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

Sept/Oct 2014

Also:

Subcellular functional imaging with µOCT

Measuring cell volume optically

Imaging spectrometers for life sciences

Semiconductor diode lasers advance medicine

Superlenses boost subwavelength microscopy

FDA approves first noninvasive colon cancer screen

Optogenetics reverses memory- linked emotion

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SEPT | OCT 2014

VOLUME 7, NO. 5

F E A T U R E S

OPTICAL COHERENCE TOMOGRAPHY

16 High-resolution micro-OCT uncovers correlations

MEDICAL AND AESTHETIC LASERS

21 Semiconductor diode laser advances

enable medical applications

CYTOMETRY/CELL ANALYSIS

26 Optical cell volume measurement

3D OPTICAL MOLECULAR IMAGING

29 Developing drugs in 3D

SPECTRAL IMAGING

31 Imaging spectrometers look at life in two ways

SUPER-RESOLUTION MICROSCOPY

35 New twists on superlenses improve

subwavelength microscopy

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Advances in lasers,

optics, and imaging

for the life sciences

Depar tment s

Editor’s Column 2

Online 4

BioOptics Breakthroughs 6

6 Raman signal boost for cell/tissue analysis

6 Low-cost fluorescence diagnoses Type 1 diabetes early

7 Optical absorption’s suppression of interference promising for biomedical imaging

8 Photoacoustic method images the small intestine

9 ‘Molecular movies’ promise life sciences discoveries

9 Direct monitoring of singlet oxygen in individual cells

11 Optical amplifier enables external transmission of bio signals

News & Notes 12

12 Pioneering products for global priorities

12 Regulatory approval takes photonics-based systems to the clinic

14 Fast microbe ID system aids multiresistance fight

15 CBORT, BiOptix each attract $1.4M

Products 38

End Result 40

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2 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

On average, a new study on optogenetics is reported every day. That’s been

the trend for the past two years. According to PubMed, 359 research papers

on optogenetics were published in 2013; this year through the end of

August, it was 229.

When you consider that until 2010, only a dozen papers were published on

optogenetics in total, that’s impressive.

Optogenetics, which uses light to control neuron activity, is highly intriguing—

and a little unnerving. As with plenty of other technological developments, it carries

opportunity for misuse. But in the history of humanity, technology has never been

required for abusive treatment of our fellow humans. And, importantly, optogenetics’

potential benefits are absolutely astounding.

Karl Deisseroth, the Stanford professor and psychiatrist who coined the term, has

been heard to comment that optogenetics’ impact on humans will be the facilitation

of drug development for treatment of psychological disorders. Indeed, the past couple

of months have brought news of studies that seem promising in this regard. We’ve seen

reports of optogenetics being used in animal studies to:

1. Learn how memories become linked with emotions—and to show that it’s possible to

reverse the positive/negative emotional association of specific memories;1

2. Understand the dynamics of cellular signaling;2

3. Clarify the operation of the thalamic reticular nucleus (TRN), which is linked to

human brain disorders such as schizophrenia, autism, and post-traumatic stress dis-

order (PTSD);3 and

4. Control muscle movement to the spinal cords of awake, alert animals.4

Clearly, each of these studies has important and exciting implications for treatment

of disorder and injury in human subjects. Equally exciting, then, is the translation of

optogenetics technology to the marketplace. As I write this, Strategies in Biophotonics

is about to take place in Boston (Sept. 9–11), and the Innovators’ Demo Stage, a late

addition to the conference program, will feature a number of biophotonics-based

technology demonstrations. Among these is Kendall Research Systems’ (Cambridge, MA)

FireFly, an ultralight, wirelessly powered and controlled headstage system for chronic,

freely behaving optogenetics research. The folks behind this company and technology are

MIT professor Ed Boyden and technology entrepreneur Christian Wentz, who earned his

PhD at MIT and founded both Kendall Research and another company, Cerenova.

Optogenetics is still in its infancy, but its hockey-stick growth trajectory portends an

exciting future that we look forward to reporting.

REFERENCES

1. R. L. Redondo et al., Nature, doi:10.1038/nature13725

(2014).

2. M. Grusch et al., EMBO J., 33, 1713–1726 (2014);

doi:10.15252/embj.201387695.

3. M. M. Halassa et al., Cell, doi:10.1016/j.cell.2014.06.025

(2014).

4. V. Caggiano, M. Sur, and E. Bizzi, PLoS One, 9, 6, e100865

(2014); doi:10.1371/journal.pone.

The rise of optogeneticsChristine A. Shaw Senior Vice President/

Group Publishing Director

(603) 891-9178; cshaw@pennwell.com

Barbara Goode Editor in Chief

(603) 891-9194; barbarag@pennwell.com

W. Conard Holton Executive Editor in Chief

(603) 891-9161; cholton@pennwell.com

Lee Dubay Associate Editor

(603) 891-9116; leed@pennwell.com

Ashley Goldstein Business Operations Analyst &

Administrator

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Tom Markley Digital Media Sales

Operations Manager

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Kimberly Ayer Marketing Manager

Angela Millay Presentation Editor

Sheila Ward Production Manager

Dan Rodd Senior Illustrator

Debbie Bouley Audience Development Manager

Alison Boyer-Murray Ad Services Manager

CONTRIBUTING EDITORS

Mike May

Susan M. Reiss

EDITORIAL OFFICES

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Barbara Goode

Editor in Chief

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4 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

online @ www.BioOpticsWorld.com

Look what’s trending...

Near-infrared imaging helps make cancer glow to improve surgical outcomes

A team of researchers has developed

a new method involving near-infrared

light to help surgeons see an

entire tumor in a patient,

increasing the likelihood of a positive

outcome.

http://bit.ly/1sR1qJA

OCT imaging of the inner ear promising for new hearing loss therapies

Optical coherence tomography (OCT), which generates

high-resolution 3D images, enables mapping of the

tissues within the cochlea, the portion of the

inner ear responsible for hearing. The work

could lead to new therapies for hearing loss.

http://bit.ly/Z0MLmy

Pen-sized device employs

spectroscopy methods to boost

skin cancer detection

A newly developed probe employs

a combination of spectroscopy methods

to reduce unnecessary biopsies by offering

a fast, noninvasive, and lower-

cost solution to detect melanoma and other

skin cancer lesions.

http://bit.ly/1kpSoE9

Visit our website to catch up on the latest developments in biophotonics—both in optical technologies and

instrumentation—by attending webcasts, watching videos, and following our continually updated news and analysis.

Correction: In the feature article “‘Painting’ tumors to guide cancer surgery” that ran in the May/June 2014 issue, the acknowledgement should have stated that Tumor Paint is a trademark of Blaze Bioscience, Inc., rather than PerkinElmer.

1409bow_4 4 9/4/14 4:57 PM

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6 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

B R E A K T H R O U G H S

BioOpticsBioOpO

ticsCELL BIOLOGY/HYPERSPECTRAL RAMAN

Dramatic boost in Raman signal facilitates cell/tissue analysis

A new form of spontaneous Raman spectroscopy deliv-

ers signals 10,000 times stronger than those obtained

from spontaneous Raman scattering, and 100 times

stronger than signals generated by comparable “coher-

ent Raman” instruments.1 Furthermore, the technique

uses a much larger portion of the vibrational spectrum

of interest to cell biologists. A version of broadband

coherent anti-Stokes Raman scattering (BCARS), the

technique is fast and accurate enough to create high-

resolution images of biological specimens. Such images

contain detailed spatial information on the specific bio-

molecules present at speeds fast enough to observe

changes and movement in living cells.

Most current coherent Raman methods obtain useful

signal only in a spectral region containing approximately

five peaks with information about carbon-hydrogen and

oxygen-hydrogen bonds. The improved method adds to

this high-quality signal from the “fingerprint” spectral

region, which has approximately 50 peaks—most of the

useful molecular ID information.

Also, conventional coherent Raman instruments must

tune two separate laser frequencies to excite and read dif-

ferent Raman vibration modes in the sample. The new

M O B I L E H E A LT H / F L U O R E S C E N C E

Portable, low-cost fluorescence biosensor diagnoses Type 1 diabetes earlyA portable, inexpensive, and microchip-based fluorescence biosensor

could improve Type 1 diabetes diagnosis and care, and help research-

ers better understand the disease.1

Evidence suggests that early detection and aggressive new therapies

may halt Type 1 diabetes’ autoimmune attack on the pancreas and pre-

serve some insulin-making ability. But distinguishing between Type 1 and

Type 2 diabetes now requires a time-consuming, expensive test that’s lim-

ited to sophisticated health-care settings. The traditional test detects the

auto-antibodies using radioactive materials, takes several days, requires

highly trained lab staff, and costs several hundred dollars per patient.

By contrast, the microchip, developed at the Stanford University School

of Medicine (Stanford, CA), uses no radioactivity, produces results in min-

utes, and requires minimal training to use. Each chip, expected to cost

about $20 to produce, can be used for upward of 15 tests. In addition,

instead of requiring a lab-based blood draw, it can be done with blood

from a finger prick.

The device relies on a fluorescence-based method, near-infrared fluo-

rescence-enhanced (NIR-FE) detection, for detecting the antibodies. The

team’s innovation is that the glass plates forming the base of each micro-

chip are coated with an array of nanoparticle-sized islands of gold, which

intensify the fluorescent signal to enable reliable antibody detection.

The test was validated with blood samples from people newly diag-

nosed with diabetes and from people without diabetes, and com-

pared with tests on the same samples using the older method.

In addition to people newly diagnosed with diabetes, the test may

benefit people who are at risk of developing Type 1 diabetes, such as

patients’ close relatives—because it will allow doctors to quickly and

cheaply track their auto-antibody levels before they show symptoms.

Because it is so inexpensive, the test may also allow the first broad

screening for diabetes auto-antibodies in the population at large.

Stanford University and the researchers have filed for a patent

on the microchip, and the researchers also are working to launch a

startup company to help get the method approved by the FDA and

bring it to market, both in the U.S. and in parts of the world where

the standard test is too expensive and difficult to use.

1. B. Zhang, R. B. Kumar, H. Dai, and B. J. Feldman, Nature Med., 20,

948–953 (2014); doi:10.1038/nm.3619.

High-speed BCARS allows detailed mapping of specific components

of tissue samples. A false-color BCARS image of mouse liver tissue

(left) picks out cell nuclei in blue, collagen in orange, and proteins

in green. An image of tumor and normal brain tissue from a mouse

(right) has been colored to show cell nuclei in blue, lipids in red, and

red blood cells in green. Images show an area about 200 µm across.

(Image courtesy of Camp/NIST)

1409bow_6 6 9/4/14 3:40 PM

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B I O M E D I C A L I M AG I N G

Optical absorption’s suppression of interference promising for biomedical imaging

A new discovery could improve med-

ical imaging within biological tissue:

Physicists from the University of Twente

(Enschede, The Netherlands) and Yale

University (New Haven, CT) found that

light traveling through a diffusing mate-

rial follows a straighter path if the mate-

rial partially absorbs the light.1

Photons traveling through a scattering

medium perform a random walk, which

resembles an uncoordinated stagger. The

researchers found that in opaque media

such as biological tissue, light absorp-

tion actually straightens this path, leading

to less diffraction by scattering and thus

improved imaging through the material.

This seems counterintuitive: Light

absorption is usually detrimental for

imaging, as it reduces the intensity of the

visible image. But the researchers dis-

covered that if enough light is absorbed,

interference is suppressed; numerical calculations showed that long, meandering light paths

are suppressed far more than short, straight paths. With increasing absorption, straight (“bal-

listic”) light paths persist while the number of scattered paths is considerably reduced.

1. S. F. Liew et al., Phys. Rev. B, 89, 25 (2014); doi:10.1103/PhysRevB.89.224202.

instrument uses ultrashort laser pulses to

simultaneously excite all vibrational modes

of interest; this “intrapulse” excitation pro-

duces its strongest signals in the finger-

print region. Because too much light will

destroy cells, “we’ve engineered a very effi-

cient way of generating our signal with lim-

ited amounts of light. We’ve been more

efficient, but also more efficient where

it counts, in the fingerprint region,” said

chemist Marcus Cicerone, one of the proj-

ect’s researchers from the National Institute

of Standards and Technology (NIST; Gaith-

ersburg, MD) working with others from the

Cleveland Clinic in Ohio.

Raman hyperspectral images are built

up by obtaining spectra, one spatial pixel

at a time. The hundred-fold improvement

in signal strength makes it possible to col-

lect individual spectral data much faster

and at much higher quality than before—a

few milliseconds per pixel for a high-quality

spectrum vs. tens of milliseconds for a mar-

ginal-quality spectrum with other coherent

Raman spectroscopies, or even seconds for

a spectrum from more conventional spon-

taneous Raman instruments. Because it’s

capable of registering many more spec-

tral peaks in the fingerprint region, each

pixel carries a wealth of data about the bio-

molecules present. This translates to high-

resolution imaging within a minute or so,

whereas, notes NIST electrical engineer

Charles Camp, Jr., “It’s not uncommon to

take 36 hours to get a low-resolution image

in spontaneous Raman spectroscopy.”

Camp adds, “There are a number of

firsts in this paper. Among other things,

we show detailed images of collagen

and elastin—not normally identified with

coherent Raman techniques—and mul-

tiple peaks attributed to different bonds

and states of nucleotides that show the

presence of DNA or RNA.”

1. C. H. Camp et al., Nature Photon., 8,

627–634 (2014); doi:10.1038/npho-

ton.2014.145.

Numerical calculations reveal the distribution of light

intensity inside an opaque diffusing medium. Light enters

the material from the left. The top image demonstrates

multiple scattering, which causes the light paths to become

random walks (blue arrows). The light exits in random

directions, which precludes imaging. The bottom image

illustrates an absorbing opaque medium. The transport of

light occurs via straighter paths, which results in a coherent

image on the right-hand side. (Image courtesy of the Dutch

Foundation for Fundamental Research on Matter)

1409bow_7 7 9/4/14 3:40 PM

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Nanoparticles suspended in liquid, ingested by

patients, promises to enable photoacoustic tomog-

raphy (PAT) imaging for a noninvasive, real-time

view of the small intestine.1 The advancement could

help doctors better identify, understand, and treat

gastrointestinal ailments such as irritable bowel syn-

drome, celiac disease, and Crohn’s disease.

To assess the small intestine, doctors typi-

cally require patients to drink a thick, chalky liq-

uid called barium. They then image using x-rays,

magnetic resonance imaging (MRI), or ultra-

sound, but these techniques are limited—in

terms of safety, accessibility, and lack of adequate contrast, respec-

tively. Also, none effectively provides real-time imaging of move-

ment such as peristalsis, the contraction of muscles that propels

food through the small intestine. Dysfunction of these movements

may not only be linked to gastrointestinal illnesses, but also be side

effects of thyroid disorders, diabetes, and Parkinson’s disease.

“Conventional imaging methods show the organ and blockages,

but this method lets you see how the small intestine operates in real

time,” says corresponding author Jonathan Lovell, Ph.D., assistant

professor of biomedical engineering at the University of Buffalo (UB),

where researchers are pursuing the work.

The researchers worked with a family of dyes

called naphthalcyanines—small molecules that

are highly light-absorbent in the near-infrared

spectrum, which is the ideal range for biological

contrast agents. However, the dyes are unsuitable

for the human body because they don’t disperse

in liquid and they can be absorbed from the intes-

tine into the bloodstream. To address these prob-

lems, the researchers formed nanoparticles called

“nanonaps” that contain the colorful dye mole-

cules and added the abilities to disperse in liquid

and move safely through the intestine.

In laboratory experiments with mice, the researchers adminis-

tered the nanojuice orally. They then used PAT, which uses pulsed

laser lights to generate pressure waves that, when measured, pro-

vide a real-time and more nuanced view of the small intestine. The

researchers plan to continue to refine the technique for human tri-

als, and move into other areas of the gastrointestinal tract.

1. Y. Zhang et al., Nature Nanotechnol., 9, 631–638 (2014);

doi:10.1038/nnano.2014.130.

P H O T OAC O U S T I C T O M O G R A P H Y/ N A N O T E C H N O L O G Y

Photoacoustic method enables noninvasive, dynamic imaging of small intestine

The combination of “nanojuice” and

photoacoustic tomography (PAT)

illuminates the intestine of a mouse.

(Image courtesy of Jonathan Lovell)

1409bow_8 8 9/4/14 3:40 PM

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F E M T O S E C O N D L A S E R S / B I O I M AG I N G

‘Molecular movie’ technology promises life sciences discoveries

A powerful new imaging technology involving femtosecond laser pulses and bio-

luminescent proteins is fast enough to observe life processes as they happen at the

molecular level, according to the researchers who devised it.1 “With this technology,

we’re going to be able to slow down the observation of living processes and under-

stand the exact sequences of biochemical reactions,” says Chong Fang, an assistant

professor of chemistry at Oregon State University (OSU; Corvallis, OR) and leader

of the research team, which also involves scientists at the University of Alberta

(Edmonton, AB, Canada). “We believe this is the first time ever that you can really

see chemistry in action inside a biosensor,” he says.

The new approach offers sufficient speed to allow scientists to “see” what is

happening at the molecular level and create whatever kind of sensor they want by

rational design. This will improve the study of, for example, cell metabolism to nerve

impulses, how a flu virus infects a person, or how a malignant tumor spreads.

The technology, for instance, can follow the proton transfer associated with the

movement of calcium ions—one of the most basic aspects of almost all living sys-

tems and also one of the fastest. This movement of protons is integral to everything

from respiration to cell metabolism and plant photosynthesis. Scientists will now be

able to identify what is going on, one step at a time, and then use that knowledge

to create customized biosensors for improved imaging of life processes.

Fang explains, “We’re making molecular movies. And with this, we’re going to

be able to create sensors that answer some important, new questions in biophysics,

biochemistry, materials science, and biomedical problems.”

1. B. G. Oscar et al., Proc. Nat. Acad. Sci., 111, 28, 10191–10196 (2014); doi:10.1073/

pnas.1403712111.

C E L L B I O L O G Y/ S P E C T R O S C O P Y

Microspectroscopy setup enables direct monitoring of singlet oxygen in individual cellsSinglet oxygen, the first excited state of molecular oxy-

gen, is a highly reactive species that plays an important

role in a wide range of biological processes, including

cell signaling, immune response, macromolecule degra-

dation, and elimination of neoplastic tissue during pho-

todynamic therapy (PDT). Now, researchers have devel-

oped an experimental setup that enables direct micro-

spectroscopic monitoring of singlet oxygen.1

The Charles University (Prague, Czech Republic)

scientists used two detection channels—visible and

near-infrared (NIR)—to perform real-time imaging of

the very weak NIR phosphorescence of singlet oxygen

and photosensitizer simultaneously with visible fluo-

rescence of the

The experimental setup for direct

monitoring of singlet oxygen in

cells includes a 2D InGaAs camera

coupled to an imaging spectrograph.

(Image courtesy of Princeton

Instruments)continued on page 11

1409bow_9 9 9/4/14 3:40 PM

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www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 11

photosensitizer. Their experimental setup

enables acquisition of spectral images

based on singlet oxygen and photosen-

sitizer luminescence from individual cells,

where one dimension of the image is spa-

tial and the other is spectral, covering a

spectral range from 500 to 1700 nm.

To achieve these results, the research-

ers coupled a 2D-array indium gallium

arsenide (InGaAs) camera with NIR sen-

sitivity to an imaging spectrograph. The

2D detection array in the camera dramati-

cally reduced acquisition times and helped

to avoid some of the problems caused

by photobleaching as compared to the

group’s previous 1D InGaAs detectors. A

back-illuminated, silicon CCD camera was

used to detect visible light in the setup.

The researchers indicate that the intro-

duction of spectral images for such stud-

ies addresses the issue of a potential spec-

tral overlap of singlet oxygen phosphores-

cence with NIR-extended luminescence of

the photosensitizer and provides a power-

ful tool for distinguishing and separating

them, which can be applied to any photo-

sensitizer manifesting NIR luminescence.

1. M. Scholz, R. Dedic, J. Valenta, T.

Breitenbach, and J. Hála, Photochem.

Photobiol. Sci., 13, 1203–1212 (2014);

doi:10.1039/C4PP00121D.

MICROSPECTROSCOPY continued from page 9

B I O L O G I C A L S I G N A L I N G

Laser-like optical amplifier portends external transmission of bio signals

“Potential applications in medicine are exciting,” says J. Gary Eden, professor of elec-

trical and computer engineering (ECE) at the University of Illinois Urbana-Champaign

(UIUC). “We have made optical systems at the microscopic scale that amplify light and

produce ultra-narrowband spectral output,” he adds, explaining a new optical amplifier

(or laser) design that paves the way for power-on-a-chip applications. Actuated by light

that penetrates human skin, the amplifiers can transmit signals—produced by cells and

biomedical sensors—to electrical and optical networks outside the body.1

The speed of currently available semiconductor electronics is limited to about

10 GHz due to heat generation and interconnect delays. Dielectric-based photon-

ics, though not limited in speed, are limited in size by the laws of diffraction. The

researchers, led by Eden and ECE associate professor Logan Liu, discovered a path

to the best of both worlds: Plasmonics—metal nanostructures—can serve as a

bridge between photonics and electronics, to combine small size and high speed.

“We have demonstrated a novel optoplasmonic system comprising plasmonic

nanoantennas and optical microcavities capable of active nanoscale field modula-

tion, frequency switching, and amplification of signals,” states Manas Ranjan Gar-

tia, lead author of an article describing the work.1 “This is an important step for-

ward for monolithically building on-chip light sources inside future chips that can

use much less energy while providing superior speed performance of the chips.”

At the heart of the amplifier is a polystyrene or glass microsphere about 10 µm in

diameter. When activated by an intense beam of light, the sphere generates inter-

nally a narrowband optical signal that is produced by a process known as Raman

scattering. Molecules tethered to the surface of the sphere by a protein amplify

the Raman signal, and in concert with a nano-structured surface in contact to the

sphere, the amplifier produces visible light having a bandwidth that matches the

internally generated signal.

Precise manipulation of light at the micro- and nano-spatial scales is necessary for

realizing physical analogs of optical processes in biology, and for pursuing applica-

tions in areas such as embedded biomedical sensors.

1. M. R. Gartia et al., Sci. Rep., 4, 6168 (2014); doi:10.1038/srep06168.

1409bow_11 11 9/4/14 4:28 PM

12 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

News Notes&s NC o m p i l e d b y BA RBA R A GOODE

Biophotonics-based systems earning recent regulatory approval

include a welcome advance in colorectal cancer screening that

gives patients a pass on the usual dietary restrictions and bowel

prep. Also included is a laser-based adjunct to balloon angioplasty

for peripheral artery disease, and a system for tattoo removal and

treatment of pigmented lesions on all skin types.

A comfortable colo-cancer screen

Exact Sciences (NASDAQ:EXAS; Madison, WI) has received FDA

approval for the first at-home, noninvasive test for colorectal can-

cer that analyzes both stool DNA and blood biomarkers using flu-

orescence. The test requires no medication, dietary restrictions,

or bowel preparation, and demonstrated effectiveness in a pro-

spective, 90-site, 10,000-patient pivotal study.1 The findings have

“proven that this noninvasive test is highly sensitive in detecting

both early-stage colorectal cancer and the most advanced pre-

cancerous polyps most likely to develop into cancer,” said David

Ahlquist, MD, a Mayo Clinic (Rochester, MN) gastroenterologist

who co-invented the test. Mayo researchers developed the tech-

nology, and licensed it to Exact Sciences.

When a physician orders the Cologuard test, the kit is mailed

directly to the patient’s home. And once the patient collects a sam-

ple, he or she sends the kit to the Exact Sciences lab. There, Colo-

guard’s Quantitative Allele-specific Real-time Target and Signal Ampli-

fication (QuARTS) technology looks for biomarkers that are shed from

the colon as part of the digestive process, and blood released in the

stool. Multiplexed QuARTS reactions are processed using a real-time

cycler, with each biomarker (NDRG4, BMP3, KRAS, and ACTB) moni-

tored separately through independent fluorescent detection channels.

The sample is prepared and analyzed for fecal occult blood in a quan-

titative Enzyme-Linked Immunosorbent Assay (ELISA) that determines

the concentration of hemoglobin in the sample.

Patients learn their results from their prescribing physician. The test is

available in the U.S. for $599. The company plans to make Cologuard

available in select countries in Europe pending CE Mark approval.

Laser-based adjunct to balloon angioplasty

Excimer laser maker Spectranetics (Colorado Springs, CO) has

received FDA approval for its laser atherectomy products to treat

in-stent restenosis (ISR; that is, return of

Pioneering products for global priorities

Regulatory approval takes photonics-based systems to the clinic

Dr. Anita Goel’s ultimate goal, to under-

stand living systems, has led her on a

20-year quest to help decentralize, mobi-

lize, and personalize medicine. Previously

named by MIT Technology Review as one

of the World’s “Top 35 under 35 science

and technology innovators,” Goel, an MD

and PhD, is a pioneer in the emerging field

of nanobiophysics, a new convergence of

physics, nanotechnology, and biomedi-

cine—and was a featured speaker at Strat-

egies in Biophotonics (Boston, MA; Sept.

9-11, 2014). These days, she carries out her

pioneering efforts as chairman and CEO of

both Nanobiosym Inc. (NBS) and Nanobio-

sym Diagnostics (NBSDx).

NBS is a technology incubator for funda-

mental research funded at various points

of time with grants from agencies such as

DARPA, DoD, and NSF. The institute/incu-

bator has a three-fold mission: 1) To create

new science and disruptive technologies that

emerge at the convergence of physics, bio-

medicine, and nanotechnology; 2) to spin off

new companies and joint ventures that cap-

ture the commercial impact of the promise

of nanotechnology; and 3) to transition these

technologies to solve pressing healthcare,

energy, and environmental challenges.

By contrast, NBSDx is NBS’s commer-

cial engine, with a focus on commercializa-

tion of its diagnostic technology platform,

which Goel explains “will empower people

worldwide with rapid, accurate, and porta-

ble diagnostic information about their own

health.” Called Gene-RADAR, the chip-

based platform, which is the size of an iPad,

“can diagnose any disease with a genetic

fingerprint”—including HIV, Ebola, tuber-

culosis, malaria, and drug-resistant muta-

N A N O T E C H N O L O G Y/ M O B I L E H E A LT H

F L U O R E S C E N C E / M E D I C A L L A S E R S

continued on page 14

Anita Goel, MD, Ph.D., who was a featured

speaker at Strategies in Biophotonics (Boston;

Sept. 9-11, 2014), pursues global health advances

as chairman and CEO of both Nanobiosym Inc.

(NBS) and Nanobiosym Diagnostics.

1409bow_12 12 9/4/14 3:40 PM

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tions thereof, Goel told BioOptics World. As

ever, early detection means the opportunity

for both better containment of commu-

nicable diseases and better treatment for

those infected. In a dramatic example, Goel

explains how Gene-RADAR’s ability to mea-

sure viral load in HIV-positive mothers and

their infants will enable real-time custom-

ization of antiretroviral therapy and moni-

toring of treatment.

Flexible and direct

Gene-RADAR uses light to measure

directly from a single drop of blood or

saliva. “Unlike approaches that are based

on antigens or antibodies, Gene-RADAR’s

approach is very simple in that it measures

directly. There is no hybridization involved;

instead, it is based on actual direct detec-

tion,” Goel explains. And it requires no lab

infrastructure, no running water or constant

electricity, and no trained healthcare staff,

so it has potentially dramatic humanitar-

ian impact in areas and situations previously

inaccessible to high tech. In 2013, Nano-

biosym was awarded the grand prize at

the Nokia Sensing XCHALLENGE for Gene-

RADAR’s advanced sensing technology.

Goel credits tools based on optics and

quantum optics—for instance, optical

tweezers and atomic force microscopes—

for enabling study at the single-molecule

level and for facilitating exploration of the

dynamics of enzymes such as polymer-

ases that operate as they read and write

DNA. Such research, she says, has pro-

vided unprecedented insight into these

structures’ context-dependent function—

insight that has become foundational

for NBSDx.

Gene-RADAR is based upon findings

that these enzymes (which Goel says can be

viewed as nanoscale bio-motors able to con-

vert chemical energy stored in nucleotides

into mechanical work) serve to replicate, tran-

scribe, or otherwise process biological infor-

mation.1 Precision control of these “molecular

engines” has broad implications for biomed-

icine (including whole-genome sequencing

with ultra-high precision and accuracy, and

molecular manufacturing of biopolymers)

and beyond (for instance, biological compu-

tation and information storage).2

The global health impact of inexpen-

sive mobile technologies such as Gene-

RADAR was the focus of Goel’s presenta-

tion at Strategies in Biophotonics. Her talk

followed the opening keynote by lauded

engineer and entrepreneur Robert Langer

of MIT. For more on the event, see www.

strategiesinbiophotonics.com.

ACKNOWLEDGEMENT

Gene-RADAR is a registered trademark of

Nanobiosym Diagnostics.

REFERENCES

1. A. Goel and V. Vogel, Nature Nanotech-

nol., 3, 465–475 (2008); doi:10.1038/

nnano.2008.190.

2. A. Goel, Sci. Am. India, 5, 12, 53–57

(2010).

1409bow_13 13 9/4/14 3:40 PM

Cleavage

Marker 1 Marker 2 Marker 3

Quencher

DNA amplification and target probe cleavage

FRET cleavage and fuorescent signal generation

5’

5’

3’3’5’

CleavageQuencher Cleavage

HEX Dye FRET Cassette

Quencher

Cleavage

5’

5’

3’3’5’

CleavageCleavage

5’

5’

3’3’5’

Fluorescent signal emitted if target is present

Marker-specific probe Marker-specific probe Marker-specific probe

Forward primer/Invasive oligo Forward primer/Invasive oligo Forward primer/Invasive oligo

Reverse primer Reverse primer Reverse primer

Quasar670 Dye FRET Cassette FAM Dye FRET Cassette

14 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

News Notes&s N

continued from page 12

blockage following stent placement) for patients with peripheral

artery disease (PAD).

The development prompts a new standard of care in ISR treat-

ment with improved clinical outcomes, and it follows clinical find-

ings of the EXCImer Laser Randomized Controlled Study for Treat-

ment of FemoropopliTEal (the arteries above and behind the

knee) In-Stent Restenosis (EXCITE ISR). The study, reportedly the

first multi-center, randomized prospective trial ever conducted for

ISR treatment, demonstrated highly superior safety and efficacy

of laser atherectomy with adjunctive percutaneous transluminal

angioplasty (PTA, or “balloon angioplasty”) compared with PTA

alone. The trial shows a 94-percent procedural success rate using

laser atherectomy with PTA vs. 83 percent with PTA alone.

In the study, the average lesion length was approximately 20 cm—

compared to various stent IDE studies with average lesion lengths of 4

to 6 cm. Additionally, a high number of complex or advanced disease-

state patients were enrolled in the trial, a fact that indicates success in

treating all types of ISR lesions, including the most complex. Complete

results from the EXCITE

trial have been submit-

ted to a peer-reviewed

medical journal.

While stents deliver

improved overall out-

comes compared to

PTA treatment, reste-

nosis is common and

stent re-obstruction or

ISR remains therapeuti-

cally challenging. Once

ISR develops, there is a 65-percent chance of recurrence after PTA,

which is considered the standard of care. With over 115,000 ISR pro-

cedures performed annually in the U.S., Spectranetics says it is posi-

tioned to capitalize on potential market opportunities of $350 million

domestically and up to $750 million worldwide.

Removing pigments from all skin types

Aesthetic laser system maker Syneron Medical (Irvine, CA) has

received CE Mark approval for its dual-wavelength picosecond laser

to remove tattoos and pigmented lesions on any skin type. Delivering

532 and 1064 nm wavelengths, the laser uses proprietary PicoWay

technology to apply high peak power and ultrashort pulses for strong

photomechanical impact. The company will begin a staged launch of

the laser in the international market during the third quarter of 2014,

and anticipates FDA clearance by the end of 2014.

REFERENCE

1. T. F. Imperiale et al., N. Engl. J. Med., 370, 1287–1297 (Apr. 3,

2014); doi:10.1056/NEJMoa1311194.

The first at-home,

noninvasive test for

colorectal cancer,

by Exact Sciences,

analyzes DNA and

biomarkers using

fluorescence.

Fast microbe ID system aids multiresistance fightThe China Food and Drug Administration

(CFDA) has given clearance to Bruker Corp.

(Billerica, MA, and Beijing, China; NAS-

DAQ: BRKR) to market and sell its in vitro

diagnostics (IVD) MALDI Biotyper system

as a medical device for identifying micro-

organisms isolated from human specimens.

Starting from a culture, the IVD MALDI

Biotyper allows for microbial identifica-

tion in a few minutes without further

incubation steps. The system features

the company’s microflex matrix-assisted

laser desorption ionization time-of-flight

(MALDI-TOF), mass spectrometry-based

identification to enable analysis of species

that are difficult to identify using other

microbiology techniques. An increas-

ing number of case reports describe the

identification of microorganisms causing

human infections, which previously have

been isolated only from environmental

sources. The company’s extensive refer-

ence library covers more than 2300 micro-

bial species of gram-negative and gram-

positive bacteria as well as anaerobes and

yeasts, and often enables the identifica-

tions of unexpected microorganisms.

The approval is expected to facilitate

clinical microbiology in China. “With the

significant reduction of the time to result

M A L D I -T O F/ M I C R O B I O L O G Y

1409bow_14 14 9/4/14 3:40 PM

High Performance Priced Right

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Designed specif cally to improve optical performance in the

most demanding life sciences applications.

News Notes&s N

for identification of pathogenic bacte-

ria, the patient outcomes will improve

and the cost of care will be reduced

significantly,” said Dr. Lisong Shen,

Director of Laboratory Medicine at Xin

Hua Hospital (affiliated with Shanghai

Jiao Tong University School of Medi-

cine) and president of the Shanghai

Society of Laboratory Medicine.

Fast species identification is important

for helping to guide selection of thera-

peutic drug(s) because of the increas-

ing threat by multiresistant bacteria caus-

ing severe infections. The system is avail-

able in most of Europe; in the Ameri-

cas in Canada, Argentina, Colombia,

Ecuador, and Mexico; and in the Asia/

Pacific region in Japan, China, Hong

Kong, Singapore, Taiwan, and Australia.

In November 2013, the MALDI Biotyper

CA System received clearance for a first

claim by the U.S. FDA.

Two entities working on photonics-based

biomedicine have recently attracted funding

in the same amount: $1.4 million.

Massachusetts General Hospital (Bos-

ton, MA) has received a National Institutes of

Health grant totaling $1,449,151 for its Cen-

ter for Biomedical OCT Research and Trans-

lation (CBORT). With CBORT, principal inves-

tigator Brett Bouma (a Strategies in Biopho-

tonics advisory board member) aims to bring

groundbreaking advances in optical coher-

ence tomography (OCT) to biomedicine. The

center seeks to address the deficient acces-

sibility of cutting-edge OCT instrumentation

and technology through innovation of new

technical capabilities that are motivated by

significant biological and clinical challenges,

and through translation, facilitated by direct

collaboration. Since CBORT’s 2011 launch,

its members have initiated six driving bio-

medical projects and have identified techni-

cal projects that will have significant biologi-

cal and clinical impact. The projects fall into

three core thematic areas: advanced struc-

tural imaging, functional and compositional

contrast, and hybrid imaging modalities.

Colorado-based optical biodetection com-

pany BiOptix (Boulder) reports that it has

received an additional $1.4 million from

investors, which they will use to expand sales

and marketing and ramp up manufactur-

ing to meet growing customer demand. The

company sees opportunity in the label-free

marketplace, which CEO Rick Whitcomb says

is looking for a reasonably priced high-per-

formance surface plasmon resonance (SPR)

instrument. They have developed and pat-

ented an ultra-sensitive detection platform

known as Enhanced Surface Plasmon Reso-

nance (ESPR), which pairs the high sensitivity

of SPR with the high stability and lower noise

of common-path interferometry.

CBORT, BiOptix each attract $1.4MO P T I C A L C O H E R E N C E T O M O G R A P H Y/ S U R FA C E P L A S M O N R E S O N A N C E

1409bow_15 15 9/4/14 3:40 PM

d)

MCT

a) b) c)

5

6 7 89

43 2

10

16 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

O P T I C A L C O H E R E N C E T O M O G R A P H Y

B y Kengyeh K . Chu , Susan E . B i rket , L inbo L iu , Steven M. Rowe, and Gui l le rmo J . Tearney

High-resolution micro-OCT uncovers correlationsWith a resolution of 1–2 µm, µOCT is the only noninvasive method able to comprehensively

and simultaneously study both the subcellular structures and functions important for diseases

such as cystic fibrosis (CF), and the correlations between them, in vivo. Thus, µOCT promises

to become an important new tool for understanding the mechanisms of such pathologies.

Anew, high-resolution

form of optical coher-

ence tomography

(OCT), micro-OCT

(µOCT) images at 1–2 µm—suf-

ficient to visualize individual cells

and even subcellular features in

vivo. Developed by the Tearney Lab

at the Massachusetts General Hospi-

tal in collaboration with Dr. Steven

Rowe at the University of Alabama

at Birmingham, µOCT was origi-

nally demonstrated for imaging ath-

erosclerosis, where microscopic fea-

tures (e.g., cholesterol crystals, mac-

rophages, platelets, and fibrin) were

revealed in arterial plaques.1 More recently,

the technique is being applied to investigate

airway surfaces, where tiny, hair-like organ-

elles known as cilia protrude approximately

7 µm and beat in a whipping motion 10 to 20

times per second to carry dirt- and bacteria-

trapping mucus away from the lungs.2 This

process, known as mucociliary clearance, is

sometimes impaired by disease, which leads

to increased incidence of infection, airway

obstruction, and decreased lung function.

Cystic fibrosis (CF) is perhaps the

best-known example of a disease associ-

ated with mucus transport impairment.3

Affecting 30,000 patients in the United

States and 70,000 globally, CF results from

mutations in the cystic fibrosis transmem-

brane conductance regulator (CFTR)

gene, which acts primarily as a chloride

and bicarbonate ion channel. The cell

membranes of CF patients are abnormally

impermeable to these anions. CF lungs

are characterized by impaired mucocili-

ary transport, highly viscous mucus, and

are prone to frequent and persistent infec-

tion. Even though the genetic defect and

the resulting pulmonary consequences

are understood, the intermediate links in

the chain—including the mechanism that

causes mucus transport to break down—

remain unknown.

Imaging cilia motion

While living cilia have been difficult to

study noninvasively because of their small

KENGYEH KEN CHU, Ph.D., is Research Fellow in Dermatology at Massachusetts General

Hospital’s Wellman Center for Photomedicine. SUSAN E. BIRKET, PharmD, Ph.D., is a

postdoctoral trainee in Pulmonary, Allergy, and Critical Care Medicine at the University of Alabama

at Birmingham (UAB). LINBO LIU, Ph.D., is Assistant Professor of Electrical and Electronic

Engineering at the Nanyang Technical University in Singapore. STEVEN M. ROWE, MD, MSPH,

is Associate Professor of Medicine, Pediatric Pulmonary Medicine, and Physiology & Biophysics at

UAB and the Director of the UAB Cystic Fibrosis Transition Clinic. GUILLERMO J. TEARNEY,

MD, Ph.D., is Mike and Sue Hazard Family MGH Research Scholar and Professor of Pathology

at Harvard Medical School and the Wellman Center for Photomedicine. Contact Dr. Tearney at

tearney@partners.org; www.tearneylab.org.

FIGURE 1. Produced by µOCT,

images of cilia (false-colored in green)

on human bronchial epithelial cells

show a low path during the recovery

stroke (a), a high path during the

effective stroke (b), and an ellipsoidal

trajectory over a complete cycle

(c), with distinguishable effective

stroke (yellow arrows) and recovery

stroke (orange arrows). Effective and

recovery stroke positions are visible

in a densely ciliated region of the

epithelium (d). Scale bars: 10 µm.

(Reprinted from Liu et al.2)

1409bow_16 16 9/4/14 3:43 PM

a)

b)

air

mu

pcl

ep

air

mu

air

mu

ep

pcl

ep

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mounted at 45 degree angles above the sample on an inverted

microscope, a third objective also can be used to view the sample

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devices so precise image stacks can be obtained.

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 17

O P T I C A L C O H E R E N C E T O M O G R A P H Y c o n t .

size and high rate of motion, µOCT offers

the resolution and speed to image them

directly. The tip of a single cilium can be

imaged as it progresses through its whip-

like motion, which can be divided into

effective and recovery strokes. Figure

1 demonstrates this: Panel A shows the

recovery stroke of the cilium; on µOCT,

the cilium tip, colored in green, traces

a path close to the cell surface as it arcs

backwards in preparation for the effective

stroke, shown in Panel B. In this stroke,

the cilium tip travels forward at a greater

height than during the recovery stroke.

Panel C illustrates the full cycle of the cil-

ium through one full period, as the com-

plete arc trajectory of the tip is seen. Panel

D shows a time-averaged region of densely

ciliated swine epithelium in which the

stroke arc extents are also observed.

For CF research, several quantitative

measures are often used to characterize the

functional health of the respiratory epithe-

lium and consequential mucus clearance.

Two such metrics are airway surface liquid

(ASL) and periciliary liquid (PCL) depth,

which together indicate the hydration status of the airway surface.

The ASL depth quantifies the total thickness of mucus overlying

the cells, and the PCL depth measures the thin sheet of liquid gel

that encompasses the cilia; it is primarily within the PCL that the

cilia beat. The ciliary beat frequency (CBF) is a measure of the cilia

stroke rate. The mucociliary transport (MCT) velocity quantifies

the objective of mucociliary clearance: the removal of mucus. MCT

rate is the speed at which mucus is conveyed over airway surface.

These four parameters (ASL depth, PCL depth, CBF, and MCT

velocity) are essential metrics for analyzing CF because diseased

airways often exhibit dehydration (depressed ASL and PCL), lag-

ging ciliary beat (reduced CBF), and of course mucus clearance

difficulty (significantly decreased MCT). Though methods are

available to measure each of these parameters in cultured cells

or tissue, no pre-existing technique is able to quantify all of these

metrics together.

Fortunately, in addition to the already described imaging of

individual cilia, µOCT is able to quantify each of these measure-

ments, as demonstrated in Fig. 2, which shows µOCT images of

cultured ciliated airway cells derived from a healthy patient.4

Because µOCT is a cross-sectional technique, the thicknesses

of the ASL and PCL can be easily measured from images. The

yellow bar in Fig. 2a is drawn between the bright line that rep-

resents the top of the mucus layer and the top of the epithe-

lium, and corresponds to the thickness of the ASL layer. The

red bar similarly indicates the PCL layer, which can be more

distinctly seen on a time-averaged image (right side). As µOCT

can image the up-and-down periodic motion of cilia tips (green

arrows in Fig. 2a), the frequency of the beat (CBF) can also be

FIGURE 2. Single-frame (left) and time-averaged (right) µOCT images of normal human bronchial

epithelial cells show depth measurements of the airway surface liquid (ASL, yellow bar) and periciliary

liquid (PCL, red bar). Cilia tips (green arrows) are seen statically in the single-frame image to the left

and as motion-blurred arcs in the time-averaged image to the right. The motion of the mucus induces

a streaking effect of the mucus in the time-averaged image (a). A µOCT time-averaged image of

bronchial epithelial cells from a CF donor reveales compacted ASL/PCL (b). Scale bar: 10 µm. (Reprinted

from Birket et al.4, with permission from the American Thoracic Society)

1409bow_17 17 9/4/14 3:43 PM

ASL (µm) PCL (µm)

CBF (Hz) MCT (mm/min)

Non-CF CF

Non-CF CF Non-CF CF

Non-CF CF

40

30

20

10

0

8

6

4

2

0

40

30

20

10

0

30

20

10

0

a) b)

c) d)

MCT (mm/min) MCT (mm/min) MCT (mm/min)

CFTR (+/+) CFTR (Ð/Ð) DNDS

PCL (μm) PCL (μm) PCL depth (μm)

15

10

5

0

15

5 10 15

10

5

0

6

5 10 15

4

2

05 10 15

a) b) c)

18 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

O P T I C A L C O H E R E N C E T O M O G R A P H Y c o n t .

directly determined from µOCT movies.

Finally, as particles in the mucus are swept

across the µOCT field of view by mucocil-

iary clearance, the speed of their motion

defines the rate of mucociliary transport

(MCT). The streaking pattern within the

mucus on the time-averaged right-hand

image of Fig. 2a results from the motion

of the particles seen within the mucus on

the left-hand image.

Figure 2b depicts an image from cells

taken from a CF patient and cultured. This

µOCT data shows very different character-

istics compared to the healthy sample. The

mucus layer is very thin and the cilia-con-

taining PCL layer is compressed as well.

The cilia also beat more slowly and mucus

transport is significantly reduced in the CF

case. Figure 3 shows the quantitative differ-

ences observed in the non-CF and CF cells.

Revealing relationships

Thus, µOCT is the only method avail-

able to comprehensively and simultane-

ously study airway hydration, ciliary func-

tion, and mucus transport, and µOCT’s

ability to derive measurements from the

same regions of the same samples at the

same time makes it possible to study cor-

relations between these factors. Because

the cause-and-effect relationship between

parameters (e.g., airway hydration and

transport rate) can be determined only

by studying them together, µOCT has the

potential to be an important new tool for

understanding the mechanisms of CF and

other ciliary disorders.

In a recent study, µOCT was used to

analyze the relationship between the

periciliary liquid (PCL) and the rate

of mucus transport (MCT), which is pos-

sible only using a method that can cap-

ture both measurements at once.4 In nor-

mal airways, it is expected that a higher

amount of liquid in the PCL is associ-

ated with higher MCT rates, reflecting

the importance of hydration for mucus

transport. And indeed, µOCT imaging

revealed a positive correlation between

PCL thickness and MCT rate in nor-

mal explanted airways.4 However, µOCT

also showed that this relationship was

disrupted and even reversed in CF sam-

ples, indicating that a factor beyond air-

way hydration alone is responsible for

delaying mucus transport in CF, and

the slight negative slope in the correla-

tion even indicates that more PCL hydra-

tion is associated with decreased mucus

FIGURE 3. Cultured non-CF and CF donor bronchial epithelial cells have statistically significant

differences in the measured depths of airway surface liquid (ASL, a) and periciliary liquid (PCL, b),

ciliary beat frequency (CBF, c) and mucociliary transport velocity (MCT, d), as demonstrated in these

µOCT images. (Reprinted from Birket et al.4, with permission from the American Thoracic Society)

FIGURE 4. Correlated µOCT image-derived measurements show that in normal swine tracheas, increased hydration of the periciliary liquid (PCL) is

associated with higher mucus transport rates (MCT; a), but in CF-model tracheas, the relationship is inverted (b). The inverted relationship observed in the

CF trachea is duplicated in normal tracheas exposed to DNDS, a bicarbonate transport inhibitor, suggesting a bicarbonate-related defect in CF tracheas (c).

(Reprinted from Birket et al.4, with permission from the American Thoracic Society)

1409bow_18 18 9/4/14 3:43 PM

O P T I C A L C O H E R E N C E T O M O G R A P H Y c o n t .

transport.4 These results suggest that

there is an innate defect in the CF mucus

itself such that more of its presence is, in

fact, counterproductive.

One possible explanation for this is

that CF mucus is inherently more vis-

cous. Though elevated viscosity of expec-

torated CF sputum is a well-established

fact, it is not known whether the viscos-

ity increase is inherent or results from

other CF-related phenomena such as

chronic infection or mucociliary clear-

ance failure. One hypothesis suggests

that a mucus viscosity anomaly in CF

can be caused by a decreased concen-

tration of bicarbonate ions (also result-

ing from CFTR dysfunction), disrupting

the ability of mucins (the long molecu-

lar chains that mesh around a watery

medium to form mucus) to unfold and

causing them instead to stick together

to form an abnormally viscous mucus.5

Using µOCT to test this hypothesis, we

analyzed the response of the PCL/MCT

relationship following application of the

bicarbonate transport inhibitor 4,4’- dini-

trostilbene-2,2’-disulfonic acid (DNDS)

to normal airways. Indeed, µOCT mea-

surements showed that the PCL/MCT

relationship is inverted when bicar-

bonate is inhibited, duplicating the CF

observation. The viscosity of mucus was

also found to be elevated in both CF and

bicarbonate-inhibited airways, which

suggests that bicarbonate inhibition

does cause a viscosity increase in situ that

could explain the newly discovered PCL/

MCT inversion.

Another revelation enabled by µOCT

was a previously unobserved phenome-

non that occurs when cilia on normal cul-

tured human airways cells are subjected

to an increased workload when encoun-

tering mucus rafts.6 Imaging with µOCT

showed that a small compression (up to

2 µm) of the cilia caused by a mucus load

stimulates the cilia to beat faster and to

transport mucus more rapidly. Further

load that compresses the cilia beyond

2 µm negates this increase, and the beat

frequency further declines in response

to additional compression. Simultane-

ous fluorescence imaging revealed a cal-

cium spike at the onset of the mucus

load, and the application of BAPTA-AM

(a chemical that binds calcium and thus

reduces its availability to the cell) pre-

vents the ciliary beat increase. These

results indicate that a calcium-signaling-

based feedback mechanism helps airways

to cope with periods of increased load by

increasing ciliary beating and that this

feedback mechanism fails when the load

is too great.

Recent and future discoveries

The ability of µOCT to measure multi-

ple facets of airway function and mucus

transport has proven to be very use-

ful, and has already generated new dis-

coveries enabled by simultaneous and

co-localized measurements of mucus

1409bow_19 19 9/4/14 3:43 PM

c)

d)e)

Baseline

Mucus-invasion

Mucus

7 μm

R

0.50.60.70.8 Baseline

Mucusinvasion

0.91.01.11.21.3

2.0

1.5

1.0

0.5

0

M-mode image

Post-invasion

E

RE

Mucus invasion

Mucus invasion

Baseline

Baseline

Time

MCT

µOCT beam

Z

Z

2X 2X

t

X

Time-averagedB-mode image

b)

4

8

6

4

3

2

1

010 20 30 40 50

Mucus invasionCBF

PCL height reduction

Baseline

PCL compression (μm)

e) Normalized CBF

a)

f) Normalized cilia tip intensity

CBF (Hz)

τ

Time (sec)

PCL height reduction (μm)

60 70

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2

Effective Recovery

20 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

O P T I C A L C O H E R E N C E T O M O G R A P H Y c o n t .

FIGURE 5. This diagram of a mucus compression experiment represents horizontal (X), vertical (Z), and time axes. A

human bronchial epithelial culture is imaged before, during, and after the application of a mucus load, during which

time the cilia are subject to compression and a change in the time spent in the recovery (R) and effective (E) strokes

(a). A single-column-over-time presentation (M-mode) shows ciliary beat before and during mucus invasion. The

cilia begin to beat more rapidly under pressure (b). A time-averaged, cross-sectional image of cilia shows unloaded

cilia beating in an uncompressed arc (c), while cilia beating under load are flattened (d). PCL compression and CBF

plotted over the same time axis show that the compression and CBF elevation occur simultaneously at the onset of

the mucus load (e). The amount of CBF elevation is dependent on the amount of PCL compression, with response

peaking at 0.8 µm compression and decreasing below baseline at about 2 µm compression (f). The effective and

recovery stroke distribution is altered under compression, with the mucus causing the cilia to spend a greater ratio of

time in the effective state (g). (Adapted from Liu et al.6, with permission from the American Thoracic Society)

volume, ciliary beating, and

transport rate. Furthering sci-

entific understanding of the

basic mechanisms of mucus

transport, including charac-

terizing the effects of diseases

such as CF, clarifies suitable

therapeutic targets for investi-

gation. For example, the find-

ing that the absence of bicar-

bonate transport is associated

with failure of mucus clear-

ance, despite adequate air-

way hydration in CF, suggests

a pathway to address the vis-

cosity or adherence of CF

mucus itself.

Furthermore, µOCT could

help identify new drugs by

detecting beneficial changes

to cultured airway cell systems

induced by investigational sub-

stances, and quantitatively

characterize the functional

therapeutic effect of drugs

that have shown promise in the

laboratory or in human trials.

µOCT can also characterize

the airway function of new CF

animal models to determine

their suitability for represent-

ing human CF airway disease

features, as recently done for

the new CF rat.7

And now, µOCT endoscopic

probes are being developed to

characterize the same bench-

marks of airway function in liv-

ing patients. In vivo imaging

will allow direct characteriza-

tion of the progression of CF

lung disease, mitigating the

need for cell culture and ani-

mal models.

In addition, µOCT has potential

as a clinical tool for diagnosing the

severity of lung disease and monitor-

ing responses to treatment. Besides

CF, other common respiratory diseases

that affect the airway epithelium or

the mucociliary transport apparatus—

for instance, primary ciliary dyskinesia

(PCD) or chronic obstructive pulmo-

nary disease (COPD)—can be investi-

gated by µOCT imaging. «

ACKNOWLEDGEMENT

Figures 2, 3, and 4 are reprinted and

Figure 5 is adapted with permission

from the American Thoracic Society.

Originally published in references 4

and 6. Copyright © 2014 American Tho-

racic Society.

REFERENCES

1. L. Liu et al., Nat Med., 17, 1010–1014

(2011).

2. L. Liu et al., PLoS One, 8, e54473 (2013).

3. S. M. Rowe, S. Miller, and E. J. Sorscher,

N. Engl. J. Med., 352, 1992–2001 (2005).

4. S. E. Birket et al., Am. J. Respir. Crit. Care.

Med., 190, 421–432 (2014).

5. P. M. Quinton, Am. J. Physiol. Cell Physiol.,

299, C1222-33 (2010).

6. L. Liu et al., “An autoregulatory mecha-

nism governing mucociliary transport is

sensitive to mucus load,” Am. J. Respir. Cell

Mol. Biol., in press (2014).

7. K. Tuggle et al., PLoS One, 9, e91253

(2014).

1409bow_20 20 9/4/14 3:43 PM

Absorption (log scale)

Wavelength (nm)

DiodeAlexandrite

300 500 700 1000 2000

Ruby

Pigmented lesion

Melanin

Oxyhemoglobin

GaN GaP GaAs InP GaSb

Bulk tissue

Nd: YAG

Water

VesselVessel

Vessel

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 21

M E D I C A L A N D A E S T H E T I C L A S E R S

B y Stewar t W. Wi l son

Semiconductor diode laser advances enable medical applicationsAdvances in semiconductor diode laser sources are facilitating the migration of medical

and aesthetic lasers to consumer markets. Semiconductor diode lasers offer advantages

over other light sources for applications in dermatology, dentistry, and more—

and continued advancements promise to make them increasingly compelling.

While medical and aes-

thetic lasers have been

largely limited to pro-

fessional markets, tech-

nological advances in semiconduc-

tor diode sources are facilitating their

migration to consumer markets. The

U.S. Food and Drug Administration

(FDA) approved the first-ever high-

power diode laser product for hair

removal in 1997 for the professional

market. Nine years later, the com-

pany that had won the approval, Palo-

mar Medical Technologies (acquired

in 2013 by Cynosure [Westford, MA]),

received FDA clearance for an over-the-

counter (OTC) product for home use.

In 2009, the company was the first to

receive FDA clearance for a semicon-

ductor laser-based OTC device for wrin-

kle removal, which it released to the

consumer market in 2010. Advances in

diode laser technology made both of

these cases possible.

Selective photothermolysis

and common chromophores

What makes a light source a good choice

for a given application is the ability of its

emitted light to interact with the tissue

so as to achieve the desired effect. This

is known as selective photothermolysis.

Selectivity is accomplished by matching a

specific wavelength of light to a chromo-

phore—that is, the light-absorbent part

of a molecule—in the tissue. During the

absorption process, an electron is raised

to its excited state from a ground state. In

biological molecules that serve to capture

light energy, the chromophore is the moi-

ety that causes a conformational change

of the molecule when hit by light. The

energy directed into the target area pro-

duces sufficient heat to damage or alter

FIGURE 1. Each of the major biological chromophores has an absorption spectra. The wavelength of

light needed to activate each of these correlates with a semiconductor laser material. Above are shown

typical solid-state lasers used for these wavelengths. Penetration depth of light is strongly dependent

on wavelength, and must be considered when determining the energy for a particular application.

STEWART W. WILSON is a principal partner of Integrated Engineering Consulting LLC,

Burlington, MA; e-mail: stewart@iecteam.com

1409bow_21 21 9/4/14 3:43 PM

• Pulsewidth ≤ TRT

TRTd2

cm2

sec

K*α

α ≈

≈

• Thermal relaxation time (TRT)

(sec)

where:

0.1

8 ≤ K ≤ 24 (Geometric factor)

Typical TRT ranges:

- 0.005 mm melanosome ≈ 0.001 ms

- 0.01 mm vessels ≈ 0.010 ms

- 0.1 mm vessels ≈ 1 ms

- Hair follicle ≈ 10 – 200 ms

- 5 mm of subcutaneous fat ≈ 1 min

( ( (Thermal diffusivity of tissue)

Penetration depth (mm)

Wavelength (nm)

10

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500

9

8

7

6

5

4

3

2

1

0

Dermis

Subcutaneous fat

22 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

M E D I C A L A N D A E S T H E T I C L A S E R S c o n t .

the target while allowing the surround-

ing area to remain relatively untouched.

There are three light-related “knobs”

to adjust when employing selective

photothermolysis: wavelength, pulse

duration, and energy level. As already

described, the wavelength chosen assists

in selective absorption of light by the tar-

get vs. the surrounding tissue. The pulse

duration should be significantly shorter

than the target’s thermal relaxation time

to avoid interaction with the area out-

side of the target. And the light energy

selected should be higher than the dam-

age threshold of the target. Proper selec-

tion of these critical parameters ensures

the desired results.

Common tissue chromophores tar-

geted in dermatology applications are

water (which makes up 70 percent of tis-

sue), hemoglobin (blood), melanin (in

the epidermis, pigmented lesions, and

hair), lipids (subcutaneous fat and seba-

ceous glands), and protein (specifically,

collagen). Each of these has a specific

absorption spectra that corresponds to

a semiconductor laser material able to

produce the matching wavelength (see

Fig. 1). And certain solid-state lasers

used correlate with the materials and

wavelengths. Because penetration depth

depends highly on the wavelength of

light, it must be considered when deter-

mining the energy needed for a particu-

lar application (see Fig. 2).

For effective treatment and to pre-

vent interaction with the surrounding

area, the pulse width should be suffi-

ciently shorter than the thermal relax-

ation time (TRT) of the targeted chro-

mophore. While there is no substitute

for testing and experimentation, Fig-

ure 3, which provides guidance for

determining approximate pulse width,

can serve as a starting point. The single

limiting factor for using semiconduc-

tor lasers is high peak power. Improve-

ments in this area make their use quite

compelling since semiconductor lasers

also offer performance in the other

parameters.

Light source effectiveness

Semiconductor laser light sources

have many advantages over other light

sources for a variety of medical appli-

cations. Emphasis is on conversion effi-

ciency, temperature sensitivity, and

cost rather than beam quality and

brightness, which tend to be the driv-

ing factors for applications outside

biomedicine.

Lamps generate polychromatic diver-

gent light, whereas lasers generate mono-

chromatic light beams. Using f lash

lamps to target chromophores requires

use of optical filters to narrow the spec-

tral width—and reduces conversion

efficiency to 10–20 percent. Addition-

ally, a flash lamp-pumped laser has a

conversion efficiency of around 1–10

percent. Semiconductor-based lasers, on

the other hand, offer electro-optic con-

version efficiency of 40–65 percent for

wavelengths between 780 and 2000 nm.

Recent advances have enabled efficien-

cies as high as 72 percent in the 780–

1100 nm range, and 80 percent is theo-

retically possible.

Using a more efficient light source

has two major advantages: The power

FIGURE 2. Depth of light penetration into tissue depends on wavelength and is limited by scattering

and absorption.

FIGURE 3. For effective treatment, the pulse duration should be sufficiently shorter than the thermal

relaxation time (TRT) of the targeted chromophore to allow selectivity from surrounding tissue.

Typical optical fluence needed for effective treatment targeting is between 1 and 100 J/cm2; the

corresponding spot size ranges from 0.1 to 10 cm2. Typical durations for common treatments include

0.001 ms for a melanosome (the organelle responsible for melanin in animal cells) to 1 min for 5 mm of

subcutaneous fat.

1409bow_22 22 9/4/14 3:43 PM

Mpa

Tooth surface after acid etching

Tooth surface after laser processing

d)

a) b)

c)

25

20

15

10

5

0Self etch

60%

Self etch and superBonding

Shear bondstrength

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www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 23

M E D I C A L A N D A E S T H E T I C L A S E R S c o n t .

supply needed to gen-

erate the optical energy

is significantly less, and

the required cooling sys-

tem is smaller because

there is less waste heat.

Both factors allow for

a more compact, cost-

effective design. As an

example, in comparing

a system having a 25-per-

cent vs. 65-percent con-

version efficiency and

producing the same

optical power, the lower

efficient system would

require ~2X more

electrical power and

produce ~6X more

waste heat.

Semiconductor lasers

can produce a variety of

monochromatic wave-

lengths and generate

significant optical power

FIGURE 4. A compact multi-wavelength dental handpiece is designed for both soft tissue treatments and hard, including

etching of dental surfaces (a). Acid etching of dental surfaces is prone to failure of dental restoration (b), while laser

etching (c) produces more reliable outcomes (d).

1409bow_23 23 9/4/14 3:43 PM

24 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

M E D I C A L A N D A E S T H E T I C L A S E R S c o n t .

in a small, compact footprint. By

contrast, sources such as lamps

tend to be not only inefficient, but

also clumsy—and unable to focus

on spot sizes smaller than 5 mm,

which practically eliminates their

use for tissue cutting and fiber

delivery. Additionally, lamps are

unable to generate pulses shorter

than 1 µs, which makes them

unsuitable for applications such as

tattoo removal.

While gallium arsenide (GaAs)-

and indium phosphide (InP)-

based semiconductor lasers

emit quite well, gaps have tradi-

tionally existed between their wave-

length ranges. In recent years, though,

more wavelengths have become

available, and, for instance, 1200 nm

(in the gap between GaAs and InP)

has been demonstrated. Likewise, a

system using gallium antimonide

(GaSb) to generate type-I lasing (that

is, typical laser operation in which

light emission is generated by transi-

tion from the conduction to the valence

band) in the 2000–3000 nm range has

been shown to produce optical power

between 0.5 and 2 W from a single

device. These wavelengths are ideal for

applications involving both soft and

hard tissue, such as epidermal ablation,

drug delivery, and dental treatment.

Lastly, with the advent of type II inter-

band and quantum cascade lasers (in

which light is generated by transitions

from a single level within the conduc-

tion band), even longer wavelengths—

beyond 3000 nm (that is, mid IR)—are

possible. It will be interesting to see

what medical applications emerge for

these longer wavelengths.

Direct and indirect confgurations

Medical applications using semiconduc-

tor lasers as the main light source can be

configured either directly (non-pump-

ing) or indirectly (pumping)—the differ-

ence being whether or not light is being

delivered directly to the treatment site.

Indirectly configured semiconduc-

tor lasers are commonly used as optical

pump sources for solid-state crystals—

known as diode-pumped solid-state

(DPSS) lasers. Another example of an

indirect configuration is a diode-pumped

fiber laser, which is also a solid-state

laser, but in this case, the pumping gain

medium is optical fiber. Choosing one of

these would depend on the gain medium

used and the wavelength needed.

A compact DPSS laser pen-like hand-

held dental device has been shown to pro-

duce multiple wavelengths simply by chang-

ing the pulse width of the semiconductor

pumping laser (see Fig. 4). It produced five

distinct wavelengths (2.66, 2.71, 2.81, 2.83

and 2.85 µm) using only a single compact

semiconductor laser pump module lasing

at one discrete wavelength: 975 nm. This is

of particular interest for soft- and hard-tis-

sue applications that typically involve five

different lasers for use in deep soft-tissue

coagulation (2.66 µm, holmium laser), cut-

ting (2.71 µm, CO2 laser), and hard- and

soft-tissue ablation (2.81, 2.83, and 2.85

µm; Er:YAG laser) with minimal thermal

damage (see Fig. 5).

An example of a hard-tissue application

would be in the creation of ultra-retentive

dental surfaces—that is, the preparation of

tooth surfaces to improve bonding with den-

tal prostheses or composites. Retentive den-

tal surfaces are conventionally formed using

acid etch, which creates inconsistent surface

structures and frequently results in the fail-

ure of dental restorations, especially those

bonded to dentine. This failure rate can

be as high as 40 percent. Using a laser for

this same application creates a super-reten-

tive bond on dentine and sclerotic dentine,

and produces a 60-percent improvement in

bond strength to dramatically reduce bond-

ing failures, especially for dentine.

Some of the first direct-diode prod-

ucts to enter the consumer market have

been demonstrated by Palomar Med-

ical Technologies (with the PaloVia

skin-renewing laser system in 2010) and

Tria Beauty (then SpectraGenics, Dub-

lin, CA, with the Tria hair removal sys-

tem in 2008). PaloVia is the first home-

use semiconductor laser device that is

FDA-cleared for removing fine lines and

wrinkles. This battery-operated, hand-

held device delivers 1410 nm in small spot

patterns to the skin, creating a fraxel

pattern of damage sites and thus initiat-

ing the body’s natural healing response

FIGURE 5. Soft- and hard-tissue applications typically involve five different lasers.

Medical applications using semiconductor lasers

as the main light source can be confgured either

directly (non-pumping) or indirectly (pumping)—

the difference being whether or not light is

being delivered directly to the treatment site.

Wavelength

(µm)

Coeffcient of

absorption (cm-1)

System

currently used

Clinical

application

2.66 210 Ho laser Deep soft-tissue

coagulation and cutting

Hard- and soft-tissue

ablation with minimal-

residue thermal damage

2.71 760 C02 laser

2.81 7260 Er:YAG

2.83 8480 Er:YAG

2.85 9470 Er:YAG

1409bow_24 24 9/4/14 3:43 PM

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MOM: 2-Photon Movable Objective Microscope

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Flexible It’s unique 3- dimensional objective movement and rotation allows the specimen to remain stationary and the scope to convert from an upright to an inverted microscope.

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www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 25

M E D I C A L A N D A E S T H E T I C L A S E R S c o n t .

to generate more collagen while at

the same time removing the wrinkles.

Although Palomar was the first to gain

FDA clearance for a home-use semicon-

ductor laser for hair removal (2006),

the purchase of Gillette by Procter &

Gamble in 2005 significantly disturbed

relationship and focus, thus enabling

Tria Beauty to become the first and

only company to deliver a semiconduc-

tor laser hair-removal system to the con-

sumer market.

Impact for the future

An example of a direct-diode laser

product that makes evident the signif-

icant improvements in semiconductor

laser technology is the Vectus Laser

designed by Palomar Medical Technol-

ogies in 2012. With Vectus, Palomar

took direct aim at Lumenis’ (Yokneam,

Israel) LightSheer hair-removal sys-

tem for the professional market (which

Palomar actually designed in 1997

and sold to Coherent in 1999). Both

systems emit at ~808 nm, but the

Vectus uses an advanced design that

requires four times fewer diode laser

bars and produces significantly more

optical energy in a wider treatment

range. In a split-body direct-compari-

son study, the Vectus significantly out-

performed LightSheer. This is not to

trivialize the success of LightSheer at

all; rather, the fact that LightSheer has

been on the market since 1997 con-

firms the importance of using semi-

conductor laser technology for medi-

cal applications.

Semiconductor diode lasers already

offer many advantages over other

light sources for medical applications.

Improvements in electro-optic con-

version efficiency, optical power, and

wavelength options make the technol-

ogy ideal for targeting chromophores.

Continued advancements will make it

increasingly compelling. «

REFERENCES

1. M. Inochkin et al., “High efficiency

diode pumped Er:YLF laser with

multi-wavelength generation,” Proc.

SPIE, 8234, 8234-4, session 1 ( Jan.

22, 2012).

2. S. Wilson et al., “Long pulse compact

and high brightness near 1-kW QCW

diode laser stack,” Proc. SPIE, 8241,

8241-14, session 3 (Jan. 22, 2012).

3. L. Shterengas et al., “High-power 2.3-

um GaSb-based linear laser array,”

IEEE Photon. Technol. Lett. (2004).

4. A. Samad-Zadeh et al., “The influ-

ence of laser-textured dentinal sur-

face on bond strength,” American

Dental Association, 2005-2007, IADR

(2009).

5. J. Lowery et al., “Comparative study

between the Vectus™ diode laser sys-

tem and LightSheer™ Duet for long-

term hair reduction in the axilla,”

Palomar Medical Technologies white

paper (2013).

1409bow_25 25 9/4/14 3:43 PM

Cell

_ +

ν

∆V

a) b) c)

Cell

t

Electrolyte

26 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

C Y T O M E T R Y / C E L L A N A L Y S I S

B y Ethan Schonbrun

Optical cell volume measurement The ability to measure cell volume enables greater understanding of biological processes, including

disease development. A new method based on light absorption enables high throughput, less sample

processing, and a simpler setup. Plus, it allows for multiplexing, which could produce greater insights.

Cell volume is a

critical parame-

ter in both biol-

ogy and med-

icine: Understanding cell

growth and death, quanti-

fying intracellular concen-

trations of ions and pro-

teins, and diagnosing most

hematological disorders all

require accurate measure-

ment of cell volume.

Optical technology offers

many benefits for cytome-

try, but obtaining an accu-

rate optical measure of cell

volume is challenging.1 Often, cells have

complex, three-dimensional shapes and

are composed of heterogeneous materi-

als that have various optical properties.

In addition, because there is consider-

able cell-to-cell variability in a popula-

tion, making assumptions based on aver-

age cell properties is of limited value.

A clever solution for finding the vol-

ume of an object with a complex shape

and composed of an unknown material

was proposed by Archimedes more than

2000 years ago. By submerging the object

in a fluid-filled container, the displaced

volume can be easily measured and the

measurement corresponds absolutely to

the object volume (see Fig. 1a). As long

as the object is not porous, the displaced

volume is nearly independent of the phys-

ical properties of the object. The volume

displacement method works effectively

even on objects with extremely complex

three-dimensional shapes that would be

incredibly difficult to quantify with any

other method. These same advantages

make volume displacement appealing for

measuring cell volume.2

Not coincidently, an electrical ana-

logue to the volume displacement prin-

ciple, called the Coulter counter, has

become the most widely used method

for quantifying cell volume. Cells are

immersed in a conducting electrolyte

and the suspension is passed through a

small aperture (see Fig. 1b). A constant

current is applied across the aperture

and voltage pulses are obtained as cells

traverse the opening. If the cells are

non-permeable to the electrolyte and

assumed to be insulators, the voltage

pulse height is proportional to the cell

volume. In order to retrieve an accurate

absolute volume, however, a shape fac-

tor needs to be used: While the method

is nearly independent of the cell’s elec-

trical properties, it is not (unlike Archi-

medes’ displacement) completely inde-

pendent of a cell’s geometry.

An optical Coulter counter

Scientists at Harvard University’s Row-

land Institute have developed a volume

displacement method based not on dis-

placed fluid volume or free electrons, but

on light absorption.

Cells are immersed in a buffer that

contains an absorbing dye, and, in direct

analogy to the electrical Coulter counter,

are passed through a microfluidic chan-

nel with a finite height. The presence of

a cell in the field of view displaces a num-

ber of dye molecules that is proportional

to the cell volume and the dye concen-

tration. The concentration of the dye

FIGURE 1. Methods of measuring displacement include Archimedes’ approach, which yields the object volume

independent of the object’s properties or geometry, as in the case of a king’s crown composed of unknown materials

(a). An electrical Coulter counter uses displaced free electrons in an electrolyte and a measurement of voltage to

determine cell volume (b). The optical Coulter counter uses the displacement of dye molecules and optical transmission

to measure cell volume (c).3

ETHAN SCHONBRUN, Ph.D., is a junior fellow and the principal investigator of the Optofluidics

Cytometry Group at Harvard University’s Rowland Institute; e-mail: schonbrun@rowland.harvard.

edu; http://www2.rowland.harvard.edu/book/ethan-schonbrun.

1409bow_26 26 9/4/14 3:43 PM

Volume (fL)

e)

c) d)

a) b) f)

Volume (fL)Height(μm)

150

8

6

4

2

0 μm

100

50

0 500

(i) (ii)

5 µm 5 µm

(iii) (iv)

(i) (ii)

(iii) (iv)

10001500 2000 2500 3000 3500

150

100

12

10

8

6

4

2

0

50

0 500 10001500 2000 2500 3000 3500

673 fL(i)

(ii) 971 fL

(i) 1046 fL

(ii) 1375 fL

N = 552

(iii) 2364 fL

(iv) 3028 fL

N = 648

(iii) 1792 fL

(iv) 2926 fL

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 27

C Y T O M E T R Y / C E L L A N A L Y S I S c o n t .

can be measured before beginning the

experiment and is the only parameter

that needs to be known a priori—unlike

the sample’s shape factor in the Coul-

ter counter.

For an object that does not scat-

ter light, a single-intensity measure-

ment would be enough to reconstruct

an accurate volume estimate from the

measured intensity. Cells do scatter

light, however, and the measured inten-

sity has scattering contributions in addi-

tion to dye exclusion. We have mini-

mized the contribution from scatter-

ing in two ways. First, we added bovine

serum albumin (BSA) to raise the

refractive index of the absorbing buffer

to approximately match that of the cell.

In addition, we image each cell with

a second color that is not absorbed by

the dye and consequently can be used

to subtract out the remaining scatter-

ing component.

In this setup, a color camera mea-

sures at a throughput of approximately

1000 cells per minute. Figure 2 shows

results for optical displacement imag-

ing of leukemia cells and a compar-

ison of volume distributions for two

different strains. Intracellular scatter-

ing is nearly eliminated (see Fig. 4d),

and the height map does not suffer

from speckle, haloing, shade-off, or

any other artifacts commonly found in

quantitative phase microscopes.

No need for sphering

The most common application of cell

volume measurements is in hematology

where complete blood cell counts quan-

tify red blood cell volume and distribu-

tion width. Using these measurements,

hematologists can diagnose whether the

patient has macrocytic (in which cells

are too large) or microcytic (cells are too

small) anemia.

Current clinical hematology ana-

lyzers use optical scattering to quan-

tify both cell volume and hemoglobin

concentration. But because scattering

couples geometry to optical proper-

ties, cells are required to be “sphered”

(that is, processed to swell from disc-

to sphere-shape) before measurement.

Departure from a spherical shape pro-

duces artifacts in measurement that

lead to inaccurate results.

Instead, we have applied the optical

displacement method to measure cell vol-

ume while cells are in their natural, non-

spherical state. In addition, we have com-

bined volume displacement with a simul-

taneous hemoglobin absorption measure-

ment to quantify single cell hemoglobin

mass.4 Our results are similar to those

obtained with a clinical hematology ana-

lyzer, Siemens Advia 2120, but we mea-

sure a smaller coefficient of variation of

hemoglobin concentration (see Fig. 3).

Studies have demonstrated that hemoglo-

bin concentration is tightly regulated in

the body5 and a more narrow distribution

of hemoglobin concentration might more

accurately represent the actual physiolog-

ical state.

Multiplex-able

Another major strength of an opti-

cal technique, compared to an elec-

trical one, is that it is straightforward

to multiplex with other optical tech-

niques. In our tests, we combined this

method with optical absorption to cre-

ate a method for measuring red blood

cell volume and hemoglobin mass—a

method that we believe is more accu-

rate than the clinical standard. In addi-

tion, cell volume measurements can be

matched with a f luorescence image or

signal to correlate volume and organ-

elle morphology.

Figure 4 shows thickness maps of neu-

trophils that are captured simultane-

ously with fluorescence images of their

nuclei. Neutrophil volume distributions

have been linked to early detection of

FIGURE 2. In optical displacement imaging, color images using blue and green illumination are collected by a color camera (a). The green channel is

used to compensate for intracellular scattering (b), while the blue channel contains contributions from both volume displacement and scattering (c). After

processing, a thickness map can be retrieved where contributions from scattering have been removed (d). Measurements enable display of the thickness

map and volume distribution for 648 HL60 leukemia cells (e) and 552 K562 leukemia cells (f).3

1409bow_27 27 9/4/14 3:43 PM

Hb mass (pg)

Volume (fL)Volume (fL)

Hb concentration (g/dL)a) c)

b)

60

50

40

30

20

10

55

45

50

40

35

30

25

20 0 50 1000

AdviaQAC

50 100 150 150

20

15

10

5

0 fg

40

30

20

10

0 fg

2.0 50403020

100 fg

3

2

1

(i)

(i)

(i)

(ii)

(ii)

(ii)

(iii)

(iii)(iii)

(iv)

(iv)

(iv)

1.51.00.50µm

3.0

2.0

1.0

0µm0µm

A1 A2

5µm 3µm 3µm3µm

3µm

AdviaQAC

5µm

28 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

C Y T O M E T R Y / C E L L A N A L Y S I S c o n t .

infection.6 Nuclear morphology of neu-

trophil cells has also been studied in

the context of infection due to the fact

that nuclei become more segmented

as the cell ages. We have begun a proj-

ect to correlate these two behaviors in

blood samples. A better measurement of

cell age could have major implications in

the early detection of infection when the

body is trying hard to make more white

blood cells.

Simpler and full of potential

By reducing and correcting for scatter-

ing, we have enabled this optical method

to be, like Archimedes’ approach, nearly

invariant to both the object’s shape and

physical properties. Unlike volume mea-

surements based on optical scattering

and interferometry, the optical Coulter

counter does not rely on a priori knowl-

edge or measurement of a cell’s refractive

index, which results in a significant sim-

plification of the system.

We are also investigating correla-

tions between cell volume and nuclear

morphology in white blood cells in the

hope of building a diagnostic system

for the early detection of infection.

The ability of optics to multiplex mea-

surable quantities will further enable

cell volume to be correlated with a host

of other biochemical and morphologi-

cal cell properties. «

REFERENCES

1. A. Tsur, J. K. Moore, P. Jorgensen, H.

M. Shapiro, and M. W. Kirschner, PLoS

One, 6, e16053 (2011).

2. W. H. Grover et al., Proc. Nat. Acad. Sci.,

108, 10992–10996 (2011).

3. E. Schonbrun, G. Di Caprio, and

D. Schaak, Opt. Exp., 21, 8793–8798

(2013).

4. E. Schonbrun, R. Malka, G. Di Caprio,

D. Schaak, and J. M. Higgins, J. Cytome-

try, 85, 332–338 (2014).

5. J. M. Higgins and L. Mahadevan,

Proc. Nat. Acad. Sci., 107, 20587–20592

(2010).

6. F. Chaves, B. Tierno, and D. Xu, Am. J.

Clin. Pathol., 124, 440–444 (2005).

FIGURE 3. Complete blood counts analyze red blood cell volume and hemoglobin mass and are one of the most frequent clinical tests. To measure both these

parameters, the system uses two-color absorption, where red light is used to retrieve cell volume and blue light retrieves hemoglobin mass (a). Color, thickness,

and mass maps represent a discoid and parachute red blood cell (b). A comparison of the optics-based system (red) and a clinical hematology analyzer (blue)

demonstrates that in addition to volume and mass, the optical system captures images of every cell, which enables study of the morphology of outliers (c).4

FIGURE 4. Images of height maps of neutrophil cells collected by the optical Coulter counter are shown in red. The nuclear morphology is also

simultaneously observed using a nucleic acid fluorescence stain, Syto16, shown in blue.

1409bow_28 28 9/4/14 3:43 PM

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 29

3 D O P T I C A L M O L E C U L A R I M A G I N G

B y Mike May

Developing drugs in 3DNew 3D imaging tools and techniques are enabling drug development with multiple, simultaneous

views; tracking of multiple targets; greater depth and resolution; multimode operation; and in vivo

monitoring. In doing so, they provide promise that tomorrow’s medicines will be safer and more effective.

Three-dimensional (3D) imag-

ing is changing biology—

and changing the world of

drug development. This

truth becomes clear in talking with 3D

imaging specialists like Jacob G. Tes-

dorpf, director of high-content instru-

ments and applications for life sciences

and technologies at PerkinElmer’s facil-

ity in Hamburg, Germany. For Tesdorpf,

biology intertwines with drug discovery,

and he sees 3D imaging used here in two

ways. A scientist can look at a single cell

or something made of many cells, from a

cell culture to a small model organism,

like a zebrafish. “If you image in 3D,” he

explains, “you can tell the spatial rela-

tionship of the components, regardless

of scale.” For example, two structures

might appear side by side in two dimen-

sions, but 3D could reveal that they actu-

ally lie far apart—at least in terms of cel-

lular space.

Even cells themselves ‘know’ that 3D is

better. That is, cells live in 3D environ-

ments in the body. As a result, 3D culture

tells scientists more about how cells really

behave. As Tesdorpf says, “3D cell culture

is a better proxy for how cells really func-

tion and how they interact with neigh-

boring cells.” To see those 3D effects in

action, scientists need tools that provide

3D images.

Tracking multiple targets

In drug development, a scientist might

want to track more than one target—say,

a couple of proteins—in 3D, and look

for interactions between those targets.

To get the most from drug-development

experiments, that researcher must quan-

tify the imaging results.

The recent Opera Phenix High

Content Screening System from

PerkinElmer provides a high-through-

put approach to 3D confocal imag-

ing to track fluorescent targets in

microplates. This platform uses a

scientific CMOS (sCMOS) camera

that captures larger structures—

three times larger than the plat-

form that it updates—in a single

image (see Fig. 1). A scientist can

even deck out this system with four

cameras to get simultaneous images

from four fluorophores. Moreover, this

system uses objectives from Carl Zeiss

(Oberkochen, Germany), which pro-

vide large apertures and water immer-

sion. “With water-immersion there’s not

as much change in the refractive index,”

says Tesdorpf, “so the resolution remains

high as you move deeper into a sample.”

How deep, though, really depends on the

sample at hand. Here, ‘deep’ might only

mean 80 µm, but that gets paired with the

ability to quickly explore many samples.

Matching modalities

In addition to tracking multiple targets,

some medical researchers want to image

a sample in more than one way. For exam-

ple, the new InSyTe series from TriFoil

Imaging (Chatsworth, CA) allows drug

developers to combine optical imaging

with other modalities, such as positron

emission tomography (PET) and com-

puted tomography (CT). As TriFoil CEO

Kevin Parnham explains, “If you can

image a subject in vivo, you can watch

the progress of a treatment or the effi-

cacy of a drug. To have any idea of the

volume that you’re looking at, you need

to image in 3D.” With the InSyTe system,

a researcher can label a chemical in the

body and then see where it gets taken up

in a sample.

Some scientists will desire different

combinations of modalities than oth-

ers. To accommodate such a variety

of uses, the InSyTe comes with a single

modality or as many as three. It can also

be upgraded later if needed. The opti-

cal modality provides 3D fluorescent

FIGURE 1. In this 3D InSight tumor microtissue

from InSphero—a supplier of 3D microtissues for

predictive drug testing—an image captured with

the Opera Phenix shows expressed GFP (green)

and Hoechst dye (blue) stains the nuclei. (Image

courtesy of InSphero)

1409bow_29 29 9/4/14 3:43 PM

pco.edge gold

from the pioneers

in sCMOS

image sensor technology

deepcooled

www.pco.de

www.pco-tech.com

37 000 : 1

intrascene dynamic

on the cuttingedge

0.8 e-

read out

noise

3 D O P T I C A L M O L E C U L A R I M A G I N G c o n t .

imaging. The system’s laser rotates

around the sample and captures image

information at 49 positions. For depth,

the system samples in 1 mm steps. The

system’s software combines the images

to build a 3D representation of the sam-

ple. Moreover, the system accommodates

organisms from neonatal mice to small

rats, up to about 300 g for the latter.

Although the InSyTe provides

advanced imaging modalities, it does not

require a highly controlled environment

for use. Scientists can place the InSyTe

on a benchtop in an ordinary lab. “It can

tolerate a wide range of temperatures,”

Parnham explains.

Creating combinations

A combination of hardware and software

makes up the LumiQuant from Aspect

Imaging (Toronto, ON, Canada). This

system consists primarily of a miniatur-

ized magnetic resonance imaging (MRI)

platform, which can be used without the

cooling and shielded rooms needed for

larger superconducting MRI systems.

The LumiQuant integrates with leading

luminescence platforms, including the

IVIS technology from PerkinElmer, to

bring in optical capabilities. For example,

the LumiQuant allows MRI plus biolumi-

nescent imaging (see Fig. 2). “The com-

bination of hardware and software in the

LumiQuant,” says Robert Sandler, senior

VP of marketing, “allows us to look at an

optical bioluminescent signal localized

in 3D with exquisite morphological ref-

erence from the compact MRI.”

LumiQuant collects an MRI and

optical signal from the same sample

by using a cassette. A mouse, for exam-

ple, can be anesthetized, placed in the

cassette, imaged with compact MRI,

and then transferred to an optical sys-

tem for bioluminescent imaging. “We

use the MRI image for 3D morphologi-

cal imaging, and then apply organ-spe-

cific filters—from an atlas—to the bio-

luminescence to detect elements of a

disease at the molecular level and with

organ-specific attenuation factors,”

Sandler explains.

Although the LumiQuant

system provides high-powered

imaging options, users do not

need extensive training to use

it. “The optical platform is lit-

erally push-button technology,”

Sandler says. “The compact MRI

side is a little more complicated,

but our users are usually biolo-

gists and not MRI experts.” He

adds, “With only marginal train-

ing, biology researchers and stu-

dents can generate and quantify

images on the Aspect Imaging

M3 compact MRI platform.”

Turning high-tech imag-

ing capabilities into far more

user-friendly tools will surely

entice more basic researchers

and industrial scientists to go

3D in medical research. By col-

lecting an added dimension in

drug research, scientists will get

a better understanding of dis-

ease mechanisms, find safer and

more effective medicines and,

thereby, create new treatments

that go far beyond today’s medi-

cal options. «

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

images from luminescence and compact MRI. (Image courtesy

of Aspect Imaging)

1409bow_30 30 9/4/14 3:43 PM

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 31

S P E C T R A L I M A G I N G

B y John Wal lace

Imaging spectrometers look at life in two waysSpectral imaging is finding more and more applications in life sciences, from

noninvasive disease diagnosis to food processing. Various imaging spectrometers

make those applications possible—and promise more for the future.

Imaging spectroscopy combines the

chemical detection capabilities of

spectroscopy with the image-mak-

ing of camera visualization to

reveal both chemical signature and spa-

tial structure. The technique—known

variously as chemical imaging, spectral

imaging, multispectral imaging, hyper-

spectral imaging, and Raman imaging—

is enabled by a subset of optical spectrom-

eters called imaging spectrometers.

Indeed, while optical spectrometers

can be classified in any number of ways

(for instance, by size, wavelength range,

or optical configuration, or whether they

work via absorption, emission, trans-

mission, or scattering), one of the most

fundamental divisions is between imag-

ing and nonimaging varieties. If you’re

analyzing a uniform sample or look-

ing at the spectral properties of a single

light source, then nonimaging is fine.

But if you need to get spectral details

for every point across a scene or a medi-

cal specimen, then you need an imaging

spectrometer.

Imaging spectrometers come in dif-

ferent forms—for example, multi- or

hyperspectral instruments, which often

characterize externally illuminated

objects such as those under a microscope;

and Raman instruments, which use an

internal laser to illuminate the object or

area under test, then analyze the result-

ing Raman-scattered (and wavelength-

shifted) light.

At least some of the technology inside

a particular spectrometer, which includes

the optics, the type of sensor array, the

electronics, and the software, is at least

partly unique to the instrument’s man-

ufacturer. Many imaging spectrome-

ters are tweaked—or even specifically

designed—for a certain application.

Because such technology is often specific

to an instrument’s maker, this article will

cover the topic of imaging spectrometry

by focusing on a few instrument manufac-

turers one at a time.

Many confgurations for science

In science, particularly the biosciences,

imaging spectroscopy finds use in micro-

spectroscopy, multichannel plasma anal-

ysis, photoluminescence and electro-

luminescence, Raman microscopy and

spectroscopy, time-domain-fluorescence

imaging microscopy, time-resolved

imaging and spectroscopy, fluorescence JOHN WALLACE is senior editor for Laser Focus World; e-mail: johnw@pennwell.com.

EDITOR’S NOTE: This article is adapted

from Laser Focus World, 50, 1, 70–76

(Jan. 2014).

FIGURE 1. The shape of Horiba’s iHR550 imaging spectrometer is dictated by its requirements.

1409bow_31 31 9/4/14 3:43 PM

32 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

S P E C T R A L I M A G I N G c o n t .

spectroscopy, and other applications.

Horiba JobinYvon (Edison, NJ) pro-

duces a range of imaging spectrometers

that are paired with the company’s scien-

tific cameras.

Horiba designs and manufactures a

range of imaging spectrometers varying

in focal length from 70 to 1250 mm. “As

diffraction gratings are a core technology

of Horiba JobinYvon, we have the ability

to optimize each design to best match

the experimental requirements of wave-

length range, resolution, throughput,

size, and cost,” notes Joanne Lowy, Hori-

ba’s marketing manager.

The necessity for a large choice of

designs is driven by applications, cost,

and size constraints set by the research-

ers, says Lowy. In an example of one

design, Horiba produces Czerny-Turner

imaging spectrometers with fully aber-

ration-corrected reflective optics that

the company says are free of redif-

fracted light (the Czerny-Turner design,

the basis for many imaging-spectrome-

ter optical layouts, has two mirrors that

image an entrance slit onto an exit slit; a

diffraction grating between the mirrors

disperses the light). In another design,

Horiba offers supercorrected, concave,

holographic-grating-based spectrom-

eters in which the concave grating per-

forms the function of collimation, disper-

sion, and focus in one element.

The shape of Horiba’s iHR550 imaging

spectrometer is dictated by the require-

ments of the spectrometer, explains Lowy

(see Fig. 1). All-oversized reflective optics

eliminate chromatic aberration (their

large size is to provide high throughput);

toroidally corrected optics reduce aberra-

tions; and careful selection of the angle

of incidence eliminates rediffracted

light. The last is of particular impor-

tance, as systems that suffer from redif-

fracted light, such as criss-cross Czerny-

Turner spectrometers, will not produce

pure spectra at their output foci, which

can lead to erroneous spectra or images.

Unlike typical imaging spectro-

graphs, which measure over only a nar-

row spectral band and (due to their

optical design) one optical output, the

iHR550 has multiple entrance ports, mul-

tiple gratings with on-

axis rotation, and

multiple array exit

ports, with wide

focal planes acces-

sible outside the

instrument’s cast-

ing. With selec-

tion of appropri-

ate gratings, the

instrument has

a spectral range

from the deep-

ultraviolet (UV;

122 nm) to the

far-infrared (IR;

40 µm).

Uses for the

iHR550 named

by Lowy include

multitrack plasma analysis (which

requires high resolution and imaging

quality); direct coupling to a microscope

for photon-starved applications such as

Raman spectroscopy; broadband-emis-

sion applications such as photolumines-

cence; and time-resolved applications

such as fluorescence-lifetime imaging

microscopy (FLIM).

Abolishing astigmatism

The Czerny-Turner configuration is not

simply a commonly used imaging-spec-

trometer design; it also serves as a spring-

board for inventive improvements. One

of these is a Czerny-Turner-based config-

uration developed by Princeton Instru-

ments (Acton, MA) that contains the

addition of a Schmidt corrector to its

optics; this is a transmissive plate with an

aspheric surface that corrects aberrations

(added to a telescope, a Schmidt correc-

tor plate is designed to eliminate spheri-

cal aberration).

The company’s imaging spectro-

graph, the IsoPlane 320, has zero astig-

matism at all wavelengths across the

entire focal plane (see Fig. 2). When

used in multichannel spectroscopy, in

which a number of fiber-optic probes

(the “channels”) are placed across the

image field to collect information,

the astigmatism-free design allows up

to 200 channels, according to Jason

McClure, chief scientist at Princeton

Instruments and designer of the optics.

The design, which is patent-pending,

also boosts sensitivity by focusing more

photons onto fewer pixels.

FIGURE 2. Princeton Instruments’

IsoPlane 320 has zero astigmatism at all

wavelengths across the entire focal plane.

FIGURE 3. BaySpec’s OCI-1000 hyperspectral imager is able to serve as a

noninvasive diagnostic tool.

1409bow_32 32 9/4/14 3:43 PM

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 33

S P E C T R A L I M A G I N G c o n t .

The IsoPlane 320 is used, for example,

in tip-enhanced Raman spectroscopy,

studies of carbon nanotubes, plasmonic

structures, and quantum dots. The ability

of the IsoPlane to image dozens of optical

fibers greatly enhances the spatial resolu-

tion available for imaging. In single-mol-

ecule tip-enhanced Raman spectroscopy

(SMTERS), both the sample and signal

are minuscule; the IsoPlane is the only

spectrograph to date that has successfully

been used in this technique, says Debby

Flint-Baum, marketing communications

manager at Princeton Instruments.

Using the IsoPlane 320 for this pur-

pose, the Van Duyne group at Northwest-

ern University has combined SMTERS

and scanning tunneling microscopy

(STM) to obtain unprecedented sensi-

tivity and spatial resolution in obtaining

Raman spectra of single rhodamine 6G

molecules, advancing the understanding

of excited-state dynamics in adsorbate-

substrate interactions.1

Handheld hyperspectral

Hyperspectral cameras divide the light

spectrum into many small wavelength

bands (compared to traditional cam-

eras, which only have three bands—red,

green, and blue); spectrometers based

on this technology thus can give very

detailed information about the mate-

rial constitution of an imaged object.

Based on its own volume-phase-grating

(VPG) technology, BaySpec (San Jose,

CA) has developed two closely related

battery-powered handheld hyperspec-

tral imaging spectrometers that enable

field-based uses such as agriculture and

food inspection.

Both are VPG-based, which gives them

an efficiency of up to 99 percent, along

with high stability and repeatability, as

there are no moving parts. The instru-

ments can be configured for any spec-

tral range within 400–1100 nm, with a

spectral resolution up to 0.01 nm. Cou-

pled with a high-speed camera, the sys-

tem can generate hyperspectral image

cubes with up to 2048 spectral bands in

a single scan.

BaySpec’s handheld hyperspectral

imager, the OCI-1000, integrates the

spectral dispersing element (which has

more than 100 bands covering the 600–

1000 nm range) on an image sensor at

the level of the chip itself, eliminating the

need for the large, expensive optics that

are used on traditional systems (see Fig.

3). The instrument weighs less than 0.5

lb. and can be installed almost anywhere,

says Lin Chandler, spectroscopy product

manager at BaySpec.

The similar (and, at 0.8 lb., just

slightly heavier) OCI-2000 snapshot

imager goes one step further, simulta-

neously dispersing 2D images into mul-

tiple spectral bands (currently 32 bands

covering the 600–1000 nm range), elim-

inating the need for mechanical scan-

ning. Because a single “snapshot” pro-

duces an entire multispectral image

data cube, true multispectral imaging

can be done at video rates.

Chandler adds that one of the novel

applications in development for these

hyperspectral imagers is as a noninva-

sive diagnostic tool. For instance, such

an instrument can achieve an accurate

hyperspectral representation of the anat-

omy of any living organism without dis-

turbing it.

Photon conservation

By ef f icient ly making use of

photons, an imaging spectrometer not

only shortens data acquisition times—

it can also minimize damage to pho-

tosensitive samples. Andor (Belfast,

Ireland) is known for its low-light sci-

entif ic cameras; the company also

produces imaging spectrometers that

conserve photons.

The company’s Czerny-Turner instru-

ments have motorized multiport, multi-

grating platforms with toroidal optics for

astigmatism correction of aberrations

inherent to Czerny-Turner design; these

spectrometers provide good multitrack

spectroscopy capabilities, as well as good

sample image relay at “zero order” in

microspectroscopy configurations (allow-

ing the sample to be visualized and spec-

tral analysis to be performed through

the same optical path with one detector),

according to Marion Mathieu, the com-

pany’s digital marketing executive.

Andor also makes instruments with a

transmission-optics configuration and

low-stray-light volume-phase-holographic

(VPH) gratings, resulting in high collec-

tion efficiency, throughput, and multi-

track-spectroscopy capabilities across a

large focal plane. The company’s third

type of instrument is hyperspectral,

based on either a “grism” (combined

grating and prism) transmission configu-

ration or an aberration-corrected, reflec-

tive diffraction-grating configuration.

The instruments can be used in trans-

mission- or reflection-based absorption

spectroscopy; Raman spectroscopy at

wavelengths from the UV to the near-IR;

micro-Raman and microfluorescence;

photon counting; single-molecule spec-

troscopy (all requiring efficient use of

photons); and for plasma studies. Appli-

cations are many, including biomedical

FIGURE 4. Headwall Photonics’ hyperspectral imaging device (right) reveals spatial and spectroscopic

features of tissues, such as these cancerous kidney cells (left).

1409bow_33 33 9/4/14 3:44 PM

34 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

S P E C T R A L I M A G I N G c o n t .

screening and diagnosis and analytical

cell biology.

As in any optical system, combining

high light throughput (low f-numbers)

with high resolution (low aberrations) is

challenging. Andor’s VPH-based trans-

missive-optics imaging spectrometer,

called the Holospec, is an on-axis, high-

throughput instrument with imaging-cor-

rected optics that allow both high-den-

sity, multi-track spectroscopy (clear sepa-

ration of individual channel images from

densely packed fiber-optic bundles) and

high throughput, says Mathieu. The f/1.8

aperture allows 100-percent light collec-

tion from fiber optics with a numerical

aperture (NA) of 0.22.

“Up to 32 100-μm-core fiber channels

can be individually resolved over a 4-mm-

high sensor with low crosstalk, despite

the high-density fiber-bundle configura-

tion,” notes Mathieu.

Applications include intrinsically pho-

ton-starved experiments such as quan-

tum-dot photoluminescence and micro-

Raman of biosamples. The Holospec is

also used when acquisition time is a con-

straint (gathering enough photons in

short periods of time while maintaining

a meaningful signal-to-noise ratio)—for

example, in microspectroscopic chemi-

cal mapping, microfluidics such as spec-

trally resolved flow cytometry, and on-

line process control. In addition, effi-

cient photon use minimizes photodam-

age of photosensitive samples such as

live cells or luminescent biotags, protect-

ing them from photodegradation and

phototoxicity.

Near and far

While spectrometers do perfectly well sit-

ting in labs and eyeing microscopic sam-

ples, they can also see the world—for

example, from the vantage point of an

unmanned aerial vehicle (UAV). Head-

wall Photonics (Fitchburg, MA) makes

instruments that do either: The company

produces both hyperspectral and Raman

imaging spectrometers, with the hyper-

spectral version the one that gets to take

the airborne route.

“The type of hyperspectral technology

we deploy is commonly known as ‘push-

broom,’ meaning that motion needs to

occur,” says David Bannon, Headwall’s

CEO. “Either the sensor moves about the

field of view (as it would in an aircraft), or

the scene moves beneath a stationary sen-

sor (as it would in an in-line inspection

deployment).”

Headwall’s hyperspectral instru-

ments have an athermalized design,

making them fully functional in

demanding environments such as those

that UAVs endure, and have almost no

moving parts—again, for reliable per-

formance while aloft. The instruments

have aberration-corrected imaging to

provide high spatial and spectral reso-

lution across a wide field of view. “They

have excellent image fidelity off to the

sides,” says Bannon.

Headwall makes its own diffraction

gratings that provide this aberration-cor-

rected imaging; this aids in designing

a package that tightly integrates optics,

electro-optics, electronics, and software,

but also aids in customization. The com-

pany also customizes its software when

needed, in some cases for the specific

application (for example, UAV platforms)

and in others for a specific customer (for

example, to identify foreign objects in

food processing).

Medical and biotech, one of four key

areas served by Headwall’s imaging spec-

trometers, includes establishing noninva-

sive diagnostic techniques as well as labo-

ratory applications for microscopy. Med-

ical analysis and interpretation includes

identifying and differentiating between

types of skin cancer, for example (see

Fig. 4). Industrial inspection, another key

area, includes poultry inspection: Hyper-

spectral imaging spectrometry can dis-

cern diseased vs. healthy poultry. Bannon

says, “Imaging spectroscopy is the next

generation of machine-vision capability.”

However, UAVs are what’s making

the news these days. “Headwall’s sensor

design is geared toward airborne deploy-

ment,” says Bannon. “Users of hyperspec-

tral sensors aren’t always white-coated

scientists. They need help optimizing

these elements so that they work well

together: 1) the sensor; 2) the hyperspec-

tral data processing unit that collects

the incoming data and manages it, also

‘instructing’ the sensor when and where

to operate; the software (Hyperspec III)

needs to not only manage the data and

sensor operation, but do so with an easy-

to-assimilate GUI; 3) GPS and inertial-

navigation system (INS); and 4) cabling/

cooling/overall integration.”

As a further step into the UAV arena,

Headwall has begun selling a package

consisting not just of its Micro-Hyper-

spec sensor in the airborne configura-

tion—but also the UAV that flies the sen-

sor system.

From UV to IR

Imaging spectrometers can be designed

for use in just about any spectral region

used in life sciences, from UV, visible

(Vis), near-IR (NIR), or short-wave, mid-

wave, and long-wave IR (SWIR, MWIR,

and LWIR). McPherson (Chelmsford,

MA) makes them all.

McPherson’s instruments collect spec-

tra from multiple, spatially distinct chan-

nels. They can be one of several differ-

ent optical designs; each is optimized for

a particular spectral range or particular

type of application, says Erik Schoeffel,

marketing and sales at McPherson.

For example, MWIR and LWIR 2D

spectroscopy applications favor low-dis-

persion prism-based systems. These dis-

perse many microns of spectral range

on an array detector and have high

throughput and no multiple-order

effects, albeit relatively low spectral res-

olution, explains Schoeffel.

In contrast, UV-Vis-NIR spectroscopy

applications gravitate towards diffrac-

tion-grating-based, high-resolution, long-

focal-length instruments. These tend to

use fiber optics. These disperse a cou-

ple of nanometers’ spectral range on the

array detector, and have high line symme-

try and spectral resolution.

This is just a sampling of the imag-

ing spectrometers now available for life

sciences applications. For more, follow

BioOptics World’s product reporting by

visiting http://www.bioopticsworld.com/

products.html. «

REFERENCE

1. M. D. Sonntag et al., J. Am. Chem.

Soc., 135, 17187 (2013); doi:10.1021/

ja408758j.

1409bow_34 34 9/4/14 3:44 PM

www.BioOpticsWorld.com SEPTEMBER/OCTOBER 2014 35

S U P E R - R E S O L U T I O N M I C R O S C O P Y

B y Jef f Hecht

New twists on superlenses improve subwavelength microscopyMetamaterials can overcome traditional limits on optical resolution, but they pose other

challenges including high losses, dependence on resonances, and limited depths of field.

Subwavelength microscopy

has come a long way from its

early days as an exotic con-

cept. The diffraction limit

still rules for conventional bulk optics,

but resolution can be pushed below

half a wavelength in a couple of ways.

Metamaterials and special instru-

ments can achieve true super-resolu-

tion by directly recording transmit-

ted, reflected, or emitted light. Alter-

natively, special processes using flu-

orescence, nonlinearities, or other

techniques can achieve functional

super-resolution.1 The latter “is more

like signal processing. You acquire an

image and try to improve [it],” says

George Eleftheriades of the Univer-

sity of Toronto (ON Canada).

So far, the most practical techniques

are functional ones, such as stimulated

emission depletion (STED) microscopy,

developed by Stefan Hell of the Max

Planck Institute for Biophysical Chem-

istry (Göttingen, Germany).2 STED

allows subwavelength resolution of a

f luorescent spot by de-exciting fluoro-

phores surrounding it. Its major appli-

cations, like those of other functional

superresolution imaging, are in bio-

medicine. For example, in June 2014,

PicoQuant (Berlin, Germany) added

STED to its time-resolved MicroTime

200 confocal microscope, improving

resolution to well below 100 nm.

Scanning near-field optical micros-

copy (SNOM) offers true super-resolu-

tion by moving a tiny aperture across

the near field, but is too time consum-

ing for many applications. Metamaterial

devices, such as the superlens devised

by Sir John Pendry of Imperial College

(London, England), can observe much

larger areas at one time.3 Thanks to that

advantage, and the flexibility of true

super-resolution, superlenses are com-

ing on fast, although they still face major

challenges, including limited bandwidth

and resolution depth and special mate-

rial requirements.

Superlenses and evanescent waves

The superlens concept is based on meta-

materials in which both electric permit-

tivity and magnetic permeability are neg-

ative at certain wavelengths. That gives

them a negative refractive index, so they

can capture evanescent waves that con-

tain information needed to produce sub-

wavelength images, but are not captured

by conventional optics. As shown in Fig. 1,

a superlens bends light entering it back-

ward, forming an image plane inside it

and another on the opposite side.

The most familiar evanescent waves are

those that leak through a surface where

total internal reflection occurs. They can

be detected very close to the surface, but

decay exponentially with distance from the

surface, so they can’t carry energy away.

Pendry’s superlens can achieve subwave-

length resolution by focusing the waves

near the surface. Negative-index materials

“can actually amplify evanescent waves and

thus restore high-resolution details which

are inaccessible by classical imaging sys-

tems,” writes Eleftheriades.4

A slab of negative-index metama-

terial can serve as a superlens, which

FIGURE 1. A superlens with a refractive index of

-1 bends light entering it backwards, producing an

image plane inside the metamaterial and another

on the opposite side of the material, which can have

super-resolution. (Adapted from Wikipedia)

Object(in near feld)

Image(near feld)

Negativerefractionat surface

Air

Metamaterial

Internalfocus

JEFF HECHT is a contributing editor for Laser Focus World; e-mail: jeff@jeffhecht.com.

1409bow_35 35 9/4/14 3:44 PM

Cr

Opticalmicroscope

Conventional lens

Objectplane

Hyperlensimage plane

Far feldimage plane

Quartz

Hyperlens

λ = 365 nmE

Ag/Al2O3multilayers

36 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

S U P E R - R E S O L U T I O N M I C R O S C O P Y c o n t .

essentially produces a time-reversed

wave with a negative frequency relative

to the original, creating a super-resolu-

tion image, says Stephane Larouche of

Duke University (Durham, NC). Nega-

tive refraction also reverses Snell’s law

and focuses rather than disperses eva-

nescent waves. The metamaterial has

subwavelength resolution over a large

area, unlike SNOM, which can address

only one point at a time.

Superlenses have their drawbacks.

Attenuation tends to be high, particularly

for metal films that have negative permit-

tivity at visible wavelengths. Metamaterial

properties depend on resonances, so the

negative index crucial for super-resolu-

tion imaging only exists across a limited

band of wavelengths. Superlenses also are

limited to the near field and cannot focus

in three dimensions.

Broadband subwavelength imaging

In 2012, Thomas Taubner of RWTH

Aachen University (Aachen, Germany)

and his colleagues proposed making a

tunable broadband superlens from gra-

phene. Graphene layers can both support

plasmons and guide them, making them

suitable for use in metamaterials. Varying

gate voltage, electric field, or chemical

doping applied to the graphene changes

its conductivity, allowing continuous tun-

ing of graphene properties at infrared

and terahertz frequencies.5

Imaging in graphene is weaker than

true superlensing because it is not

strongly resonant, but Taubner’s study

showed that graphene sheets would

enhance evanescent waves for subwave-

length imaging. The RWTH team pre-

dicted that two graphene layers could

achieve resolution of about one-seventh

wave (λ/7), and multiple layers could

reach about λ/10. Most important, the

team predicted that a graphene lens

could focus to below the diffraction

limit from the mid-infrared to the tera-

hertz band. Tauber and his colleagues

also say their concept could be general-

ized to a two-dimensional “conducting

sheet lens” of semiconductor hetero-

structures. Results of experiments with

their graphene multifrequency super-

lenses have yet to be published.

Hyperlenses and metalenses

Evanescent waves can be

focused into the far f ield

with a hyperlens, based on

an anisotropic layered meta-

material. The simplest type is

a stack of alternating metal/

dielectric layers much thin-

ner than the optical wave-

length. The original proposal

called for hyperbolic disper-

sion in the metamaterial, but

eccentric elliptical dispersion

is another alternative, write

Dylan Lu and Zhaowei Liu of

the University of California at

San Diego (La Jolla, CA) in a

review paper.6

Far-field imaging was a sur-

prise to many, but several types

have been demonstrated. Fig-

ure 2 shows how a hyperlens

with hyperbolic dispersion can

focus evanescent waves radially,

effectively converting them into

propagating waves that carry

subwavelength information in the eva-

nescent waves into the far field. The flat

layers are bent so light travels radially

through them and magnifies an object

placed at the inner radial surface so it

can be viewed on the outer surface with

subwavelength resolution. The ratio of

the radii at the inner and outer curved

layers gives the magnification at the

outer surface.

A hyperlens cannot do such standard

optical tasks as focusing plane waves or

performing Fourier transforms. That

requires a metalens, a dense array of

identical resonators fabricated in the

plane of a metamaterial to provide the

phase compensation missing in a hyper-

lens. Metalenses built from such ele-

ments as “meta-atoms,” plasmonic metal-

insulator-metal waveguide couplers, or

graded index patterns can provide “an

exceptional combination of super-resolu-

tion and desirable functions of conven-

tional lenses,” write Lu and Liu.6

Many metalens designs are under

study. Liu proposed using arrays

of bidirectional planar plasmonic

waveguide couplers, as shown in Fig-

ure 3, but has yet to demonstrate it.

Nikolay Zheludev at the University of

Southampton (England) used meta-

atoms that form a metalens to focus

800 nm light onto an array of sub-

wavelength 160 nm hot spots beyond

the near field of the lens.7 Interfer-

ence among light waves propagating

through the meta-atoms caused an

effect called “superoscillation” to gen-

erate the spots.

Vladimir Shalaev’s group at Purdue

University (West Lafayette, IN) developed

a different planar nanostructure; a 4 µm

lens with concentric rings of nanoholes

machined in a 30 nm gold film. The lens

focused 676 nm light only a spot 2.5 µm

away; 476 nm light was focused at 7 to 10

µm from the lens. Their proposed design

can be machined onto the end of an opti-

cal fiber.8

Camille Jouvaud and colleagues at

the Langevin Institute (Paris, France)

made magnetic metalenses from arrays

of split-ring oscillators with different res-

onant frequencies. Microwave experi-

ments produced localized modes instead

of extended ones, and they predict their

approach would allow subwavelength

imaging in the visible and infrared.9

FIGURE 2. Curved stack of layers in a hyperlens captures

evanescent waves from object (inside curvature at top), then

transfers them radially to the outside curvature at bottom

before they are focused with subwavelength resolution.

(Courtesy of Zhaowei Liu)

1409bow_36 36 9/4/14 3:44 PM

a) b) c)

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New directions for

subwavelength microscopy

Super-resolution imaging is st ill

young, and new ideas continue to

emerge. One that particularly intrigues

Larouche is assembling optical meta-

materials by a self-organizing bottom-

up process rather than using the top-

down approach of photolithography.

His lab recently made a near-infrared

metamaterial with 30 nm elements.

“We used electron-beam lithography,

which is a great approach for research,”

he said, but not for mass production.

“That particular device took weeks

to fabricate.”

He was intrigued by a recent proposal

by Zsolt Szabo of the Budapest Univer-

sity of Technology (Hungary) and col-

leagues to embed silver nanospheres in

silica to make a composite metamate-

rial for subwavelength imaging. They

calculated that with self-organizing

unit cells smaller than 20 nm, a single-

or multi-layer metal-dielectric compos-

ite metamaterial could achieve 100 nm

resolution.10

Subwavelength imaging is still

young, and development is so

diverse that there is no room to cover

it all, particularly the many variations

on functional super-resolution. Big

challenges remain, but real progress

is being made in seeing the previously

unseeable. «

ACKNOWLEDGEMENT

This article originally appeared in Laser

Focus World, 50, 8, 33–37 (2014).

REFERENCES

1. A. Neice, “Methods and limitations of

subwavelength imaging,” in Advances

in Imaging and Electron Physics, pp.

117–140 (2010).

2. S. Hell and J. Wichmann, “Break-

ing the diffraction resolution limit

by stimulated emission: Stimulated-

emission-depletion f luorescence

microscopy,” Opt. Lett., 19, 780 (1994);

doi:10.1364/OL.19.000780.

3. J. Pendry, “Negative refraction makes

a perfect lens,” Phys. Rev. Lett., 85,

3966 (Oct. 2000).

4. A.M.H. Wong and G. V. Eleftheriades,

“Advances in imaging beyond the dif-

fraction limit,” IEEE Photon. J., 4, 586

(Apr. 2012).

5. P. Li and T. Taubner, “Broadband

subwavelength imaging using a tun-

able graphene lens,” ACS Nano,

6, 11 10107 (2012); doi: 10.1021/

nn303845a.

6. D. Lu and Z. Liu, “Hyperlenses and

metalenses for far-field super-resolu-

tion imaging,” Nat. Commun., 3, 1205

(2012); doi: 10.1038/ncomms2176.

7. T. Ray, E.T.F. Rogers, and N. I. Zhe-

ludev, “Sub-wavelength focusing

meta-lens,” Opt. Express, 21, 7577

(Mar. 2013).

8. X. Ni, S. Ishii, A. V. Kildishev, and V.

M. Shalaev, “Ultra-thin planar Babi-

net-inverted plasmonic nanolenses,”

Light: Sci. Appl., 2, e72 (2013); doi:

10.1038/lsa.2013.28.

9. C. Jourvad, A. Ourir, and J. de

Rosny, “Far-field imaging with a

multi-frequency metalens,” Appl.

Phys. Lett., 104, 243507 (2014); doi:

10.1063/1.4882277.

10. Z. Szabo, Y. Kiasat, and E. P. Li.,

“Subwavelength imaging with com-

posite metamaterials,” J. Opt. Soc. Am.

B, 31, 1298 (June 2014); doi:10.1364/

JOSAB.31.001298.

FIGURE 3. Proposed metalens design uses an array of waveguides—vertical in the “metal” layer—

to provide phase compensation so it can focus plane waves. (Courtesy of Zhaowei Liu)

1409bow_37 37 9/4/14 3:44 PM

38 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

PRODUCTSComponents Systems&s S

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40 SEPTEMBER/OCTOBER 2014 www.BioOpticsWorld.com

B y LEE DUBAYEnd Result

University of Cincinnati (Ohio)

philosophy and psychology

graduate assistant Luis Favela studies

how people perceive their environment,

and how those perceptions inform

their judgments. Inspired by the CDC

prediction that more than 6 million

Americans aged 40 and older will be

affected by blindness or low vision by

2030, Favela wondered whether an

infrared (IR) light-based handheld device,

developed by University of Reading

(Reading, Berkshire,

England) cybernetics

students Tom Froese and

Adam Spiers1, could help

such people.

The device, called the

Enactive Torch (ET), uses

two nonlinear-response

IR rangefinders (one is

a Sharp GP2D12, which

covers a range of 8–80

cm, and the other is a

Sharp GP2Y0A02YK0F,

which covers 20–150

cm) located at the

front to detect

distance information.

The information is

then transferred to

a vibrational motor

that is attached to the

wearer’s wrist and emits

a vibration similar to a cellphone alert.

This enables the user to feel whether

something is in front of the device and

then how near or far that something is

based on the intensity of the vibrations,

Favela told BioOptics World. The gentle

buzz increases in intensity as the torch

nears the object, letting the user make

judgments about where to move based

on a virtual touch.

Comparative test

Favela conducted

an experiment

to determine the

value of such a

tool. He asked

27 participants

with normal or

corrected-to-

normal vision to

make perceptual

judgments about

their ability to

pass through an

opening a few

feet in front of

them without

needing to shift their normal posture. He

tested the judgments they made in three

modes: using only their vision, using a

cane while blindfolded, and using the

ET while blindfolded. The idea was to

compare judgments made with vision

against those made by touch.

Favela thought that vision-based

judgments would be the most accurate

because vision tends to be most

people’s dominant perceptual modality.

But the data revealed that all three

modalities were equally accurate.

“People can carry out actions just about

to the same degree whether they’re

using their vision or their sense of

touch. I was really surprised,” he says.

Favela plans on additional

experiments with the ET that require

more complicated judgments, such as

the ability to step over an obstacle or to

climb stairs. He, Froese, and Spiers have

also discussed the possibility of smaller

versions of the ET, considering devices

with other forms of feedback such as

skin stretch feedback, he says.

Favela presented his research

“Augmenting the Sensory Judgment

Abilities of the Visually Impaired” at the

American Psychological Association’s

(APA) annual convention, held Aug.

7-10, 2014, in Washington, DC. «

REFERENCE

1. T. Froese, M. McGann, W. Bigge, A.

Spiers, and A. Seth, IEEE Trans. Haptics, 5,

365–375 (2012); doi:10.1109/ToH.2011.57.

Handheld IR device could be game-changer for the visually impaired

The Enactive Torch uses infrared sensors

to detect objects in front of it and, upon

detection, send a vibrational warning to

the wearer. (Both photos courtesy of Colleen

Kelley, University of Cincinnati)

Luis Favela observes Amon as she uses the Enactive Torch during a

demonstration of Favela’s experiment.

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