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November/December 2011

Worldwide Coverage: Optics, Lasers, Imaging, Fiber Optics, Electro-Optics, Photonics Component Manufacturing

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www.photonics.com10 BIOSCANBioPhotonicseditors curate the most significant headlines

of the month for photonics in the life sciences and take

you deeper inside the news. Featured stories include:

DNA nanosensors pave way for cancer tests, drugs STED microscopy reveals how immune system

attacks infected cells

Laser technique produces synthetic tissue

for regenerative medicine

21 BUSINESSSCANPhotonics-related projects score NIH grants

NEWS

35

BioPhotonics November/December 2011

Volume 18 Issue 9

t TABLE OF CONTENTS

24 TO LABEL OR NOT?by Dr. Neil Anderson, Semrock Inc., a unit of Idex Corp.

The emerging field of multimodal nonlinear optical imaging, which

integrates label-based and label-free methods, could become the

definitive diagnostic tool of the future.

28 PHOTOCOAGULATION: HEALING THROUGH LASER DAMAGEby Marie Freebody, Contributing Editor

Used in almost all surgical specialties, this decades-old method of containing

tissue damage has become especially important to ophthalmologists.

31 DIFFRACTION-IMAGING FLOW CYTOMETRY ENABLES RAPID CELL ASSAYby Xin-Hua Hu, East Carolina University and Wavmed Technologies Corp.;

Yuanming Feng, Tianjin University; and Jun Qing Lu, East Carolina University

A novel method of combining flow cytometry with diffraction rather than fluores-

cence imaging enables 3-D visualization and suggests new applications in medicine.

35 CSI EXPERTS FIND CLUES FASTER WITH MICROSCOPYby Marie Freebody, Contributing Editor

The field of forensics is being catalyzed by the efficiencies of digital imaging

and by combining optical techniques with spectroscopy and spectrophotometry.

38 INVERTED FLUORESCENCE MICROSCOPY AIDS MICROSEPARATION STUDIESby Dr. Yolanda Fintschenko, Labsmith Inc., and Dr. Blanca Lapizco-Encinas,

Tennessee Technological University

This microscope has the stationary stage and software required by researchers

working with micro- or nanofluidic experiments.

FEATURES

NEWS

7 EDITORIAL

8 LETTERS

43 BREAKTHROUGHPRODUCTS

48 APPOINTMENTSUpcoming Courses and Shows

49 ADVERTISER INDEX

50 POST SCRIPTSby Laura S. Marshall

Genetic approach shows bright promise against AIDS

DEPARTMENTS

PHOTONICS

The technology of generating and harnessing light and other forms of radiant energy whosequantum unit is the photon. The range of applications of photonics extends from energy generation

to detection to communications and information processing.

BIOPHOTONICS

The application of photonic products and techniques to solve problems for researchers,product developers, clinical users, physicians and others in the fields of medicine,

biology and biotechnology.

This months cover is basedon an article about multimodalnonlinear optical imaging.Written by Dr. Neil Andersonof Semrock Inc., it beginson page 24. Design by Art

Director Suzanne L. Schmidt.
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www.photonics.comGroup Publisher Karen A. Newman

Editorial Staff

Managing Editor Laura S. Marshall

Senior Editor Melinda A. Rose

Features Editor Lynn M. Savage

News Editors Gary Boas, Caren B. Les, Ashley N. Paddock,Krista Zanolli

Contributing Editors Hank Hogan, Marie Freebody

Copy Editors Judith E. Storie, Patricia A. Vincent,

Margaret W. Bushee

Creative Staff

Senior Art Director Lisa N. Comstock

BioPhotonics Art Director Suzanne L. Schmidt

Designer Janice R. Tynan

Director of Publishing Operations Kathleen A. Alibozek

Electronic Media Staff

Director Charley RoseMultimedia Services & Marketing

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

Corporate Staff

Chairman/CEO Teddi C. Laurin

President Thomas F. Laurin

Director of Sales Ken Tyburski

Controller Mollie M. Armstrong

Accounting Manager Lynne Lemanski

Accounts Receivable Manager Mary C. Gniadek

Business Manager Elaine M. Filiault

Human Resources Coordinator Carol J. Atwater

Business Staff

Advertising Production Coordinator Kristina A. Laurin

Trade Show Coordinator Allison M. Mikaniewicz

Computer Systems Manager Deborah J. Lindsey

Computer Assistant Angel L. MartinezCirculation Manager Heidi L. Miller

Assistant Circulation Manager Melissa J. Liebenow

Circulation Assistants Alice M. White, Kimberly M. LaFleur,

Theresa A. Horn

Subscriptions Janice L. Butler

Distribution Manager George A. Houghtlin

Traffic Manager Daniel P. Weslowski

EDITORIAL MAIN OFFICELaurin Publishing, Berkshire Common, 2 South St.

PO Box 4949, Pittsfield, MA 01202-4949+1 (413) 499-0514; fax: +1 (413) 442-3180; e-mail: [email protected]

Subscription Policy BioPhotonics ISSN-1081-8693, (USPS 013913) is published 9 times per year by LaurinPublishing Co. Inc. TITLE reg. in US Library of Congress. The issues will be as follows: January, February,March, April, May/June, July/August, September, October, November/ December. Copyright 2011 by Lau-rin Publishing Co. Inc. All rights reserved. POSTMASTER: Periodicals postage paid at Pittsfield, MA, and at ad-ditional mailing offices. Postmaster: Send form 3579 to BioPhotonics, Berkshire Common, PO Box 4949,Pittsfield, MA 01202-4949, +1 (413) 499-0514. CIRCULATION POLICY: BioPhotonics is distributed without charge

to qualified researchers, engineers, practitioners, technicians and management personnel working with thefields of medicine or biotechnology. Eligibility requests must be returned with your business card or organi-zations letterhead. Rates for others as follows: $45 domestic and $56.25 outside US per year prepaid. Over-seas postage: $30 airmail per year. Publisher reserves the right to refuse nonqualified subscriptions. ARTICLESFOR PUBLICATION: Individuals wishing to submit an article for possible publication in BioPhotonics shouldcontact Laurin Publishing Co. Inc., Berkshire Common, PO Box 4949, Pittsfield, MA 01202-4949; phone: +1 (413)499-0514; fax: +1 (413) 442-3180; e-mail: [email protected]. Contributed statements and opinions ex-

pressed in BioPhotonics are those of the contributors the publisher assumes no responsibility for them.

6 BioPhotonics November/December 2011
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Enhance, Disrupt, Revolutionize, Repeat

In his introduction to a plenary session called Material

Nanoscience, Photonics and Technologies for Revolutionary

Innovation at LIAs ICALEO (International Congress on

Applications of Lasers & Electro-Optics) last month in Orlando,

Fla., session chairman Kunihiko Washio of Paradigm Laser

Research Ltd. in Tokyo said, Although incremental innovation

within a known systematic framework is very important for

steady technology progress, disruptive innovation is often desired

to push the limits of the imaginable and to leap forward to attain

revolutionary innovation.

Disruptive innovator Steve Jobs, the former Apple CEO,

changed the world with his computers and phones, and just one

day before he died last month at the age of 56, a press release

from the University of California, Davis, described an adaptation

to his iPhone that turned it into a microscope capable of bothrevealing vital medical information and transmitting images for

analysis and diagnosis. I wonder if Jobs knew about the adapta-

tion. I wonder what he thought, if he knew, about his disruptive

phone being turned into a potentially game-changing device to

bring a new level of health care to the world.

The group at UC-Davis was not the first to build a smartphone

microscope, and it certainly wont be the last to add some good,

incremental innovation to an already great idea, whatever it may

be. And thats good for all of us. (You can read more about the

UC-Davis enhanced iPhone on photonics.com, http://www.

photonics.com/a48604.) Innovation of all kinds keeps an industry

growing, and thats pretty good, too.

In his introduction at ICALEO, Washio went on to say, Mate-

rials science, particularly material nanoscience, is believed to be

the treasure house of the seeds of desired disruptive innovations.

The keynote presentation that followed, The Story and Prospects

of Carbon Nanoscience and Technologies for Future Exciting

Applications by Stanford researcher Hongjie Dai, presented a

look at carbon nanotubes and their unique intrinsic physical

and chemical properties, which can be exploited for biological

and biomedical applications including detection, diagnostics,

imaging and novel therapy.

We cant wait to see where the next disruptive and incremental

biophotonics innovations will come from. In a small change of

our own, were going to focus a couple of articles in every issue

around a specific topic, exploring aspects of the subject that are

bringing real change to the way we think about biophotonics,

its applications and our world.

You might see an update on the smartphone microscope in

our September issue coverage of biophotonics and global health,

while Dais work on carbon nanotubes may make its way into

our March issue, with its focus on biophotonics and cancer. With

so much interesting work going on in photonics and the lifesciences, we wanted to organize it and bring it to you with some

sense of the impact it could have on the way we live. Well ex-

plore photonics in dentistry in January. In February, well take a

closer look at biophotonics in the pharmaceutical industry. Other

topics will be covered, too, and all the things you like about Bio-

Photonics will still be there, just enhanced. We hope you like it.

In the meantime, enjoy this issue, and make plans to visit us

at our booths at BiOS (8327) and Photonics West (323) in San

Francisco in January.

7

EDITORIAL

Karen A. [email protected]

http://photonics.com/http://www.photonics.com/a48604http://www.photonics.com/a48604mailto:[email protected]://www.photonics.com/a48604mailto:[email protected]://www.photonics.com/a48604http://photonics.com/


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The limits of diffraction limits

In your recent article on high-resolution

microscopy (July/August 2011, p. 31),

the heading asks, Can optical micros-

copy further shred the Abbe diffraction

limit? Although the statement aboutshredding the Abbe diffraction limit often

is perpetuated by those working in the

field of STED [stimulated emission

depletion] microscopy, it is basically

wrong. The Abbe resolution criterion

(or Rayleigh, for that matter) is valid only for real imaging

systems like a microscope lens. It basically teaches us that, even

if the best optical materials are used in the lens manufacturing,

the resolution of the lens will be limited by diffraction of the

lens aperture.

The new, and impressive, scanning techniques like STED and

PALM [photoactivated localization microscopy] rely on a clever

interplay between fluorophores and sophisticated laser scanning

methods and, as such, are more related to raster scanning known

in scanning electron microscopes.

It is my hope that the erroneous comparison of classical imag-

ing and laser scanning methods will be avoided in the future.Lars Ren Lindvold

Senior Scientist, Radiation Research Div.Ris National Laboratory for Sustainable Energy

Technical University of Denmark

Letters


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9/52BioPhotonics November/December 2011

Photonics Medias industry-leading site features the latest industry news and events

from around the world.

Welcome to

Web Exclusive:Optogenetics using light to control geneti-

cally altered living cells began as a way

to probe neural code and reveal the secrets

of disorders in the still-mysterious human

brain. But teams at two universities are taking

their optogenetic work to heart, literally,

with the goal of replacing electrical pace-

makers. News Editor Caren Les asked

Stanfords Oscar Abilez and Stony Brook

University Medical Centers Emilia Entchevaabout the challenges they face and whats

next for their research. For more, visit:

www.photonics.com/webexclusives

2011 Prism Awards FinalistsFinalists for the 2011 Prism Awards for photonic innovation have

been chosen! Upon reviewing nearly 150 cutting-edge product

entries from around the world, our prestigious panel of judges

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BIOSCAN

SANTA BARBARA, Calif. Custom

DNA molecules can be used to make sen-

sors that can quickly detect a broad class

of proteins and could be used to person-alize cancer treatment and even to monitor

the quality of stem cells.

Researchers from the University of Cal-

ifornia, Santa Barbara (UCSB), and the

University of Rome Tor Vergata developed

the new nanosensors, which monitor the

activity of proteins called transcription

factors, then read the genome and translate

it into instructions for synthesizing the

various molecules that compose and con-

trol the cell. This information could deter-

mine which transcription factors in a pa-

tients cancer cells are activated or

repressed, enabling physicians to prescribe

the right combination of drugs for each

patient, according to Alexis Valle-Blisle,

a postdoctoral researcher in UCSBs de-

partment of chemistry and biochemistry.

Andrew Bonham, a postdoctoral scholar

at UCSB and co-first author of the study,explains that the teams approach to read-

ing transcription factors is quick and con-

venient. By adding their sensors to the

mashed-up cells, they can measure the

samples level of fluorescence.

The international research effort or-

ganized by senior authors Kevin Plaxco

and Francesco Ricci started when Ricci

realized that all of the information neces-

sary to detect transcription factor activities

is already encrypted in the human genome

and could be used to build sensors. Once

activated, each of the thousands of differ-

ent transcription factors can bind to its

own specific target DNA sequence, he ex-

plained. These sequences were used as the

foundation for building the nanosensors.

The key to the technology came from

studies of the natural biosensors inside

cells. The scientists expounded upon thefact that all creatures monitor their sur-

roundings with biomolecular switches

composed of RNA or proteins, which are

small enough to operate inside a cell and

could work in complex environments. In-

spired by the efficiency of these natural

nanosensors, the researchers teamed with

professor Norbert Reich of UCSB to build

synthetic switching nanosensors using

DNA rather than proteins or RNA.

Specifically, they re-engineered three

naturally occurring DNA sequences, each

recognizing a different transcription factor,

into molecular switches that become fluo-

rescent when they bind to their intended

targets. Using these nanosensors, the re-

searchers can determine transcription fac-

tor activity directly in cellular extracts by

measuring their fluorescence level. The

work was described in an online article

published Aug. 4 in the Journal of the

American Chemical Society (doi: 10.1021/

ja204775k).

This strategy ultimately will allow biol-

ogists to monitor the activation of thou-

sands of transcription factors, leading to a

better understanding of the mechanismsunderlying cell division and development.

The nanosensors could also be used to

screen and test new drugs that could in-

hibit the transcription factor binding activ-

ity responsible for tumor cell growth.

DNA nanosensors pave way for cancer tests, drugs


A structure-switching nanosensor made from DNA (blue and purple) detects a specific transcription factor(green). Using these nanosensors, researchers have demonstrated the direct detection of transcription factors incellular extracts. They believe that their strategies will allow biologists to monitor the activity of thousands of

transcription factors and will lead to more efficient cancer testing and medications. Courtesy of Peter Allen.

Alexis Valle-Blisle (left) and Andrew Bonham(right). Courtesy of George Foulsham, Office of

Public Affairs, UCSB.
http://pubs.acs.org/doi/abs/10.1021/ja204775khttp://pubs.acs.org/doi/abs/10.1021/ja204775khttp://pubs.acs.org/doi/abs/10.1021/ja204775khttp://pubs.acs.org/doi/abs/10.1021/ja204775k


11/52BioPhotonics November/December 2011 11

STED microscopy reveals how immune system attacks infected cells

PHILADELPHIA A new stimulated

emission depletion (STED) microscope

has yielded unprecedented views of the

immune system in action.

The superresolution microscope shows

how granules from natural killer (NK)

cells pass through openings in the dy-

namic cell skeleton to destroy their tar-

gets: tumor and virus-infected cells. Un-

derstanding these biological events better

may soon allow researchers to devise

more effective treatments for inherited

diseases that leave the immune system

compromised.

From a biological perspective, the

work defines a previously unappreciated

regulatory hurdle that an NK cell mustovercome in order to access essential host

defense functions, said Dr. Jordan S.

Orange of the Childrens Hospital of

Philadelphia. This of course presents

opportunities for exploiting this process

in order to obtain more or less of this par-

ticular variety of secretion. This could

have very relevant clinical and therapeutic

implications.

Previously, conventional fluorescence

light microscopes could not resolve ob-

jects smaller than 200 nm. The new STED

technique uses a unique arrangement oflasers and fluorescence to image fine

structures such as protein filaments

smaller than 60 nm.

Orange has long researched the biology

of NK cells at the immunological synapse

the site where the NK cell attaches to its

target cell and delivers cell-killing mole-

cules. A crucial component of this highly

regulated process is filamentous actin

(F-actin), a protein in NK cells that forms

a dense network through which cell-killing

molecules move into the synapse.

It was thought that F-actin is not present

at the center of the network, where the

granules fuse with the cell surface. The

current study reveals under superresolu-

tion, however, that F-actin pervades the

synapse but leaves openings just large

enough to allow granules to pass through.

The scientists observed that F-actin ap-

peared to be dynamically interacting with

the granules to move them toward their

targets.

Orange compared the F-actin filamentsto the rails of a roller coaster that quickly

rearranges itself to guide a rider through a

narrow tunnel. He explained that further

studies of NK function will investigate en-

ergy use and biological mechanisms that

allow the lytic granules to navigate the im-

munological synapse.

The patterning and coordination of the

pervasive actin network is telling a story

one that is likely to underscore how the

critical host defense function mediated by

NK cells is accessed, Orange said. We

certainly need to sort this out as well aswhat underlies the interaction between

NK cell lytic organelles and hypodense

regions within the pervasive actin at the

synaptic interface.

The study was published Sept. 13 in the

online open-access journalPLoS Biology

(doi: 10.1371/journal.pbio.1001151).

Platinum rotary electron micrograph of a naturalkiller cell cortex colorized to depict actin filaments(blue) and a single intercalated lytic granule

(yellow). Courtesy of Dr. Jordan Orange.

Superresolution stimulated emission depletion (STED)fluorescence micrograph of an activated humannatural killer cell demonstrating actin filaments(green) and perforin-containing lytic granules(red). Courtesy of Dr. Emily Mace.

AACHEN, Germany Tissue

that has been damaged by dis-ease or trauma often cannot

repair itself. However, a new

picosecond laser technique pro-

duces biomimetic matrices that

allow the body to regenerate it-

self using the patients own

cells.

The process was developed

by researchers at the Fraunhofer

Institute for Laser Technology

ILT and other Fraunhofer insti-

tutes. The biomimetic scaffolds

closely emulate endogenous tis-

sue and enable fabrication of

specialized model systems forstudying 3-D cell growth. The

researchers combined organic

substances with polymers to

produce 3-D structures that are

suitable for building artificial

tissue.

Tissue engineering is a

highly interesting and versatile

research area with huge appli-

cation potential, said Sascha

Engelhardt, project manager at

ILT. However, scaffolds devel-

oped for tissue engineering are

not yet fully able to mimic their

complex natural models.Normally, scaffold produc-

tion is focused either on

structural, mechanical or bio-

chemical aspects, but Engel-

hardt explained that all three

must be considered in a single

scaffold because they all have

been shown to influence cell

behavior significantly.

By controlling structure,

mechanics and biochemistry in

a single experiment, the com-

plexity of the natural model can

Laser technique produces synthetic tissue for regenerative medicine

Capillaries of an artificial resilient poly-mer with a diameter of 20 m. Imagescourtesy of Fraunhofer Institute for

Laser Technology ILT.
http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001151http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001151


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EDMONTON, Alberta, Canada The

palette of fluorescent highlighters used

to track the movement of messengers in-

side single cells has been dramatically ex-

panded to include red and blue fluorescent

indicators, which provide researchers a

vivid full-color view of calcium ions mov-

ing about in their role as key intracellular

signaling messengers.

Until recently, cellular imaging of the

calcium ion required the use of a green

fluorescent indicator. Accordingly, imag-

ing of calcium ions produced monochro-

matic images and movies in shades of

green. Now, scientists at the University of

Alberta have added red and blue indica-

tors.

The well known selection of fluores-

cence protein colors has proved useful for

live-cell imaging of multiple organelles

and proteins, according to doctoral candi-

date Yongxin Zhao and Robert Campbell,

associate professor of chemistry. They ex-

plained that a similar palette of calcium

indicators will enable researchers to start

designing experiments that involve visual-

izing multiple dynamic biochemical pa-

rameters simultaneously.

Imaging of the calcium ion is com-

monly used by researchers to monitor cel-

lular activity such as the firing of neurons.

However, since calcium ions are colorless,

it is necessary to introduce colored indica-

tor proteins into the cell, which can either

increase in fluorescent brightness or

change the fluorescence color when they

bind to the ions. These changes in bright-ness can be visualized easily using appro-

priate microscopy equipment.

Examining the dynamics of calcium

ions inside a single cell in better detail

could help pharmaceutical researchers de-

termine whether a drug designed to affect

a specific cell is hitting its target. In addi-

tion, it could help scientists better visual-

ize neuronal activity in model organisms

such as transgenic worms or mice.

The team, led by Zhao, engineered the

new genetically encoded indicator proteins

using bacterial cells. The indicator genes


b BIOSCAN

be reduced to its essentials, he

said. From this gained knowl-

edge, a translation in a

technical solution will be

more feasible.

The researchers used dis-

solved endogenous proteins such as albumin, collagen and

fibronectin and polymers irra-

diated with laser light and cross-

linked by photolytic processes.

To achieve this, they deployed

picosecond laser pulses from a

low-cost microchip laser to trig-

ger a multiphoton polymeriza-

tion process. The short pulse

duration caused almost no heat

damage to the material. Ultra-

fast megawatt-range pulses

drove a massive number ofprotons into the laser focus in

an extremely short time, trigger-

ing a nonlinear effect.

The molecules in the liquid

absorbed several photons simul-

taneously, causing free radicals

to form. This multiphoton poly-merization process triggered

chemical reactions between the

surrounding molecules, forming

solids from the liquid. CAD

data helped the system guide

the position of the laser beam

through a microscope with the

precision of a few hundred

nanometers in such a way that

micrometer-fine, stable volumeelements of cross-linked mate-

rial gradually formed.

With resolution of approxi-

mately 1 m, the process en-

abled the team to produce cell

scaffolds directly from dis-

solved proteins and polymers,

Engelhardt said. Offering

much greater stability, the scaf-

folds can be seeded with the

patients own cells in a med-

ical laboratory, and then the

colonized scaffolds can be ex-pected to produce good im-

plant growth in the patients

body. The long-term aim is to

produce not only individual

cell colonies but also artificial

tailor-made organs.

Our next aim will be to de-

sign and develop an in vitro

assay in the form of a con-

trolled 3-D microenvironmentbased on our technology, En-

gelhardt said. Of course, this

assay will not be realized alone

by our institute, but within an

interdisciplinary cooperation

of research partners. Our long-

term goal is to upscale our

approach by increasing the

process speed.

To reduce the time and

cost of producing tailor-made

supporting structures for syn-

thetic tissue, the team wantsto combine its fabrication

process with other rapid proto-

typing methods.

Test matrix consisting of a polymersupport structure and a proteinfunctional structure.

Vibrant palette of fluorescent highlighters tracks cells

Multicolor imaging with GECOs. HeLa cells transfected with nucleus-localized R-GECO1, cytoplasmicG-GECO1, and mitochondria-localized GEM-GECO1. (Top left) Red fluorescence. (Top right) Greenfluorescence with cyan (~470 nm) excitation. (Bottom left) Pseudocolored ratio of blue to green fluorescence

with UV (~380-nm) excitation. (Bottom right) Merge of the three images, with GEM-GECO1 ratio in magenta.


13/52

were programmed to be sent to the outside

of the bacterial cytoplasmic membrane.

The calcium ion concentration then could

be experimentally altered to find the indi-

cator variants that had the desired color

and largest changes in brightness. Using

this approach, the researchers performeddirected laboratory evolution to ultimately

provide the optimized indicator proteins.

Campbell and Zhao said that their next

priority is to expand the color palette of

the indicators to include the yellow, or-

ange and far-red regions of the spectrum.

In addition, they plan to distribute the

genes to as many research groups as

possible through Addgene, a nonprofit

plasmid repository.

The research appeared online Sept. 8

in Science (doi: 10.1126/science.1208592).


bBIOSCAN

Three-color fluorescence imaging of HeLa cellstransfected with plasmids encoding R-GECO1(red indicator) targeted to the nucleus, G-GECO1targeted to the cytoplasm and GEM-GECO1 to themitochondria. Courtesy of Robert E. Campbell et al.

New microscope delves beneath skin to detect cancer

ROME A new type of laser-scanning

confocal microscope provides more infor-

mation than previous versions and holds

promise for skin cancer diagnostics.

Unlike typical laser-scanning confocal

microscopes that take 3-D images of thick

tissue samples by visualizing thin slices

one layer at a time, the new device gathers

spectrographic information at a wide spec-tral range approximately 0.5 to 2.5 m

for every point in the sample, and all in a

single scan. This spectroscopic fingerprint-

ing was detailed online Aug. 18 in AIP

Advances (doi: 10.1063/1.3631661).

Physicists at Consiglio Nazionale delle

Ricerche (CNR), in collaboration with a

dermatologist, demonstrated the technol-

ogy by taking high-resolution images of

the edge of a silicon wafer and of metallic

letters painted onto a piece of silicon less

than a half-millimeter wide. They also

demonstrated that it is possible to apply

this technique to a tissue sample in thiscase, chicken skin without destroying it.

The main aim of our effort is not only

to produce typical histologic results with-

out tissue removal and specific prepara-

tion, but also to obtain reliable functional

An illustration of the CNR confocal microscope showing the 80-m-wide images from a silicon/silicon dioxidecalibration sample. The 10-m periodical structure is made of 100-nm-thick SiO

2squares over a silicon

substrate. The left image is obtained with the 580-nm wavelength. The scientists can compute the full-colorimage (right) by averaging the reflectivity intensities around the red, green and blue regions and associatinga suitable RGB color map. The greatest reflectivity for silicon is depicted by the green-blue portion ofthe spectrum, while the greatest contribution of the SiO

2 island is depicted by the red-infrared part.

Courtesy of F.R. Bertani, L. Ferrari, S. Selci, ISC-CNR, unpublished results.
http://www.sciencemag.org/content/333/6051/1888http://aipadvances.aip.org/resource/1/aaidbi/v1/i3/p032143_s1?bypassSSO=1http://www.lumencor.com/http://www.lumencor.com/http://aipadvances.aip.org/resource/1/aaidbi/v1/i3/p032143_s1?bypassSSO=1http://www.sciencemag.org/content/333/6051/1888


14/52

data on the tissue from spectral analysis,

said Dr. Stefano Selci of CNR. The po-

tential impact can be wide and deep.

Because the spectral region is vast,

Selci explained, the innovation could

affect many applications and contexts, in-

cluding dermatology, cosmetology, en-doscopy for recessed diagnosis of general

tissues as well as semiconductors and any

materials science research.

With further testing, the microscope

could be used to detect early signs of

melanoma, Selci said. We are examining

now a wide range of human skin samples

ex vivo to accumulate experience on sig-

nificant samples and to correlate confocal

spectroscopy data to specific cell typesand skin structures, he said. On the tech-

nical side, we have mainly to speed up ac-

quisition times as needed for biological

specimens: Our microscope has been real-

ized from scratch to avoid any compro-

mise, and we have to invent new solutions

for our particular optical setup to transport

the brilliant white laser beam.

Until then, he said, it is suitable for

nonmedical applications such as inspect-ing semiconductor surfaces.


b BIOSCAN

Sensor enables cheap, portable blood testingTOLEDO, Ohio A low-cost, portable

technique quickly and reliably detects spe-

cific proteins in a sample of human blood,

which could benefit a range of medical

sensing applications, including diagnosisof cancer and diabetes long before the

onset of clinical symptoms.

University of Toledo scientists attached

artificially created molecules called ap-

tamers to free-floating proteins in the

blood. Aptamers are commercially avail-

able, custom-made short strands of nucleic

acid that resemble antibodies found in the

body because they connect to one type of

molecule only. Specific aptamers can be

used to search for target compounds rang-

ing from small molecules such as drugs

and dyes to complex biological moleculessuch as enzymes, peptides and proteins.

However, aptamers have advantages

over antibodies in clinical testing. They

can tolerate a wide range of pH and salt

concentrations, they have high heat stabil-

ity, and they are easily synthesized and

cost-efficient. Aptamer sensors also can be

reversibly denatured, meaning that they

can easily release their target molecules,

making them perfect receptors for biosens-

ing applications.

To demonstrate the applicability of the

technique, the scientists chose thrombin

a naturally occurring protein in humans

that plays a role in clotting and throm-

bin-binding aptamers, which they attached

to a sensor surface, in this case a glass

slide coated with a nanoscale layer of

gold. As they applied the blood sample to

the testing surface, the aptamer and corre-

sponding proteins latched together.

They then used surface plasmon reso-

nance to determine whether the pairing

had been successful. If the protein was

present and had bound to the aptamer,

conditions for which resonance would

occur at the gold layer would have

changed. This change can be detected

through a simple reflectance technique

that is coupled to a linear detector.

The demonstrated surface plasmon res-

onance sensing modality is well suited for

the commercialization of portable hand-

held devices, and the aptamer-based func-

tionalization technique provides targeted

selectivity for specific protein detection,

said Dr. Brent Cameron of the department

of bioengineering at the university.

A current process based off systematic

evolution of ligands by exponential ampli-

fication (SELEX) is used by our group as

a convenient method for identifying and

optimizing unique aptamers specific to a

given target (i.e., type of protein or modi-

fied protein). Therefore, the sensing possi-

bilities are endless.

The new technique, which Cameron

said could be commercially available in

three to five years, was described in the

Aug. 31 issue of the Optical Society of

Americas open-access journal, Biomed-

ical Optics Express (doi: 10.1364/

BOE.2.002731).

Dr. Brent Cameron (right) and doctoral student Rui Zheng (left) evaluate a custom-developedfunctionalized localized surface plasmon resonance microchip used in the detection and measurementof albumin protein via a fiber optic light delivery/detection spectral probe. (Inset) A scanning electronmicrograph of the gold- based functionalized nanoparticle array used in localized surface plasmonresonance measurements. Courtesy of Brent Cameron.
http://www.opticsinfobase.org/abstract.cfm?URI=boe-2-9-2731http://www.opticsinfobase.org/abstract.cfm?URI=boe-2-9-2731http://www.opticsinfobase.org/abstract.cfm?URI=boe-2-9-2731http://www.opticsinfobase.org/abstract.cfm?URI=boe-2-9-2731


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LAUSANNE, Switzerland Digital holo-graphic microscopy (DHM) allows obser-vation of neuronal activity in real time andin three dimensions with up to 50 timesgreater resolution than ever before showing promise in testing new drugs tofight neurodegenerative diseases such asParkinsons and Alzheimers.

DHM is a noninvasive approach thatenables extended observation of neuralprocesses without electrodes or dyes thatdamage cells. The method yields impor-tant information about the shape, dynam-ics and activity of neurons, and creates3-D navigable images down to 10-nm pre-cision, according to senior team memberPierre Marquet of cole PolytechniqueFdrale de Lausanne (EPFL).

The method consists of pointing a sin-gle-wavelength laser beam at neurons,collecting the distorted wave on the otherside and comparing it to a reference beam.A computer then numerically reconstructs

a 3-D image of the neurons using an algo-rithm developed by the researchers. Thelaser beam travels through the transparentcells to obtain important information abouttheir internal composition.

Although DHM normally is used to de-tect minute defects in materials, the teamdecided to try using it for neurobiologicalapplications. In the study, the group in-duced an electric charge in a culture ofneurons using glutamate, the main neuro-transmitter in the brain. This charge trans-fer carries water inside the neurons and

changes their optical properties in a way

that can be detected only by DHM. Thus,the technique accurately visualized theelectrical activities of hundreds of neuronssimultaneously, in real time, withoutharming them with electrodes, which canrecord activity from only a few neuronsat a time. The groups findings appearedin the Aug. 17 issue of The Journal ofNeuroscience (doi: 10.1523/jneurosci.0286-11.2011).

This work has permitted us to obtainan intrinsic optical signature of the activityof many neurons in culture simultane-ously, in real time and in a noninvasivemanner, Marquet said. Practically, thehigh phase measurement accuracy allowsus to rapidly observe the effects of variouspharmacological substances mediated bythe activation of specific membrane recep-tors/transponders.

Within this framework, DHM repre-sents a very promising approach to de-velop a unique, label-free, high-content

screening technique which is highly rele-vant for the discovery of new drugs.

This advance in high-content screeninghas important ramifications for the discov-ery of new drugs that combat or preventneurodegenerative diseases, since newmolecules can be tested more quickly andin greater numbers. The researchers havefound that lab work that previously took12 hours can now be done in 15 to 30minutes.

The success of DHM for high-contentscreening has resulted in a startup com-

pany called Lynce Tec SA.


bBIOSCAN

Microscopy, holography team upto reveal neuronal activity

A 3-D representation of alive mouse cortical neuron inculture. Each pixel representsa color-coded quantitativemeasurement of the phase shift

induced by the neuron on thetransmitted wavefront. Courtesyof Pierre Marquet, EPFL.

Lasers | Lenses | Mirrors | Assemb

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16/52

CHAMPAIGN, Ill. A new imaging

technique called spatial light interference

microscopy (SLIM) soon may answer the

much debated question as to whether

cells grow at a constant rate or exponen-

tially.

An extremely sensitive method, SLIM

can quantitatively measure mass withfemtogram accuracy using two beams of

light. It can gauge the growth of a single

cell and even mass transport within the

cell. It also can measure all cell types,

including bacteria, mammalian cells,

adherent and nonadherent cells, and popu-

lations, according to Mustafa Mir, a grad-

uate student at the University of

Illinois. The method was reported in

the July 25 issue ofProceedings of the

National Academy of Sciences (doi:

10.1073/pnas.1100506108).

Combining phase-contrast microscopy

and holography, SLIM does not require

staining or any other special preparation.

The noninvasive method uses white light

and can be used with more traditional


b BIOSCAN

University of Illinois researchers have developed animaging technique called spatial light interferencemicroscopy (SLIM) that can quantitatively measurecell mass with light. Courtesy of Quantitative LightImaging Laboratory.

Artists rendition of a SLIM measurement of a pairof human osteosarcoma cells (real data). The colorson the image correspond to the dry mass density ateach point. Courtesy of Quantitative Light ImagingLaboratory and the Imaging Technology Group

Visualization Laboratory, Beckman Institute forAdvanced Science and Technology.

SLIM method measures cell growth
http://www.pnas.org/content/108/32/13124http://www.pnas.org/content/108/32/13124http://www.pnas.org/content/108/32/13124http://www.sutter.com/http://www.sutter.com/mailto:[email protected]://www.sydor.com/http://www.sydor.com/mailto:[email protected]://www.pnas.org/content/108/32/13124


17/52

microscopy techniques, such as fluores-

cence, to monitor a cell as it grows.

Thanks to the methods sensitivity, re-

searchers at the university were able to

monitor cell growth through the phases

of the cell cycle. They discovered that

mammalian cells show clear exponentialgrowth only during the G2 phase after

the DNA replicates and before the cell di-

vides. This information has great implica-

tions not only for basic biology but also

for diagnostics, tissue engineering and

drug development.

The researchers hope to apply their new

findings on cell growth to various disease

models. For example, they plan to use

SLIM to see how growth varies between

normal cells and cancer, and to determine

the effects of treatments on growth rate.

Of all the current growth measurement

techniques, SLIM is unique in its capabili-

ties to study these interactions in typical

culture conditions, said Gabriel Popescu,

a member of the Beckman Institute for

Advanced Science and Technology at the

university. Since SLIM is designed as anadd-on module to a commercial phase mi-

croscope, it requires minimal retraining to

use and can readily be implemented in any

biology laboratory. We expect that these

capabilities will allow others to explore

other fundamental questions in the field of

cell growth.

Popescu has established SLIM as a

shared resource on the universitys cam-

pus, hoping to harness its flexibility for

basic and clinical research in a number

of areas.

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bBIOSCAN

Cells open wide for nanothermometers

PRINCETON, N.J. Nanoscale thermometers have revealed for the first time that

individual cells inside the human body register various temperatures and do not ad-

here to the familiar 98.6 F norm.

For many years, scientists have suspected that temperatures vary inside individual

cells because many chemical reactions and physical changes take place there, pro-

ducing energy and heat. Some cells are more active than others, and the unused

energy is discharged as heat. Parts of an individual cell may be warmer because

they harbor biochemical power plants, known as mitochondria.

We have been very interested in understanding molecular reactivity inside livingcells, said Haw Yang, associate professor at Princeton University. One key aspect

in chemical reactions is temperature. Considering the highly compartmentalized and

heterogeneous nature of intracellular space, one might expect that temperature re-

sponse be nonuniform. Yet, before our work, there was no experimental evidence to

show whether it is true.

To measure the temperature of individual cells, Yang and Liwei Lin of the Univer-

sity of California, Berkeley, developed nanothermometers consisting of quantum

dots, in this case cadmium and selenium, which emit different wavelengths of light

depending on the temperature.

They inserted the dots into some mouse cells growing in lab dishes and found a

range of temperatures throughout the cell. Yang and his colleagues stimulated cellular

activity to watch the changes, reporting a difference of a few degrees Fahrenheit

in some cells. The team does not yet have enough data to give exact calculations,however.

The temperature changes could have a major impact on how cells work and sur-

vive, Yang said. Temperature increases could affect how DNA works, for instance,

and change how protein molecular machines operate. Yang also hypothesized that

the cells may use differences in temperature as a means of communication. The team

is working to determine how cellular temperature is regulated, with the goal of ap-

plying the information to improving prevention, diagnosis and treatment of diseases.

We hope that this experiment will change the view of intracellular thermodynam-

ics, encouraging researchers to ask questions about temperature gradient and its pos-

sible roles in signaling, Yang said.

The research was presented Aug. 28 at the American Chemical Societys annual

meeting in Denver.
http://allthingsphotonic.com/http://allthingsphotonic.com/http://allthingsphotonic.com/


18/52

PASADENA, Calif. High-resolution 3-D

imaging of a cells nucleus undergoing

cell division is now possible, thanks to a

combination of plunge-freezing and a newmethod of sample slicing. The findings

open up a biological mystery because they

indicate that some cells take one of the

characteristic steps of mitosis significantly

differently from others.

Traditionally, two sets of chromosomes

pair up at the center of the cells nucleus

during mitosis. Then hollow rods of pro-

tein microtubules composed of a cellular

structure called the spindle apparatus

grab onto the chromosomes and essen-

tially pull each set away from the center in

opposite directions, ensuring that each cellreceives a full copy of the genetic mate-

rial. Typically, in the cells of fungi, plants

and many animals, one or more micro-

tubules attach to each chromosome before

the spindle separates the sets of chromo-

somes from one another.

However, when researchers at Califor-

nia Institute of Technology observed this

step using their new technique, what they

saw was not the usual cell division. In-

stead, they discovered a cell with fewermicrotubules used than chromosomes.

The group used electron cryotomogra-

phy (ECT) to image biological samples.

Unlike traditional electron microscopy,

ECT involves plunge-freezing samples

so quickly that they become trapped in a

near-native state within a layer of transpar-

ent glasslike ice. High-resolution images

can then be captured as the sample is ro-

tated, usually one degree at a time. Beam-

penetration issues have limited ECT to

samples less than 500 nm thick, such as

small bacteria and viruses.

Observing eukaryotic cellsNow, however, the Caltech group has

extended the technique to observe eukary-

otic cells, which typically are much big-

ger. The investigators located the smallest

known eukaryote, Ostreococcus tauri, a

cell with 20 chromosomes, and imaged it

with ECT. Next, they set out to observe

the eukaryotes cell division, but even its

tiny size exceeded the 500-nm limit whenit underwent mitosis.

They used a diamond knife to

cryosect cut a frozen sample into

slices enabling them to look at them

through dividing cells in a near-native hy-

drated state. They made detailed observa-

tions of mitosis in O. tauri.

Contrary to expectations, nowhere near

40 microtubules attached to the two sets of

chromosomes during mitosis. Instead, they

discovered only about 10 small, incom-

plete microtubles, suggesting that the

chromosomes may link together to form abundle that can then be segregated all at

once by a smaller number of microtubules.

The findings appeared in the Sept. 8

issue of Current Biology (doi: 10.1016/

j.cub.2011.08.021).

The researchers are now using the same

method to try to image human cells.


b BIOSCAN

Mitosis: New techniques reveal cell division surprise
http://www.cell.com/current-biology/retrieve/pii/S0960982211008979http://www.cell.com/current-biology/retrieve/pii/S0960982211008979http://www.cell.com/current-biology/retrieve/pii/S0960982211008979http://www.cell.com/current-biology/retrieve/pii/S0960982211008979http://www.raptorphotonics.com/http://www.raptorphotonics.com/mailto:[email protected]://www.mightexsystems.com/mailto:[email protected]://www.mightexsystems.com/http://www.mightex.com/http://www.cell.com/current-biology/retrieve/pii/S0960982211008979


19/52

EAST LANSING, Mich. An inexpensive

handheld cancer diagnostic device may

soon help physicians in developing nations

with limited resources to detect and diag-nose the disease and to provide treatment

before its too late.

Cancer is emerging as a leading cause

of death in underdeveloped and develop-

ing countries, and screening for it is al-

most nonexistent because current diagnos-

tics and rapid screening methods are not

suitable for low-income and resource-

limited countries.

Syed Hashsham, a professor of civil and

environmental engineering at Michigan

State University, concentrated his efforts

on developing a low-cost, mobile device.The instrument, called the Gene-Z, oper-

ates using an iPod Touch or Android-based

tablet and analyzes microRNAs and other

genetic markers. MicroRNAs are single-

stranded molecules that regulate genes;

changes in certain microRNAs have been

linked to cancer and other diseases.

The Gene-Z is battery-operated and

solar-chargeable, making it a suitable fit

for areas where there are limited resources

and mobility is important. The devicecan screen for established markers of

cancer at extremely low cost in the field,

Hashsham said.

Until recently, little effort had been

placed on cancer detection for developing

countries. Early detection could lead to

more affordable management of cancers

with the aid of new screening and diag-

nostic technologies that could overcome

global health care disparities, said Reza

Nassiri, director of MSUs Institute of

International Health.

Hashsham, who showcased the testwith colleagues at the National Institutes

of Healths first Cancer Detection and

Diagnostics Conference in August, is

working with Nassiri on the devices

medical capabilities and is establishing

connections with physicians worldwide.

bBIOSCAN

The handheld Gene-Z, which is operated usingan Android-based tablet or iPod Touch, performsgenetic analysis on microRNAs and other geneticmarkers to detect and diagnose cancer.

Handheld units detect cancer on the go
http://www.blueskyresearch.com/


20/52

SAN DIEGO Scientists have long

known that plants and animals are gov-

erned by circadian rhythm a 24-hour

cycle of alternating light and darkness toguide biological processes. Although we

know that our bodies are entrained, or

synchronized, by light and would drift out

of phase if left in the dark, we have yet to

understand exactly how this process works

at the molecular level.

To find out, biologists and bioengineersat the University of California developed a

model biological system that is simpler

than that of an organism. Led by biology

professor Jeff Hasty, they created a simple

circadian system using a model consisting

of glowing, blinkingE. coli. The system

was detailed in the Sept. 2 issue of Sci-ence (doi: 10.1126/science.1205369).

Combining techniques from synthetic

biology, microfluidic technology and com-

putational modeling, the researchers built

a microfluidic chip containing chambers

withE. coli. Within each bacterium, the

genetic machinery responsible for the bio-

logical clock oscillations was green fluo-

rescent protein, which caused the bacteria

to periodically fluoresce.

The researchers modified the bacteria

to glow and blink whenever arabinose a

chemical that triggered the oscillatoryclock mechanisms of the bacteria was

flushed through the microfluidic chip. This

enabled them to simulate day and night

cycles over a period of minutes rather than

days to better understand how a popula-

tion of cells synchronizes its biological

clocks.

We studied quantitatively how a mini-

mal genetic oscillator in single cells is

capable of picking up the phase of an os-

cillatory external input and relay it to

drive the expression of a gene, Hasty

said. In contrast, the timekeeping systems

of natural organisms use coordinated

groups of complex cell oscillators for gen-

erating the daily rhythms and processing

the environmental inputs to produce the

multiple phased signals that coordinate es-

sential cellular and organismal processes.

Hasty said a similar microfluidic system

could be constructed, in principle, with

mammalian cells to study how human

cells synchronize with light and darkness.

In future research, they hope to integrate

synthetic clocks with cell processes such

as metabolism and cell division to under-

stand how this is achieved in naturalsettings.

Genetic models such as this could be

important because circadian rhythm dis-

ruptions have been linked to medical is-

sues such as sleep disorders and diabetes.

While fluorescence microscopy will

continue to be the central tool for the in-

vestigation of biological clocks, the use of

light to control gene expression in individ-

ual cells (optogenetics) will be, without a

doubt, an important new resource to learn

about the dynamics of synthetic multicel-

lular systems, Hasty said.


b BIOSCAN

Glowing bacteria shed light on what makes biological clocks tick
http://www.sciencemag.org/content/333/6047/1315http://www.edmundoptics.com/we-make-ithttp://www.edmundoptics.com/we-make-ithttp://www.sciencemag.org/content/333/6047/1315


21/52

BUSINESSSCAN

BETHESDA, Md. The US National

Institutes of Health recently awarded

$143.8 million in grants to speed up thetranslation of research projects into im-

proved health, and several of the recog-

nized projects rely on biophotonics tech-

nologies or techniques.

These grants were awarded under three

programs supported by the NIH Common

Fund: the NIH Directors Pioneer, New

Innovator and Transformative Research

Projects Awards. This year, approximately

$10.4 million will go to Pioneer awardees,

$117.5 million to New Innovators and

$15.9 million to Transformative Research

Projects.These programs reinvigorate the bio-

medical workforce by providing unique

opportunities to conduct research that is

neither incremental nor conventional,

said Dr. James M. Anderson, director of

the Division of Program Coordination,

Planning and Strategic Initiatives, who

guides the Common Funds High-Risk

Research program.

The NIH Directors Award Program so

far has funded 406 high-risk research

awards: 111 Pioneer Awards since 2004,

216 New Innovator Awards since 2007

and 79 Transformative Research Projects

Awards since 2009. These numbers in-

clude this years 13 Pioneer Awards, 49

New Innovator Awards and 17 Transfor-

mative Research Projects Awards.

Pioneer AwardPioneer Award recipient Jean Bennett

of the University of Pennsylvanias Scheie

Eye Institute of the Perelman School of

Medicine and her research team were

granted $4 million for the next five years

to use gene therapy to treat inherited

forms of blindness, which can be causedby mutations in any of hundreds of differ-

ent genes.

The researchers plan to resensitize the

blind eye using optogenetic techniques in

which light-sensitive molecules are deliv-

ered to any remaining retinal cells. Pre-

clinical studies in blind animals have

demonstrated that this strategy is effective,

and a new clinical study would test the

safety and efficacy of this approach in

blind patients.

Project results could improve the qual-

ity of life for millions of individuals and

also could pave the way for development

of novel gene therapy approaches to the

treatment of other sensory diseases.

New InnovatorsNew Innovator awards went to several

photonics-focused researchers. Arjun Raj

of the University of Pennsylvanias School

of Engineering and Applied Science will

receive $1.5 million over five years. His

research involves the development and

application of new microscopic imaging

tools to reveal how the physical organiza-

tion of the genetic code determines the

manner in which the cell reads the code

itself. The development of these methods

will establish a nuclear GPS that should

permit researchers to directly visualize

genetic organization in single cells. Under-

standing this organization will be impor-

tant for discovering how defects in trans-

lating the genetic code contribute todiseases such as cancer.

Other New Innovators named this year

include Bo Huang of the University of

California, San Francisco, who plans to

use superresolution microscopy to study

macromolecular complex architecture in

situ. Huang and colleagues hope that their

research will provide insights into prob-

lems in structural and neuron cell biology,

and generate tools for other life sciences

fields as well.

Long Cai of California Institute of Tech-

nology is exploring systems biology in sin-

gle cells using superresolution bar coding.

The IR-LAMP project, led by Julie C.

Canman of Columbia University, will use

optogenetic technology to spatially manip-

ulate the function of proteins in vivo.

Hongrui Jiang of the University of Wis-

consin-Madison, another New Innovator,

is developing a contact lens to correct

presbyopia by changing its focal length

to offer far-ranging and close-up vision

in a single lens.

Andrea M. Kasko of the University of

California, Los Angeles, will use photo-

tunable biomaterials to engineer complex

3-D cell microenvironments, and Haining

Zhong of Oregon Health and Science

University will examine the architecture

of brain tissue synapses at nanometer

resolution.

Transformative Research Projects

A $7 million five-year TransformativeResearch Project Award was given to a

team of investigators from the Perelman

School of Medicine and from Emory

University and Georgia Institute of Tech-

nology, both in Atlanta. The researchers

include Sunil Singhal, director of the

Thoracic Surgery Research Laboratory at

the University of Pennsylvania. If a tumor

is more visible and easier to distinguish

from surrounding tissues, surgeons are

more likely to be able to remove it com-

pletely. At present, a significant number

of patients who undergo surgery leave the

Photonics-related projects score NIH grants

BioPhotonics November/December 2011 21


22/52

operating room without total tumor re-

moval.

To address the problem, the researchers

developed fluorescent nanoparticle probes

that target cancer cells. Their main goals

are to help surgeons distinguish tumor

boundaries, identify diseased lymph nodesand determine whether a tumor has been

completely removed. The grant-funded

project includes plans for tests of the

nanoparticles in animal models and a clin-

ical trial for patients with lung cancer. The

proposed technologies could be broadly

applicable to many types of solid tumors.

Other Transformative Research Projects

awardees include Adela Ben-Yakar andJonathan T. Pierce-Shimomura of the Uni-

versity of Texas at Austin, who will use

high-speed optofluidics to study the entire

nervous system as it relates to aging and

disease.

The awards are intended to catalyze

giant leaps forward for any area of bio-

medical research, allowing investigators to

go in entirely new directions, Andersonsaid.

Compiled by BioPhotonics staff


b BUSINESSSCAN

BUSINESSBRIEFS

Avantes BV, a manufacturer of instruments,

light sources, software and accessories for spec-

troscopic applications, has moved to a state-of-

the-art 2700-sq-m facility in Apeldoorn, Nether-

lands. The energy-efficient facility will provide

increased production capacity, enabling shorter

lead times. It also will provide for extended en-

gineering operations to support planned R&D

projects. Avantes released nine new spectrome-

ters in 2011 and plans to release several new

instruments and to announce improvements to

its existing product lines.

Synoptics has established the Advanced Tech-

nology Group, a new division within the com-

pany that will work with life sciences companies

and academic clients to deliver custom imaging

equipment to improve bench-based research,

quality control and clinical development

processes. Comprising the Syngene, Synbiosis

and Syncroscopy divisions, Synoptics provides

products for life scientists to enhance the quality

and speed of their research. Part of the Scien-

tific Digital Imaging Group of companies

based in Cambridge, UK, Synoptics develops

and manufactures digital imaging systems, and

systems and software to improve biological

quality control and research processes.

In Oregon, a collaboration between industry

and academia has resulted in the creation of

a laboratory that will provide researchers with

several state-of-the-art electron microscopes

to advance the understanding and treatment

of cancer, AIDS and other diseases. The OHSU/

FEI Living Lab for Cell Biology, a joint effort

ofOregon Health & Science University

(OHSU) of Portland, where the lab is based,

and FEI Co. of Hillsboro, will be equipped with

a variety of instruments made by the latter.

Among them are a Titan Krios transmission

electron microscope and a Helios NanoLab

DualBeam instrument, which features both

scanning electron and focused ion beam

microscopy functions.

Photonic Solutions of Edinburgh, UK, an-

nounced that, with the addition of the Pharos

and Orpheus ultrafast laser systems, it has

expanded its range of Lithuania-based Light

Conversions products distributed in the UK

and Ireland. The new agreement strengthens an

existing long-term relationship based on sales

of Light Conversions original ultrafast products.

STATEMENT OF OWNERSHIP

MANAGEMENT AND CIRCULATION

of

BioPhotonics

September 30, 2011

(This statement is published in compliance with

the Act of October 23, 1962.)

Published monthly.Publisher: Karen A. NewmanEditor: Melinda A. RoseManaging Editor: Laura S. Marshall

The owners are Thomas F. Laurin and Ralph Cianflone,Berkshire Common, PO Box 4949, Pittsfield, MA 01202-4949.

Known bondholders, mortgagees and other security holdersowning or holding 1 percent or more of total amount of bonds,mortgages or other securities: None.

The average number of copies of the publication during the 12months preceding the date shown above is: Printed 23,915;paid and/or requested subscription by mail 22,186; total paidand/or requested circulation 22,186; free distribution by mail,carrier or other means, samples, complimentary and other freecopies 419; total nonrequested distribution 507; office use, leftover, unaccounted, spoiled after printing 1,222; total above 23,915. Actual number of copies of single issue published nearestto filing date: Printed 21,194; paid and/or requested subscriptionby mail 19,997; total paid and/or requested circulation 19,997;free distribution by mail, carrier or other means, samples, compli-mentary and other free copies 350; total nonrequested distri-bution 385; office use, left over, unaccounted, spoiled afterprinting 812; total above 21,194.

I certify that the above statements made by me are correctand complete.

Thomas F. LaurinPresident
http://www.madcitylabs.com/mailto:[email protected]://www.madcitylabs.com/


23/52

Laser diode manufacturer and distributor

Frankfurt Laser Co. of Friedrichsdorf, Ger-

many, has announced that it will distribute

Focuslight Technologies Co. Ltd.s high-

power laser diodes. Focuslight, of Xian, China,

provides lasers for applications including pump-

ing, materials processing, display and projection

technologies, printing and the medical field.The diodes are available from 635 to 1550 nm,

and in both continuous- and pulsed-mode

operation.

Precision positioning systems manufacturer

Physik Instrumente (PI) LP of Karlsruhe, Ger-

many, announced its expansion into the Asian

market with the opening of a direct office in Sin-

gapore. Before opening the office in June, PI was

active in the region through a distributor. PI Sin-

gapore LLPwill provide more dedicated service

to its customers, allowing it to develop new busi-

ness in Singapore and greater Southeast Asia.

The Chicago-basedJohn D. and Catherine

T. MacArthur Foundation recently announced

22 new MacArthur fellows for 2011. Recipients

of the Genius Awards receive $500,000 in

no-strings-attached support over the next five

years. They are selected for creativity, originality

and potential to make important contributions

in the future. William Seeley, an associate pro-

fessor of neurology at the Memory & Aging

Center at the University of California, San Fran-

cisco, received an award for his contributions

to neurology. He integrates microscopy, MRI

and clinical examination to explore the struc-

tural, functional and behavioral aspects of

human neurodegenerative disease, concentrat-

ing on frontotemporal dementia.

GE Healthcare of Chalfont St. Giles, UK, is

set to dedicate $1 billion of its total R&D budgetover the next five years to expanding its ad-

vanced cancer diagnostic and molecular imag-

ing capabilities as well as its technologies for

biopharmaceuticals manufacturing and for

cancer research. Announced alongside a $100

million open innovation challenge in New York,

the billion-dollar investment crosses all lines

of GE Healthcares global business and will

enable the company to bring the most promis-

ing cancer ideas to market. The companys

solutions combine medical imaging, molecular

diagnostics and health care information tech-

nology.

Mad City Labs Inc. of Madison, Wis., has

opened a direct sales office in Zurich. The com-pany said it established Mad City Labs GmbH

to enhance service and support for its European

customers and to strengthen its relationships

with its distribution network and business part-

ners. The Wisconsin company manufactures

piezoactuated, closed-loop nanopositioning

systems for metrology, photonics and micros-

copy applications.

Cosmetic laser makerPalomar Medical Tech-

nologies Inc. of Burlington, Mass., has an-

nounced the receipt of $31 million plus royalties

from aesthetic device companySyneron Med-

ical Ltd. of Yokneam, Israel, in a settlement of

their patent infringement dispute over hair re-

moval systems. The agreement includes two

nonexclusive patent license deals. Under the

first, Palomar granted Syneron and its Candela

unit a worldwide, irrevocable license to US

Patent Nos. 5,735,844 and 5,595,568 for pro-

fessional laser- and lamp-based hair removal

technology. Under the second, Palomar granted

Syneron and affiliates a nonexclusive license in

the US to the same two patents for consumerhome use lamp-based hair removal products.

A supplier of electron microscopes and scientific

instrumentation,JEOL USAof Peabody, Mass.,

and its parent companyJEOL Ltd. of Akishima,

Japan, have openedJEOL Brasil Instrumentos

Cientificos Ltda. in Sao Paulo to support the

growing installed base there, and they have

relocated the personnel to a new facility. The

company has a 40-year history in Brazil, much

of it through its agent, Fugiwara Enterprises

IC Ltda.As the number of customers has con-tinued to grow in the region, the company

decided to provide direct support through its

own service engineers, and administrative and

sales personnel.

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mailto:[email protected]://www.picoquant.com/http://www.picoquant.com/http://www.picoquant.com/mailto:[email protected]


24/52

Multimodal nonlinear optical

(NLO) imaging is an emerging

microscopy approach gainingwidespread use in a variety of biomedical

applications. It harnesses and integrates

the unique capabilities of nonlinear

processes such as multiphoton fluores-

cence, second- and third-harmonic genera-

tion (SHG and THG), and coherent Raman

scattering (CRS) and combines them

seamlessly into a single, unified micros-

copy platform. A combination of label-

based and label-free imaging modalities

enables simultaneous acquisition of com-

plementary structural information within

individual cells and elucidates the health

of biological tissue at the submicron level.

The emergence of multimodal NLO

imaging has been facilitated by advancesin ultrafast laser technology; high-perfor-

mance specialized optical filters; and

high-sensitivity detectors.

The development of in vivo diagnostic

tools and techniques that can be used to

advance our understanding of the molecu-

lar, morphological and functional changes

that occur during disease progression is

critical for human health. Today, compre-

hensive interrogation of the structure and

function of biological samples exploits ad-

vances in various optical microscopy tech-

niques and modalities. Wide-field and

laser-scanning confocal fluorescence mi-

croscopy have been used extensively to

elucidate the form and function of biologi-

cal entities at the submicron level. More

recently, however, advances in ultrafast

laser technologies have allowed the ex-

ploitation of several well-known nonlinear

optical processes for use in biomedical

imaging applications.

Two common, well-known nonlinear

imaging techniques are multiphoton fluo-

rescence1 and coherent Raman scattering2

microscopy. The former typically uses

femtosecond near-infrared laser pulses,

while the latter, based on either coherent

anti-Stokes Raman scattering (CARS) or

stimulated Raman scattering (SRS), uses

two separate picosecond near-infrared

lasers to generate a CRS signal. As a re-

sult, it is not natural or obvious to com-bine these two imaging techniques into a

single system.

Advances in ultrafast laser technology;

specialized optical filters for transmitting,

reflecting and blocking various light sig-

nals; and high-sensitivity detectors now

permit the construction of a single multi-

modal nonlinear optical microscope plat-

form that offers both label-based and

label-free imaging strategies with which

to interrogate biological samples.

Multimodal NLO microscopy combines

several imaging modalities into a single

To Label or Not?


Figure 1: Multimodal nonlinear optical (NLO) imaging of a mouse kidney: (a) CARS, (b) two-photonautofluorescence and (c) second-harmonic generation. All three signals are displayed in a single,

co-registered image in (d). The series of images of sections of mouse kidney has helped researchersunderstand the impact of fat content on cardiovascular disease and diabetic conditions in animal

models. Images courtesy of professor Eric O. Potma, University of California, Irvine.

Just as research-based studieslaid the path for clinical

investigations withintravascular OCT and

near-infraredreflectance spectroscopy,

todays pioneering researchlikely will result in a future

in which multimodal

nonlinear optical imagingwill serve as a potent weaponwith which to clinicallyinvestigate, understand

and diagnose a variety ofhuman health factors.

BY DR. NEIL ANDERSON, SEMROCK INC., A UNIT OF IDEX CORP.


25/52

unified platform and affords a myriad of

benefits. Chief among these are: inherent

3-D sectioning capability; the ability to

image deeper into samples, such as tissue,

using NIR (700- to 1100-nm) light com-

pared with linear microscopy, where visi-

ble (400- to 700-nm) light is used; the use

of lower laser average power to prevent

photodamage and phototoxicity compared

with laser-scanning confocal microscopy;

and the ability to exploit and combine

label and label-free imaging modalities.

In Figure 1, the utility of multimodal

NLO imaging shows representative com-

posite images of a section of kidney tissue

from a mouse model. The image contrast

is provided by collecting the (a) anti-

Stokes Raman light associated with the

CARS process, (b) two-photon autofluo-

rescence emission and (c) the SHG signal.

A composite image of all three signals is

shown in (d) and demonstrates how each

co-registered signal can be combined to

provide a comprehensive portrait of thesample.

The most common modalities used in

multimodal NLO imaging are multiphoton

fluorescence, SHG and THG, and CRS.

Each provides unique and complementary

benefits. Two- and three-photon fluores-

cence microscopy exploit the intrinsic

two- and three-photon absorption-induced

excitation of various organic dyes, fluores-

cent proteins and quantum dots that are

typically used to label samples fluores-

cently. Long-wavelength ultrafast laser

pulses have sufficient peak intensity to

enable a high probability that two or three

photons arrive at the molecule simulta-

neously, raising the fluorescent molecule

into an excited state. The fluorophore then

relaxes back to the ground state, produc-

ing fluorescence emission.

A key limitation of this approach is that

it requires the addition of exogenous fluo-

rophores. Label-free two-photon imaging

is possible by selectively targeting intrin-

sic cellular molecules, such as NADH

and flavins.3 This approach is commonly

called two-photon autofluorescence imag-

ing. Label-free multiphoton imaging also

is possible via three-photon processes and

can be used to image tryptophan and sero-

tonin, for example.3

Alternative label-free approaches to cel-

lular-level imaging exploit both SHG and

THG nonlinear processes within the sam-

ple. Collagen proteins exhibit noncentro-

symmetric molecular organization and

thus can be imaged by collecting the emit-

ted SHG signal under laser excitation.4

THG occurs even in centrosymmetric

structures and is useful for imaging inter-

face heterogeneities as well as myelin

sheaths in studies of the nervous system in

animal models.5 Another benefit of both

SHG and THG imaging is that, because

neither process involves the coupling of

electronic levels, photobleaching effects

can be suppressed, enabling structures to

be observed over extended periods.

Chemically specific imaging also is

possible and can be achieved by incorpo-

rating CRS into the same microscope plat-

form. CRS microscopy comes in two

unique forms: CARS and SRS.6 Both offer

Raman imaging with a better signal-to-

noise ratio and higher sensitivity than

spontaneous Raman microscopy. CARS

microscopy is the most commonly imple-

mented of the two in multimodal NLO

imaging, but SRS is rapidly growing in

popularity.

A typical multimodal NLO imaging sys-

tem consists of a laser-scanning confocal

microscope platform equipped with two

ultrafast near-infrared laser sources; vari-

ous interference filters for transmitting,

reflecting and blocking light signals; and

multiple high-sensitivity detectors to col-

lect each of the independent light signals.

One possible configuration for a multi-

modal NLO microscope for simultaneous

two-photon fluorescence, SHG and CARS

microscopy is shown in Figure 3.

In this example, two-photon fluores-

cence and SHG imaging can be performed

using a single widely tunable near-infraredfemtosecond or picosecond laser. Intro-

ducing a second picosecond laser com-

monly referred to as the Stokes beam (at

the optical frequency Stokes

) enables

CARS microscopy. When this beam is

spatially and temporally overlapped with

the beam of the first laser commonly re-

ferred to as the pump laser (at frequency

pump

) the beams interact within the sam-

ple via a four-wave mixing process to gen-

erate the CARS signal at the new optical

frequency CARS

= 2pump

Stokes

. Both

the Stokes and pump beams are collinearly


Figure 2: Energy level diagrams that describe the nonlinear processes exploited in multimodal nonlinear optical imaging: (left to right) third-harmonicgeneration (THG), second-harmonic generation (SHG), two-photon (2P) and coherent anti-Stokes Raman scattering (CARS). In the THG process, three photons

at wavelength are required to generate the THG signal at wavelength /3. In the SHG process, two input photons () are required to generate theSHG signal (at/2). In two-photon fluorescence, an excitation source at wavelength with sufficient peak intensity is used, such that there is a high

probability of absorbing two photons simultaneously, thus producing fluorescence at a wavelength just longer than the /2. The CARS processis a nonlinear four-wave mixing process that involves three input photons to generate the CARS signal at CARS = 2 pump Stokes.


26/52

overlapped and directed to a microscope

head via a specially designed notch

dichroic beamsplitter positioned at 45.

Through judicious wavelength selec-

tion, direct coupling of the characteristic

vibrational modes that uniquely identify

structures of interest within the sample can

be achieved. In CARS imaging, the Stokes

beam (Stokes

) is typically fixed at a wave-

length of 1064 nm, while the pump beam

(pump

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