2 SiPM to be
orders of magnitude lower than those of
previous models, while the PDE is higher
than that of currently available commercial devices (Figure 4). Nevertheless, more
work is needed on the overall performance

of this product for it to be widely established commercially in applications such
as cell sorting and sizing, protein analysis
and biomolecular diagnostics.
Other frontiers of the G-SAPD singlephoton detector are being explored.
Among these are pushes for a fully
integrated digital SiPM variant that can
detect single and multiple photon hits as
well as their timing and intensity, and
single and compact monolithic G-SAPD
arrays with much higher PDE for photon
counting in the red and some even in the
near-infrared. Ultimately, the G-SAPD
single-photon-counting detector of the
future must be a combination of all the
sought-after features of the PMT and
solid-state single-photon-counting
technologies and must meet the growing
needs of biomedical and life sciences
applications.
Meet the author
Bernicy Fong is an applications specialist at
Excelitas Technologies Corp. in Vaudreuil,
Qubec; e-mail: bernicy.fong@excelitas.com.
Simultaneous Multichannel Acquisition

The OptoSplit is a convenient, easy-to-use platform for simultaneous twoor three-channel fluorescence imaging. The simple yet powerful design makes
the OptoSplit the perfect solution for dual polarization imaging, FRET and
other quantitative ratiometric analyses. Though optimized for coupling to
a microscope, the OptoSplit can also be used with camera lenses for
macro imaging applications.
(802) 881-0302
sales@89north.com
www.89north.com
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2126 January 2012
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Exhibition dates
BiOS Expo: 2122 January
Photonics West: 2426 January
41
BREAKTHROUGHPRODUCTS
Femtosecond Laser
Jenoptik Laser GmbHs Lasers & Material Processing Div. has released the JenLas D2.fs femtosecond
laser based on diode-pumped disk laser technology. The device provides good parameter stability
suitable for use in industrial environments. The working temperature range is from 15 to 35 C. It emits
high pulse energies of 40 J, maximum, at a 100-kHz repetition rate, and operates in the 30- to 200-kHz
range. The beam quality of M2 = 1.25 is near the theoretical limit, and pulse duration is 400 fs. It has been
tested to guarantee safe functioning under typical transportation, storage and operating conditions.
The turnkey laser is easily integrated into complex machines and plants. The actuation signal can be
released by software or hardware. Applications include processing of dental ceramics.
Jenoptik Laser GmbH
info.lm@jenoptik.com
CCD USB 2.0 Camera
Lumenera Corp. has released the Infinity2-5, a digital

CCD USB 2.0 camera engineered for qualitative image
archiving in life sciences, clinical and materials science
applications that require high resolution and sensitivity.
The 5-megapixel camera employs the Sony Super HAD
ICX655 23-in. sensor featuring 3.45-m square pixels.
An 8-bit output is available for capturing fast video
of live events, or a 14-bit for quantifying data. The
high-speed output combined with a high dynamic
range results in a versatile entry-level research microscopy camera. Features include good color reproduction in clinical-stained samples for high-quality images; full color subwindowing for rapid focus and scanning of samples; and operation at 8.5
fps at full 2448 2048-pixel resolution (up to 23 fps at 640 480 with region of interest).
Lumenera Corp.
info@lumenera.com
Laser Scanning Microscopes
The VK-X series 3-D laser scanning microscopes from

Keyence Corp. of America combine the capabilities of
scanning electron microscopes and noncontact roughness gauges with the simplicity of an optical microscope. The systems deliver 0.5-nm Z-axis resolution with
a magnification range from 200 to 24,000. Ease of
use has been improved with the addition of the AI-Scan
function, allowing users to image and measure a target
with a click of the mouse. With high-resolution color
imaging and nanometer-level profile measurement
functions, the microscopes have been designed to
overcome the inadequacies of conventional imaging and profiling technologies. A short-wavelength
laser scans across a target to provide noncontact profile, roughness and thickness measurements,
even on targets with highly angular surfaces.
Keyence Corp. of America
keyencepr@keyence.com
Microscopy Software
Bandpass Filters
Infrared OD 4 bandpass filters that provide

the high transmission and deep rejection
necessary to isolate narrow spectral regions
have been unveiled by Edmund Optics. They
are durable, and their single-substrate dielectric construction ensures easy maintenance.
The bandpass interference filters have a
diameter of 25 mm and provide center wavelengths of 2.7 to 5.3 m. They selectively
transmit a narrow range of wavelengths while
blocking out all others. The filters feature
OD 4 blocking with center wavelengths
optimized for analyzing water vapor, ethanol,
formaldehyde, CO2, CO and NO. They are
suitable for use in environmental monitoring,
gas analysis, biotech, biomedical, quantitative
chemical and forward-looking infrared applications. Interference filters can be used in
instrumentation and for clinical chemistry,
elemental and laser line separation, fluorescence and immunoassays.
Edmund Optics
sales@edmundoptics.com
Bitplane, part of Andor Technology plc, has released its Imaris 7.3 image processing, analysis and visualization software. New capabilities include novel segmentation functions in ImarisCell, advanced filters in
Imaris MeasurementPro and further expansion of ImarisXT. The new features enhance the performance
of Imaris systems, rendering them suitable for 3- and 4-D microscopy visualization and analysis. The new
version extends the analysis of relationships between cells and cellular components via plasma membranes for segmentation of cells. With cellular organelles treated as biologically meaningful units, users
can detect and analyze multiple populations of vesicles inside cells. The software provides a high degree
of interaction and an ability to process very large image data sets by segmenting and analyzing cellular
components within individual cells.
Bitplane
ussales@bitplane.com
Raman Analyzers
BaySpec Inc. has introduced its next-generation FirstGuard dispersive multiwavelength handheld Raman
analyzers. Features include excitation wavelengths of 532, 785 and 1064 nm; a high-power ~490-mW
narrowband laser (on 785- and 1064-nm units, ~50 mW for 532 nm); high-throughput volume phase
grating technology; optimally cooled detector arrays for low-light sensitivity; a rugged package; and
single-trigger activation. The analyzers also include an integrated vial holder or direct point-and-shoot;
a bar-code scanner or a radio-frequency identification technology reader with batch reporting and print
functions; an extended long-life 5-h rechargeable battery; and Micro2020 21 CRF Part 11-compliant analysis software.
The instruments perform rapid, direct, on-site analysis in applications including food safety, forensics, and
chemical, biochemical and pharmaceutical/neutraceutical incoming raw material identification.
BaySpec Inc.
sales@bayspec.com
42
Low-GVD Broadband Polarizers
goods and pharmaceutical industries. The

code reader also can be used for counterfeit
detection and identification of single components and polychlorinated biphenyls.
Sick Inc.
info@sick.com
Achromatic Beam Shaping Optics
Precision Photonics Corp. has released a lowgroup-velocity dispersion (GVD) high-energy

polarizer for broadband femtosecond applications. The company uses its advanced ionbeam-sputtering coatings and a Telcordiaqualified, chemically activated direct-bonding,
epoxy-free technique to provide low-loss optical components. The polarizer maintains a
>2000:1 extinction ratio from 700 to 1100 nm
without sacrificing the overall transmission
(Tp >96%) or damage threshold (initial testing
to >10 J/cm2). The fused silica beamsplitters
are offered with a standard -in. input
face, and custom prism polarizers are available in sizes from 1 to >20 mm. The shape
of the prisms allows ultrabroadband pulses
to travel in and out of the polarizer without
refraction, avoiding spatial dispersion of
the broadband pulse. Applications include
OCT, microscopy and imaging.
Precision Photonics Corp.
sales@precisionphotonics.com
Code Reader
Launched by AdlOptica GmbH, the Shaper

6_6 series achromatic refractive field-mapping
beam shapers includes the Shaper 6_6_NUV,
the 6_6_VIS and the 6_6_NIR. Optimized to operate, respectively, in the near-UV from 335 to
560 nm, in the visible from 420 to 680 nm and
in the near-IR from 1100 to 1700 nm, they convert, with nearly 100% efficiency, a Gaussian
laser beam into a flattop beam with low divergence and a uniform intensity profile over a
large working distance. The achromatic design
provides the same conditions of beam shaping at any wavelength of the working spectral
band and enables simultaneous use of several
lasers of different wavelengths. The systems
provide illumination for confocal microscopy,
flow cytometry, mass spectrometry and various fluorescence applications.
AdlOptica GmbH
alex@adloptica.com
Microscope Stage
Sick Inc. has introduced the Lector620, an

image-based reader for 1- and 2-D codes that
is suitable for track and traceability applications. It offers intelligent setup and intuitive
operation, including teach-in with autofocus.
This allows operators to mount the scanner at
any distance from the code and to automatically optimize settings for the best possible
read. There is no need to use a PC to set up
the scanner. Multiple onboard interfaces, such
as Ethernet, TCP/IP, CAN, USB and RS-232,
ensure flexibility. The Lector620 series offers
real-time decoding of all images. Applications
include individual production control and
traceability of components in the automotive,
packaging, food and beverage, consumer
Prior Scientific Inc. has unveiled the

H117P2NN flattop motorized stage for
the Nikon Ti series inverted microscopes.
Although suitable for use in high-precision
biomedical and materials science scanning
operations, it is designed for prolonged livecell studies. The stage maximizes access to
the nosepiece for correction collar adjustment.
Miniaturized drive boxes occupy a fraction of
the space of previous models and provide
easy access to the nosepiece area of the microscope. Because the drive components are
mounted below the top plate, the stage also
provides easy access for micromanipulators,
environmental chambers and robotic loaders.
It enables scanning via a broad range of sample holders. Stages can be driven by proprietary motor controllers or compatible systems
in existing OEM configurations.
Prior Scientific Inc.
ddoherty@prior.com
End-Face Interferometer
Arden Photonics Ltd. has launched the VFI1200 optical fiber end-face interferometer,
based on the companys VF-20 Trio. The interferometric inspection system is designed for
checking the surface quality and flatness of
cleaved, polished or lensed fibers. Crisp endface images show defects, and high-contrast
fringe patterns make it easy to estimate end
angles. The 5-megapixel camera shows high
detail and has a 6 digital zoom. Proprietary
fiber holders render the instrument accurate
and easy to use. Third-party adapter plates
facilitate integration into cleaving and polishing operations. The holders can accommodate
fibers with diameters from 125 to 1200 m.
Software enables users to grab, save and print
images, and to record quality assurance data
and generate reports. Applications include
medical device manufacturing and fiber R&D.
Arden Photonics Ltd.
sales@ardenphotonics.com
Ultrafast Intensified CCD

Andor Technology plc has added the DH320T
model to its iStar range of scientific-grade intensified CCDs for spectroscopy applications.
It offers high sensitivity, 40 C thermoelectric
cooling, high-quantum-efficiency photocathodes, high spectral acquisition rates of up to
38,000 Hz and precise timing control through
low-jitter electronics. It provides a comprehensive software control interface and fast gating
of <2 ns. The fully integrated digital delay generator delivers low insertion delay and good
timing accuracy down to a few tens of picoseconds, allowing precise synchronization of
complex experiments through a range of
input/output triggering options. Combined
with the Czerny-Turner Shamrock spectrograph range, the DH320T provides a detection
and analysis tool for plasma study, laserinduced breakdown spectroscopy, transient
absorption spectroscopy and fluorescence lifetime study.
Andor Technology plc
marketing@andor.com
552-nm OPSLs
Coherent Inc. has announced a family of
commercial, all-solid-state optically pumped
semiconductor lasers (OPSLs) with output at
552 nm. The Sapphire 552 LP lasers offer continuous-wave output of 50, 75, 100, 150 and
200 mW, rendering them useful for applications in life sciences including flow cytometry,
confocal microscopy and drug discovery. They

produce a high-quality, diffraction-limited
TEM00 beam with M2 <1.1, pointing stability of
<5 rad/C, high-power stability of <2% and
low noise of <0.25% rms from 20 Hz to 2 MHz.
The laser head measures 125 70 34 mm,
independent of wavelength and power class.
This simplifies integration, including wavelength addition and substitution for OEMs and
end users. The devices are equipped with
USB, RS-232 and analog interface ports.
Coherent Inc.
tech.sales@coherent.com
Streak Cameras
For ultraviolet to near-infrared measurements

of extremely fast light phenomena, Hamamatsu Photonics has introduced the C10910
streak cameras. The series simultaneously delivers intensity vs. time vs. position (or wavelength) information with single-photon sensitivity and temporal resolution down to 1 ps.
The cameras are suitable for measuring fast
fluorescence lifetimes, response times of
quantum devices, electron bunch length and
stability for synchrotron and linear accelerator
applications, and photonic applications requiring high temporal resolution. They are ultrahigh-speed detectors and sensitive at a wide
range of wavelengths, depending upon the
streak tubes photocathode selection. They can
be configured with various plug-in modules to
optimize specific measurement needs. Users
can detect one-shot events or repetitive phenomena in the gigahertz domain by selecting
the appropriate sweep plug-in module.
Hamamatsu Photonics
europe@hamamatsu.com
Megahertz OPA
The Europa optical parametric amplifier (OPA)
from Elliot Scientific Ltd. is directly pumped by
submicrojoule pulses provided by the FemtoSource high-energy oscillators such as the XL
500 and XL 650. The complete package of the
FemtoSource XL, the PulseFinder programmable pulse picker and the Europa constitutes a
tunable laser source for delivering high-pulse
energy at repetition rates of up to 5.1 MHz
44
across the visible to the mid-infrared spectral

range. The OPAs signal output delivers ultrashort pulses in the mid-IR with pulse energy
exceeding 20 nJ. An optional second-harmonic-generation stage provides pulses covering the visible spectral region. The compact
system is suitable for use in spectroscopy
and microscopy applications that require
a high signal-to-noise ratio, such as timeresolved and biochemical spectroscopy,
and multiphoton microscopy.
Elliot Scientific Ltd.
sales@elliotscientific.com
version. They are plug-and-play-compatible

with standard photon counters available on
the market.
Laser Components GmbH
info@lasercomponents.com
Bioanalysis Lighting
accomplished on a compact, 3-D support

through the integration of multiple solidstate excitation sources, spectral bandpass
filters, microscope objective Z-control, a field
diaphragm, a collimating lens, a polyband
dichroic and a multiband emission filter,
an objective and tube lens, a camera mount
and control electronics.
Lumencor Inc.
info@lumencor.com
Cell Imaging System
Vacuum Stages
Lumencor Inc. has launched Core, a bioanalysis illumination and imaging system that offers equipment manufacturers a streamlined
optical block based on the companys solidstate excitation subsystems. The system includes an excitation subsystem as well as the
illumination and imaging optics needed in a
fluorescence scanner. It enables efficient light
delivery within a customizable unit that functions as the heart of a fluorescence reader. It
illuminates fluorescent samples and images
the resulting emissions. This functionality is
The cell imaging and analysis instrument Cell

Metric from Solentim Ltd. is engineered to
provide a high-throughput work flow solution
to screen for colonies from single-cell cloning
experiments. It uses proprietary non-imagebased focusing to locate all possible colonies
by imaging the whole well, right up to the
edge. The platform provides complete and
accurate measurement of cells without remov-
Steinmeyer Inc. offers a range of precision

stages suitable for ultrahigh-vacuum applications. The model MT105-LM-HV is one example. A compact two-axis (X-Z) version has a
travel range choice of 30 or 50 mm. Positioning accuracy is 3 m, with repeatability of
0.2 m. This product can be provided as a
classic X-Y stage without adapter plate or can
be configured as an X-Y-Z system. Driven by a
piezo motor, each stage includes stainless
steel crossed roller linear bearings for smooth
motion and robust load capacity. Both base
and saddle are manufactured from high-stiffness aluminum. All other components and
hardware are selected for ultrahigh-vacuum
use. Exhibiting dynamic performance, the
stages are suitable for demanding positioning
applications, including scanning electron
microscopy.
Steinmeyer Inc.
jskaltsas@steinmeyer.com
Single-Photon-Counting Modules
The COUNTblue series single-photon-counting
modules manufactured by Laser Components
GmbH achieve a quantum efficiency of 60%
in the blue spectral range and 70% in the yellow and green wavelengths. The heart of the
series is a blue-enhanced silicon Geiger-mode
low-noise avalanche photodiode that, together
with optimized electronics, produces dark
count rates of 10 to 250 counts per second.
The modules are suitable for use in fluorescence measurement. They are available with
an FC connection for optical fibers with a core
diameter of up to 105 m, or in a free-beam
45
ing them from the culture plate, and without

addition of stains or dyes. This ensures that
cells are suitable for use in downstream experiments. The system can identify every healthy,
viable cell clone from large multiwell screening projects and can track the visual history of
the colonies back to single cells. Its imaging
technology overcomes the problems of shadowing and edge effects.
Solentim Ltd.
ian.taylor@solentim.com
Microfabrication Workstation
in custom or commercial photoresists include

photonics, microelectronics and microelectromechanical systems. Materials that can be
used in ablation and surface structuring include biological targets. Waveguides and
microfluidics can be volumetrically written in
glasses and polymers. Nanosurgery and
microdissection can be performed in vivo for
subcellular investigations of model organisms.
The Laser FAB can be configured for use
with femtosecond laser oscillators, amplifiers,
optoelectronic pulse amplifiers and other
types of lasers in the visible to near-infrared
range.
Newport Corp.
ruben.zadoyan@newport.com
Deep Tissue Imaging
ers nearly double the tuning range of existing

ultrafast lasers, according to the company,
and provides seamless access to long-infrared
wavelengths for deep in vivo imaging. Robust
and fully automated, it provides hands-free
operation. It offers 680- to 1300-nm continuous tuning from a single source, short 100-fs
pulse widths and high peak power levels into
the infrared, where imaging penetration depth
is maximized. The DeepSee dispersion precompensator delivers the short pulses through
a microscope to the sample for maximum
fluorescence. A dual-wavelength option is
available for advanced imaging techniques, including uncaging, coherent anti-Stokes Raman
scattering imaging and multimodal imaging.
Spectra-Physics
herman.chui@newport.com
Slide Scanner
Newport Corp. has introduced the Laser FAB,

a tabletop laser microfabrication workstation
for research applications. It is designed for use
in additive and subtractive processes, including 3-D microfabrication by two-photon polymerization (TPP), laser ablation and surface
structuring of materials, volumetric writing of
waveguides and microfluidics, nanosurgery
and microdissection. Applications using TPP
For deep tissue imaging, Spectra-Physics, a

Newport Corp. brand, has released the InSight
DeepSee ultrafast laser system. Based on proprietary technology, the turnkey device deliv-
The SCN400 F from Leica Microsystems Inc. is

an all-in-one digital pathology slide scanner
for high-end bright-field and fluorescence applications. It combines good bright-field scanning and multichannel fluorescence imaging
on a single platform. With its specially designed optical system, scanning method and
Z-stack functionality, it captures sharp images.
Users can bring fluorescence out of the darkroom and scan slides with multiple fluorescent
labels quickly and efficiently. They can capture
whole slide scans and review crisp fluorescent
images from their computers. Rapid switching
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Photonics in Neuroscience
e-newsletter; Sneak Preview: Society
for Neuroscience; Bonus distribution
at Society for Neuroscience
7)
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46
Coming in November/December
Photocoagulation, Forensic Microscopy, Live-Cell
Imaging, Surface Plasmon Resonance Spectroscopy;
Photonics in Cell Biology e-newsletter; Bonus
distribution: ASCB Annual Meeting
Call Sales at (413) 499-0514

between bright-field and fluorescence illumination methods facilitates scanning of multiple

sample types. The scanner captures fluorescence samples with multiple markers through
a combination of tri-band cubes and excitation
filters. It delivers high-quality fluorescence
imaging and optimal dye separation.
Leica Microsystems Inc.
valerie.nicolas@leica-microsystems.com
Camera Software
Toshiba Imaging Systems Div. has announced

a customized software interface for its IK-TF7
three-chip color cameras that enhances fluorescence imaging in living retinal tissue. The
release of Streampix 5 software by Norpix Inc.
for Phoenix Research Laboratories Inc. features a built-in, long-integration button with
a window for frame time selection that enhances live-animal retinal imaging studies.
The recently designed software has been successfully used with Toshibas three-CCD cameras by Phoenix Research to capture imagery
from the retinas of mice and rats highly sensitive in vivo microscopy research. Using
Toshibas IK-TF7 three-chip cameras with long
integration time and low dark noise, the new
software enables collection of photons for
several seconds or longer while imaging even
fainter fluorescence in living retinal tissue.
Toshiba Imaging Systems Div.
gary.pitre@tais.toshiba.com
Transmission Filter
Fianium Ltd. has released its SuperChrome

high-power transmission filter accessory for
use with its supercontinuum laser sources and
fiber delivery systems. The single-channel filter enables the user to select the wavelength
and to tune the bandwidth with a switching
time of <1 s. It operates over the entire visible
spectrum from below 400 to >750 nm, realizing the blue-enhanced region available from
the companys SC400 supercontinuum series.
It offers typical transmission of >80%, with
maximum performance achieved using filter
bandwidths ranging from 8 to >50 nm, which
is useful for fluorescence imaging, spectroscopy and superresolution microscopy.
When the filter is coupled to the SC450-8-VE
supercontinuum source, more than 100 mW
of power, tunable across the visible range,
is available in a 25-nm bandwidth.
Fianium Ltd.
info@fianium.com
Glass-Bottom Microplates
Manufactured to be extremely flat (15 m
across the base), Krystal glass-bottom microplates from Porvair Sciences Ltd. are suitable for use in whole-plate CCD imaging and
laser detection applications. Available in 24-,
96- and 384-well formats, the microplates
combine the benefits of the optical properties
of glass low background and low birefringence with the versatility of a microplate.
According to the company, they demonstrate
higher performance than standard polystyrene
plates for fluorescence assays, luminescence
detection, scintillation counting and highresolution microscopy using confocal
imaging. They are available tissue culturetreated or without surface modification. The
high-clarity bottom of each plate offers exact
positioning when used with microscopes.
Based upon a standard SBS/ANSI microplate
format, they are compatible with automated
liquid handling and robotic devices.
Porvair Sciences Ltd.
int.sales@porvair-sciences.com
Spectrofluorometers
Building upon the capabilities of the FP-6000

series, the FP-8000 series spectrofluorometers
from Jasco Inc. provide additional features
and innovations. They incorporate high sensitivity, fast spectral scanning and good analysis-oriented functionality, offering integrated
solutions for advanced materials research and
biochemical analysis applications. To meet
stringent analysis demands, a variety of accessories are available for integration with the
control and analysis applications offered with
the Spectra Manager II software, yielding a
flexible platform for any luminescence application. Features include sensitivity of >5000:1
rms, a scan speed of 60,000 nm/min, a wide
dynamic range of >6.5 orders of magnitude,
auto gain and an auto sensitivity control system. The instruments also offer an automatic
higher-order diffraction cut filter and perform
rapid 3-D spectral measurements.
Jasco Inc.
sales@jascoinc.com
p BREAKTHROUGHPRODUCTS
Shine the spotlight on your Breakthrough
Product in a display ad in BioPhotonics.
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or at advertising@photonics.com
APPOINTMENTS
CALL FOR PAPERS
Optics in Cardiology December 1-2
Deadline: Poster abstracts, October 10, 10:00 a.m. CET
Rotterdam, Netherlands. Researchers are encouraged to present their
work in poster format at this symposium, which will address breakthroughs in applications of light in cardiovascular medicine, including
clinical, technological and translational advances. Among the areas to
be considered are optical devices and sensors; optics for therapy; and
contrast mechanisms, functional imaging and image processing.
Contact: Mieke Pruijsten
m.pruijsten@erasmusmc.nl
Erasmus MC, Thoraxcenter
www.opticsincardiology.org
+31 10 704 4030
BHI 2012 January 5-7

Deadline: Paper submission, October 14
Hong Kong and Shenzhen, China. The IEEE Engineering in Medicine
and Biology Society invites papers for the IEEE-EMBS International
Conference on Biomedical and Health Informatics. Among areas to be
OCTOBER
FACSS 2011 (Oct. 2-6) Reno, Nev. 38th
Federation of Analytical Chemistry and
Spectroscopy Societies Annual Meeting.
Contact: FACSS International Office, +1 (505)
820-1648; facss@facss.org; facss.org
10th International Symposium on Scanning
Probe Microscopy & Optical Tweezers in Life
Sciences (Oct. 5-6) Berlin Contact: Claudia
Bttcher, +49 30 5331 12070; info@nanobio
views.net; www.nanobioviews.net
Bio-IT World Europe Conference & Expo 2011
(Oct. 11-13) Hannover, Germany Contact:
Ming Guo, Cambridge Healthtech Institute,
+1 (781) 972-5439; mguo@healthtech.com;
www.bio-itworldexpoeurope.com
2011 BMES (Oct. 12-15) Hartford, Conn.
Annual Meeting of the Biomedical Engineering
Society. Contact: BMES, +1 (301) 459-1999
or +1 (877) 871-2637; info@bmes.org;
www.bmes.org
Frontiers in Optics 2011/Laser Science XXVII
(Oct. 16-20) San Jose, Calif. Contact:
The Optical Society of America, +1 (202)
416-1907; custserv@osa.org; www.frontiers
inoptics.com
Second International Conference on Photonics
2011 (Oct. 17-19) Kota Kinabalu, Malaysia
Contact: Secretariat of ICP2011, +6012 485
1740; info@icp2011.org; icp2011.org
Photonex (Oct. 18-19) Coventry, UK
Contact: Xmark Media Ltd., +44 1372 750
555; info@enlightenmeetings.com; www.
photonex.org
considered are medical informatics, including real time, multimodal

and molecular imaging. The conference will be held in conjunction
with the Eighth International Symposium on Medical Devices and
Biosensors and the Seventh International Symposium on
Biomedical and Health Engineering.
Contact: Laura J. Wolf
l.wolf@ieee.org
+1 (732) 981-3433
bhi2012.embs.org
2012 ASLMS Annual Conference April 18-22

Deadline: Abstracts, October 17
Kissimmee, Florida. The American Society for Laser Medicine and
Surgery Inc. seeks papers for this conference, which is held for those
who work with medical lasers in a clinical, research or business environment. The goal of the society is to advance biomedical applications
of lasers worldwide in the interest of better patient care.
Contact: ASLMS
information@aslms.org
+1 (715) 845-9283
www.aslms.org
Ukraine Contact: SPO Organizing Committee,

+380 44 526 22 96; spo_tsknu@ukr.net;
spo.univ.kiev.ua
MOC 11: 17th Microoptics Conference
(Oct. 30-Nov. 2) Sendai, Japan Contact: MOC
11 Registration Desk, +81 3 3831 2601; reg
desk@moc2011.com; www.comemoc.com/
moc11
AVS 58th International Symposium &
Exhibition (Oct. 30-Nov. 4) Nashville, Tenn.
AVS was formerly named the American
Vacuum Society. Contact: Della Miller, AVS
West Office, +1 (530) 896-0477; della@avs.org;
www2.avs.org
NOVEMBER
3D Microstructure Meeting (Nov. 2-4)
Saarbrcken, Germany Contact: Michael
Engstler, Conference Management, info@
3D-microstructure-meeting.de; www.3Dmicrostructure-meeting.de
Laser Florence 2011 (Nov. 4-5) Florence, Italy
25th International Laser Medicine Congress.
Contact: Laser Florence, ILM sas Congress
Service, +39 055 234 2330; info@laser
florence.org; www.laserflorence.org
Seventh International Symposium on
Multispectral Image Processing & Pattern
Recognition (Nov. 4-6) GuiLin, China Contact:
Fa-xiong Zhang, +86 27 8754 0131; mippr@
126.com; iprai.hust.edu.cn/mippr
2011 International Conference on
Optical Instrument and Technology (OIT)
(Nov. 6-9) Beijing Contact: Liquan Dong, +86
10 6891 2559; kylind@bit.edu.cn; www.oeoem.org/oit
Sixth LFD Workshop in Advanced

Fluorescence Imaging and Dynamics
(Oct. 24-28) Irvine, Calif. An event of the
Laboratory for Fluorescence Dynamics,
University of California, Irvine. Contact:
Milka Stakic, +1 (949) 824-7085; lfd@uci.edu;
www.lfd.uci.edu
ISCAN 2011: International Symposium

on Clusters and Nano-Structures (Nov. 7-10)
Richmond, Va. Contact: ISCAN, c/o Professor
Puru Jena, Virginia Commonwealth University, +1 (804) 828-8991; iscan@vcu.edu;
www.iscan.vcu.edu
12th International Young Scientists

Conference: Optics and High Technology
Material Science SPO 2011 (Oct. 27-30) Kiev,
Neuroscience 2011 (Nov. 12-16) Washington

Contact: Society for Neuroscience, +1 (202)
962-4000; info@sfn.org; www.sfn.org
48
Asia Communications and Photonics

Conference and Exhibition (ACP)
(Nov. 13-16) Shanghai, China Contact:
SPIE, +1 (360) 676-3290; customerservice
@spie.org; www.acp-ce.org
DECEMBER
Optics in Cardiology (Dec. 1-2) Rotterdam,
Netherlands Contact: Mieke Pruijsten,
Erasmus MC, +31 10 704 4030; m.pruijsten@
erasmusmc.nl; www.opticsincardiology.org
Functional Optical Imaging 2011
(Dec. 3-4) Ningbo, China Contact: Laura
Nightingale, University of Nottingham,
+44 115 84 68504; ibios@nottingham.ac.uk;
www.nottingham.ac.uk/ibios
2011 ASCB Annual Meeting (Dec. 3-7)
Denver Contact: American Society for Cell
Biology, +1 (301) 347-9300; ascbinfo@
ascb.org; www.ascb.org
SPIE Smart Nano+Micro Materials and
Devices (Dec. 5-7) Melbourne, Australia
Contact: SPIE, +1 (360) 676-3290; customer
service@spie.org; spie.org
Scientific Meeting of Photonics4Life
(Dec. 7-9) Karlsruhe, Germany Contact:
Juergen Popp, +49 3641 206 300; juergen.popp
@ipht-jena.de; www.photonics4life.eu
Society of Electron Microscopy Technology
(SEMT) Meeting 2011 (Dec. 12) London
Contact: David McCarthy, University of
London, +44 20 7753 5806; david.mccarthy@
pharmacy.ac.uk; www.semt.org.uk
JANUARY
SPIE Photonics West (Jan. 21-26)
San Francisco Includes the symposia
BiOSBiomedical Optics; LASELasers and
Applications; OPTOOptoelectronic Materials
and Devices; and MOEMS-MEMSMicro and
Nanofabrication. Contact: SPIE, +1 (360) 6763290; customerservice@spie.org; spie.org
For complete listings, visit

www.photonics.com/calendar
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89 North .....................................................................................................................................................41
www.89north.com
The American Society for Cell Biology......................................................................................................5

www.ascb.org/meetings
Andor Technology .....................................................................................................................................13
www.andor.com
Applied Scientific Instrumentation ............................................................................................................9
www.asiimaging.com
B&W Tek ................................... ..............................................................................................................CV3

www.bwtek.com
Cargille Laboratories.................................................................................................................................40
www.cargille.com
CVI Melles Griot...................................................................................................................................15, 30
www.cvimellesgriot.com
Edmund Optics ..........................................................................................................................................19

www.edmundoptics.com
Esco Products Inc. .....................................................................................................................................46
www.escoproducts.com
Finger Lakes Instrumentation...................................................................................................................44

www.flicamera.com
g
Gooch & Housego .....................................................................................................................................45
www.goochandhousego.com
Incom Inc................................... ..............................................................................................................CV4

www.incomusa.com
Iridian Spectral Technologies...................................................................................................................47
www.iridian.ca
Lumencor Inc. ..............................................................................................................................................6

www.lumencor.com
Mad City Labs ............................................................................................................................................40

www.madcitylabs.com
Newport Corp. .......................... ..............................................................................................................CV2

www.newport.com
Optical Building Blocks Corp. .....................................................................................................................3

www.obb1.com
Optimax Systems Inc. ...............................................................................................................................16
www.optimaxsi.com
p
Photon Technology International...............................................................................................................3
www.pti-nj.com
PI (Physik Instrumente) L.P. ......................................................................................................................20
www.pi.ws
Picoquant GmbH........................................................................................................................................34
www.picoquant.com
Prior Scientific Inc. ....................................................................................................................................23
www.prior.com
Semrock Inc. ..............................................................................................................................................17

www.semrock.com
SPIE International Society for Optical Engineering................................................................................41
www.spie.org
Toptica Photonics Inc. ...............................................................................................................................43

www.toptica.com
X Mark Media ............................................................................................................................................36

www.photonex.org
49
POSTSCRIPTS
Seeing the fluorescence for the trees
he subtle fluorescence that plants

emit during photosynthesis invisible
to the naked eye could provide a
rapid picture of how healthy the worlds
land vegetation really is.
Using satellites instead of the groundbased or airborne instruments commonly
used to measure the fluorescence scientists from NASA Goddard Space Flight
Center in Greenbelt, Md., have developed
global maps of land plant fluorescence
that could help identify plant life that is
under environmental stress. Sensing this
fluorescence remotely from space
could perhaps help farmers and aid workers to more quickly address critical issues
such as crop failures and famine.
Satellites have up until now used
greenness indicators based on reflected
light rather than fluorescence to help monitor land plants. The problem with this
method is that there is a time lag of days
or weeks before a diminishing level of
plant greenness caused by frost, drought
or the changing seasons, for example

can be detected by satellite.
Chlorophyll fluorescence enables scientists to determine immediately whether
plants are under stress before the leaves
show outward signs of decline, said Elizabeth Middleton, a biologist at NASA and
part of the team that created the maps.
The chlorophyll fluorescence from
green foliage is produced at red and farred wavelengths. Background light overwhelms it. When sunlight strikes a leaf,
the leafs chloroplast structures absorb a
good portion of the light and convert it
into carbohydrates through photosynthesis.
About 2 percent of this light is re-emitted
at the longer, redder wavelengths.
Scientists now can use lasers to measure
chlorophyll fluorescence across the Earths
surface, whereas, previously, they could
measure it only on a much smaller scale.
High spectral resolution data was gathered from the Thermal And Near-Infrared
Sensor for carbon Observation Fourier
Transform Spectrometer on the Japanese

Greenhouse gases Observing SATellite
(GOSAT). The researchers compared their
information with greenness data provided by the satellite-based Moderate
Resolution Imaging Spectroradiometer
Enhanced Vegetation Index.
To make their maps, they analyzed a
dark section of the infrared portion of the
solar spectrum embedded with a Fraunhofer line. There is little background light
at the line on which they focused at
about 770 nm making it possible to
distinguish the faint fluorescent signal.
The researchers hope that their maps
will help scientists understand carbon
cycles through ecosystems.
Caren B. Les
caren.les@photonics.com
A newly developed global map of land plant chlorophyll fluorescence shows stronger photosynthetic activity in
the Northern Hemisphere in July (above) and the reverse in December (below). The data was collected in 2009
by a spectrometer aboard the Japanese satellite GOSAT. Images courtesy of NASAs Earth Observatory.
50
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September 2011

Photoacoustic Microscopy Fluorescence Imaging FRET

BioPhotonics editors curate the most significant

FEATURES 24 PHOTOACOUSTIC MICROSCOPY UNLOCKS SECRETS

28 INTRINSIC FLUORESCENCE LIGHTS UP CELLULAR COMPONENTS

31 FLUORESCENCE IMAGING PROGRESSING FROM CELLS TO TISSUE

by Marie Freebody, Contributing Editor

35 MULTIPLEXED SENSING WITH QD-BASED FRET

38 PHOTON BY PHOTON: THE EVOLUTION OF THE GEIGER-MODE

BioPhotonics September 2011

Electronic Media Staff

.NET Developers Brian L. LeMire, Alan W. Shepherd

EDITORIAL MAIN OFFICE

BioPhotonics September 2011

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Submit by 16 September 2011

Good news for biophotonics

hether you follow the political or economic stories

Also in this issue:

And, finally, Chi Zhang, Konstantin Maslov and Lihong V.

BioPhotonics September 2011

Biophotonics News: A roundup of the industrys top research headlines.

Biophotonic products: The latest products for biophotonic applications.

Popular Topics: Check out the most-viewed stories from Photonics.com.

BioPhotonics September 2011

nary artery samples, including subcellular

intact tissues detailed information in three

Semiconductor nanowire laser tech could kill viruses, purify water

cost and power. This breakthrough in zinc

ductors. Now, the researchers have doped

BioPhotonics September 2011

From left to right, scientists Guoping Wang, a

Cornell dots characterized, ready for human trials

Kettering Cancer Center,

tumor cells, organic molecules

Nodal mapping is now being

GFP helps turn single cell into living laser

BioPhotonics September 2011

Microscope image of a single-cell living laser in action. The

15 to 20 m in size, into a microcavity

light pulses. The findings were reported in

Birds-eye view used to craft

BioPhotonics September 2011

Scientists reach for

BioPhotonics September 2011

Unnatural amino acids probe mysteries of neural stem cells

method to incorporate the Uaas into proteins expressed in

Reward-seeking behavior regulated with optogenetics

Nerve cells in the nucleus accumbens (red) receive

CHAPEL HILL, N.C. Scientists have

Using the new technique, scientists

BioPhotonics September 2011

processes, the scientists trained the mice

the sugar-producing vessel, while the

Glowing gland could reduce endocrine surgery risk

BioPhotonics September 2011

Parathyroid tissue in the upper right emits a natural

Our new higher power solid-state

newer version, which will include a filter

Lasers | Lenses | Mirrors | Assemblies

New gold nanoparticles serve as biomedical test bed

GAITHERSBURG, Md. Gold nanoparticles could be used as a

BioPhotonics September 2011