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LA SER S OPT IC S IM AGIN G S P E C TROS CO PY MICR OS COPY

May/June 2015

Femtosecond Pulse Control SMLM Camera Technology Disease Detection & Obstacles

Femtosecond Pulses:
Control Is Key to New Discoveries

M/J 15

www.Photonics.com

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COHR_LifeSciences_Biophotonics0515_print.indd
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1:11 AM
PM

Model Fluorescence Accurately With Realistic Materials


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More Optical and Illumination Design Muscle

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Test drive it now at zemax.com/demo

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4/28/15 8:58 AM

LA S ER S OPTICS IMAGIN G SP E C T R O S CO PY MICR O SCO PY

Volume 22 Issue 4

www.photonics.com

NEWS
12 BIOSCAN




BioPhotonics editors curate the most significant headlines


of the month for photonics in the life sciences and take you
deeper inside the news. Featured stories include:
Chalcogenide fibers mimic brain functions
Optogenetics could counteract erectile dysfunction

21 RAPIDSCAN



Biolases top executive optimistic after tumultuous year


Edmund, Luxexcel partner on 3-D-printed optics
Headwall partners with Ocean Spray on spectral imaging
for agriculture

FEATURES
25 FEMTOSECOND PULSES: CONTROL IS KEY

TO NEW DISCOVERIES


by Marie Freebody, Contributing Editor


The precision of femtosecond lasers is transforming the life sciences in
applications such as imaging, microsurgery and micromanipulation.

32 NANODIAMONDS SHINE NEW LIGHT ON



BIO APPLICATIONS


by Dr. Olga Shenderova, Admas Nanotechnologies Inc.


The color properties of nanodiamonds show potential for exciting
new applications in the biosciences.

37 SPECTROSCOPY AIDS DISEASE DETECTION,



FACES OBSTACLES
25

by Rodd M. Pedrotti, BioPhotonics Editor


Despite hurdles beyond the research realm, spectroscopy is advancing
disease detection and treatment.

40 SINGLE-MOLECULE LOCALIZATION MICROSCOPY


WITH sCMOS CAMERAS
THE COVER
The precision and control
of a femtosecond laser
enabled the artistry of
this stent. Cover design
by Art Director Suzanne L.
Schmidt.

by Ruisheng Lin, Alex Clowsley, Isuru Jayasinghe


and Christian Soeller, University of Exeter
Tailored algorithms are propelling sCMOS cameras to the fore
as suitable alternatives for superresolution microscopy.

DEPARTMENTS
7 EDITORIAL
45 BREAKTHROUGHPRODUCTS
48 APPOINTMENTS

Upcoming Courses and Shows

49 ADVERTISER INDEX
50 POST SCRIPTS

Now available as a
FREE mobile app
for subscribers:
www.photonics.com/apps

515BI_Contents.indd 4

Lighting stunts aphids edibles

PHOTONICS
The technology of generating and harnessing light and other forms of radiant energy whose
quantum unit is the photon. The range of applications of photonics extends from energy generation
to detection to communications and information processing.
BIOPHOTONICS
The application of photonic products and techniques to solve problems for researchers,
product developers, clinical users, physicians and others in the fields of medicine,
biology and biotechnology.

BioPhotonics May/June 2015

4/29/15 9:28 AM

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LAS ERS OPTICS IMAGING S PEC TROS COPY MICR OS COPY

www.photonics.com

Group Publisher

Karen A. Newman

Editorial Staff
Editor
Web Managing Editor
Associate Editor
Contributing Editors
Copy Editors

Rodd M. Pedrotti
James F. Lowe
Caren B. Les
Hank Hogan, Marie Freebody
Margaret W. Bushee, Bonny Betancourt,
Julia Germaine

Creative Staff
Senior Art Director
BioPhotonics Art Director
Designer
Video Production Manager
Director of Publishing Operations

Lisa N. Comstock
Suzanne L. Schmidt
Janice R. Tynan
Bryan Faas
Kathleen A. Alibozek

Digital Media & IT Staff


Digital & IT Development Manager
Digital Project Manager
Digital Developer & IT Support
Computer Specialist & Digital Support

Brian L. LeMire
Alan W. Shepherd
Brian A. Bilodeau
Angel L. Martinez

Corporate Staff
Chairman/Founder
President
Vice President
Vice President
Controller
Accounting Manager
Accounts Receivable Manager
Business Manager
Human Resources Coordinator
Administrative Assistant

Teddi C. Laurin
Thomas F. Laurin
Kristina A. Laurin
Ryan F. Laurin
Mollie M. Armstrong
Lynne Lemanski
Kathleen G. Paczosa
Elaine M. Filiault
Carol J. Atwater
Marge Rivard

Business Staff
Associate Director of Sales
Trade Show Coordinator
Circulation Manager
Assistant Circulation Manager
Circulation Assistants
Subscriptions
Traffic Manager

Rebecca L. Pontier
Joey Esposito
Heidi L. Miller
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Alice M. White, Kimberly M. LaFleur,
Theresa A. Horn
Janice L. Butler
Daniel P. Weslowski

Editorial Main Office

Laurin Publishing, 100 West Street


PO Box 4949, Pittsfield, MA 01202-4949
+1 (413) 499-0514; fax: +1 (413) 442-3180; email: editorial@photonics.com

Product and Company information

Gould Fiber Optics


410.987.5600
info@gouldfo.com

www.GOULDFO.com
6

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Subscription Policy BioPhotonics ISSN-1081-8693 (USPS 013913) is published 8 times per year by
Laurin Publishing Co. Inc., 100 West Street, Pittsfield, MA 01201. TITLE reg. in US Library of Congress.
The issues will be as follows: January, February/March, April, May/June, July/August, September, October and November/December. Copyright 2015 by Laurin Publishing Co. Inc. All rights reserved. POSTMASTER: Periodicals postage paid at Pittsfield, MA, and at additional mailing offices.
Postmaster: Send form 3579 to BioPhotonics, 100 West Street, 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 the fields of medicine or biotechnology. Eligibility requests must be returned with your business card or organizations
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postage: $30 airmail per year. Publisher reserves the right to refuse nonqualified subscriptions. ARTICLES FOR PUBLICATION: Individuals wishing to submit an article for possible publication in BioPhotonics should contact Laurin Publishing Co. Inc., 100 West Street, PO Box 4949, Pittsfield, MA 01202-4949;
phone: +1 (413) 499-0514; fax: +1 (413) 442-3180; email: editorial@photonics.com. Contributed statements
and opinions expressed in BioPhotonics are those of the contributors the publisher assumes no
responsibility for them.

BioPhotonics May/June 2015

4/28/15 8:53 AM

EDITORIAL

New Digital Conference


Explores Biophotonics
Imaging

hotonics Media has been producing its successful webinar series for more than four
years, and our editors are always looking for exciting speakers to present new topics
and interesting research. I am pleased to announce the latest addition to the series;
it comes with a format twist I think youll like.
Biophotonic Imaging for Medicine: A Digital Conference, will be presented from 1 to
5 p.m. EDT on Thursday, June 11, 2015. Presentations will focus on a range of lightbased imaging and microscopy techniques for diagnosing and assessing illness, and for
studying other attributes and functions of biological tissue in a medical context. Topics
include wavefront engineering for in vivo deep tissue engineering, deep-UV excitation
microscopy for slide-free pathology, quantitative phase digital holographic microscopy,
photoacoustic analysis, multimode fiber endoscopy, surface-enhanced Raman scattering
(SERS) microscopy and lens-free microscopy.
The afternoon-long series of presentations will be hosted by Photonics.com Editor,
James Lowe, and BioPhotonics Editor, Rodd Pedrotti. The online event will feature a
series of 15- and 30-minute talks, each of which will be followed by a brief question-andanswer period.
Aydogan Ozcan, of the University of California, Los Angeles, will deliver the keynote
presentation, Democratization of next-generation imaging, sensing and diagnostics tools
through computational photonics.
We have an outstanding lineup of speakers from around the world and a really diverse
mix of approaches to imaging that could all advance medicine, said Lowe. The best
part is that people will be able to see all these fantastic presentations without paying for a
conference registration or getting on a plane.
For a complete list of speakers and topics plus details on how the event will unfold
please visit Photonics.com/webinars. You can register there as well, and check out the
entire series of archived webinars that you can view at any time.
In this issues cover story, contributing editor Marie Freebody explores current and
emerging applications for ultrafast laser pulses. In Femtosecond Pulses: Control Is Key
to New Discoveries, she describes how these very short pulses are broadening their
reach throughout the research world and reminds us that control is critical to realizing
their full potential. Read the feature, beginning on page 25.

BioPhotonics Editorial Advisory Board

Mark A. Anastasio, PhD


Professor of Biomedical Engineering
Washington University in St. Louis

Stephen A. Boppart, MD, PhD


Bliss Professor of Engineering
Electrical and Computer
Engineering, Bioengineering and Medicine
Beckman Institute for Advanced
Science and Technology
University of Illinois at Urbana-Champaign

David Benaron, MD
Professor, Medicine (consulting)
Founder, Stanford Biomedical Optics program
Stanford University School of Medicine
CEO, Spectros Corp.

Aydogan Ozcan, PhD


Associate Professor
Electrical Engineering, Bioengineering
University of California, Los Angeles

Adam Wax, PhD


Theodore Kennedy Associate Professor
Director of Masters Studies at the Department
of Biomedical Engineering, Duke University
Chairman and Founder, Oncoscope Inc.

Also in this issue:


Nanodiamonds Shine New Light on Bio Applications, by Dr. Olga Shenderova,
Admas Nanotechnologies, beginning on page 32;
Spectroscopy Aids Disease Detection, Faces Obstacles, by Editor Rodd M. Pedrotti,
beginning on page 37; and,
Single-Molecule Localization Microscopy with sCMOS Cameras, by Ruisheng Lin,
Alex Clowsley, Isuru Jayasinghe and Christian Soeller, Biomedical Physics, University
of Exeter, beginning on page 40.
Enjoy the issue.

Karen A. Newman
karen.newman@photonics.com

BioPhotonics May/June 2015

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CONTRIBUTORS
A doctoral student at the
University of Exeter in the U.K.,
Alex Clowsley is part of the
Biomedical Physics Research
Group working on improving
superresolution microscopy.
Page 40.
Contributing editor Marie
Freebody is a freelance journalist with a masters degree
in physics from the University
of Surrey, England. Page 25.

Dr. Isuru Jayasinghe is an


associate research fellow
at the University of Exeter
in the U.K., specializing in
the application of dSTORM
(direct stochastical optical
reconstruction microscopy) to
image heart and skeletal muscle. Page 40.
Dr. Ruisheng Lin is an associate research fellow at
the University of Exeter in the
U.K., working on developing
and improving superresolution
microscopy. Page 40.
Editor Rodd M. Pedrotti
brings more than 10 years
of experience in editing,
communications, project management and TV news to his
work with Photonics Media.
Rodd integrates his passion
for science and technology into his role as editor
of BioPhotonics and Industrial Photonics magazines. He also is seen reporting industry-specic
segments on our weekly newscast, Light Matters.
Page 37.
Dr. Olga Shenderova is president of Admas Nanotechnologies Inc. in Raleigh, N.C.
She received her doctorate
in materials science from the
St. Petersburg State Technical
University of Russia in 1991.
Her areas of expertise include nanodiamond (ND)
particle surface modication, production of uorescent NDs, ND-based additives to lubricants,
polymer composites, biological applications
of NDs, and atomistic simulations of carbon
nanostructures. She has authored over 140
papers, 15 book chapters, and 20 patents, and
has edited ve books related to nanodiamonds.
Page 32.
Dr. Christian Soeller heads
the Laboratory for Biophysics and Biophotonics in the
Biomedical Physics Group at
the University of Exeter in the
U.K. His laboratory develops
and applies superresolution
imaging techniques to investigate the biophysics
of a range of cell systems. Page 40.

515BI_Online & Contributors.indd 8

Welcome to

The online companion to BioPhotonics magazine

Whats Online:

The industrys only weekly


newscast has a great new
look, a broader range of stories
and a great cast of light-loving
characters. Join your host,
Web Managing Editor James
Lowe, editors Justine Murphy,
Rodd Pedrotti and Sarina Tracy,
and contributing editor Joey
Esposito for an upbeat weekly
dash through stories about light
that will leave you glowing.

Visit photonics.com/lightmatters.

IYL2015 Playlist
The International Year of Light is a 365-day party, and weve
got the soundtrack! Our IYL2015 Playlist features four hours
worth of songs with a photonics twist. Find it on Spotify or at
tinyurl.com/IYL2015.

Join us for a free webinar on Thursday, June 11, at 1 p.m. EDT!


Biophotonic Imaging for Medicine: A Digital Conference
From the editors of Photonics.com and BioPhotonics magazine.

REGISTE
R
NOW

Researchers from around the world will present their


latest work at Biophotonic Imaging for Medicine:
Biophotonic Imaging
A Digital Conference. The online event will feature
for Medicine:
a series of 15- and 30-minute talks, each followed
A Digital Conference
From the editors of Photonics.com and BioPhotonics magazine.
by a brief question-and-answer period.
Presentations will focus on a range of light-based
imaging and microscopy techniques for diagnosing
and assessing illness, and for studying other attributes and functions of biological tissue in a medical
context. Topics include computational 3-D lens-free
microscopy, wavefront engineering for in vivo deep tissue engineering, deep-UV excitation microscopy for slide-free pathology, quantitative phase digital holographic microscopy, photoacoustic analysis, multimode ber endoscopy and surface-enhanced
Raman scattering microscopy.

BioPhotonics May/June 2015

4/28/15 9:01 AM

International year of Light 2015

The Month in Light


MAY

May 4, 2015
Light Week! begins at MOTAT (Museum of Transport and Technology) in Auckland, New Zealand,
where students will experiment with color mixing,
refraction, reflection and shadows.
May 5, 1921
Birth date of Arthur Leonard Schawlow. In 1981,
he won the Nobel Prize in physics for work on laser
spectroscopy, sharing the honor with Nicolaas
Bloembergen and Kai Siegbahn.
May 9, 2015
The CIE (Commission Internationale de lclairage)
invites light research laboratories worldwide to
participate in Global Open Lab Days (GOLD)
through May 25.
May 14, 2015
Attend The Light Project All that Glitters: Egyptian Light in London at Cleopatras Needle on the
Victoria Embankment to explore ancient Egyptian
beliefs about light.
May 15, 1859
Birth date of Pierre Curie. He won the 1903 Nobel
Prize in physics for research on radiation, sharing
the win with his wife, Marie Curie, and with Henri
Becquerel.
May 20, 2015
The annual World Metrology Day has adopted the
theme Measurements and Light, focusing on the
role of measurement in advancing and applying
light-based technologies.
May 26, 2015
The meeting Light Pollution: Theory, Modelling and
Measurements begins at the Centre de Villgiature Jouvence, located in the Eastern Townships,
Qubec.
May 29, 1919
Astrophysicist Arthur Eddington, observing the
total solar eclipse on this date, confirmed Albert
Einsteins theory that light bends.

BioPhotonics May/June 2015

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JUNE

June 5, 1900
Birth date of Dennis Gabor, who won the 1971
Nobel Prize in physics for the invention and
development of holography.
June 9, 1781
Birth date of civil and mechanical engineer George
Stephenson. He devised a miners safety lamp
that would burn without causing an explosion.
The lamp design featured tiny holes for air and a
glass cylinder.
June 11, 1867
Birth date of French physicist Charles Fabry, who
discovered an explanation for the phenomenon
of interference fringes within the field of optics.
He and Alfred Prot invented the Fabry-Prot
interferometer.
June 13, 1831
Birth date of James Clerk Maxwell. He formulated
the classical theory of electromagnetic radiation,
which brings together light, electricity and magnetism as manifestations of the same phenomena.
June 24, 2015
The start of the two-day Smart City Lighting Event
2015 in Eindhoven, the Netherlands, where lightingindustry professionals will gather to discuss developments and creative solutions.
June 26, 2015
The two-day New York Blue Light Symposium
begins. Scientists and clinicians will discuss how
blue light emitted from LED devices may affect
human health.
June 27, 2015
The two-day annual meeting of the Society for Light
Treatment and Biological Rhythms will take place
in San Diego.
June 29, 1818
Birth date of Angelo Secchi. A pioneer in astronomical spectroscopy, he was one of the first scientists
to authoritatively state that the sun is a star.

4/28/15 9:02 AM

SAVE the
DATE
Over 26% of attendees in 2014 were speakers selected from submitted abstracts
Submit an abstract by August 4 to be considered for a speaking spot:

Minisymposium Standard Talk


(15-minute talk, 5-minute Q&A)
Minisymposium Lightning Talk
(5 minute talk, 1-2-minute Q&A)
Microsymposium Talk
(5-minute talk plus electronic poster)
The 2015 ASCB Annual Meeting is designed around the
big data revolution and how you can take advantage of it
for your own research. Explore the relevance of cell biology
research that integrates disciplines on different scales, from
intracellular all the way to systems, organisms, ecosystems,
and the macrocosmic. Discover how cell biology is
relelvant to researchers in neuroscience, immunology,
cancer biology, biophysics, and molecular medicine.
See the future of cell biology. Be the future of cell biology.

EXHIBIT BOOTHS AVAILABLE

www.ascb.org/2015meeting
/ascbiology

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@ascbiology

ascb

4/28/15 9:02 AM

Biophotonic Imaging for Medicine:


A DIGITAL CONFERENCE
Thursday, June 11, 2015, 1 to 5 p.m. EDT

Researchers from around the world will


present their latest work at Biophotonic
Imaging for Medicine: A Digital Conference,
hosted by Photonics Media. This free online
event will feature a series of 15- and 30-minute
talks, each of which will be followed by a brief
question-and-answer period.

KEYNOTE

Democratization of Next-Generation
Imaging, Sensing and Diagnostics
Tools through Computational
Photonics
Aydogan Ozcan,
University of California, Los Angeles

FEATURED PRESENTATIONS
Wavefront Engineering for
In Vivo Deep Tissue Imaging
Lingjie Kong,
HHMI Janelia Research Campus

Presentations will focus on a range of lightbased imaging and microscopy techniques


for diagnosing and assessing illness, and for
studying other attributes and functions of
biological tissue in a medical context. A list
of presentation titles and speakers follows.

Deep UV Surface Excitation


Microscopy for Imaging
Unsectioned Tissue: Realizing
Slide-Free Pathology
Richard Levenson,

You can read the full abstracts and speaker


biographies, as well as sign up for the free digital
conference, at photonics.com/bioconference.

Applications of Multiphoton
Microscopy in Urology
Manu Jain,
Weill Cornell Medical College

Severity of Inammation
in Inammatory Bowel
Diseases Assessed by
Quantitative Phase Digital
Holographic Microscopy
Bjrn Kemper,
University of Muenster

Advancing Optical Time


Stretch for High-Throughput
Imaging
Kevin Tsia,
University of Hong Kong

University of California, Davis

A Novel Photoacoustic
Camera for Medical Imaging
Navalgund Rao,
Rochester Institute
of Technology

Photoacoustic Imaging
of Breast Cancer
Srirang Manohar,
University of Twente

Raman Microscope System


for the Detection of B-cell
Malignancies Using SurfaceEnhanced Raman Scattering
Nathan Israelsen,
Utah State University

Photoacoustic and Ultrasound


Tomography of the Human
Finger: Towards the
Assessment of Inammatory
Joint Diseases
Peter van Es,
University of Twente

Quantitative Analysis
of Multimode Fiber
Endoscopes
Antonio M. Caravaca Aguirre,
University of Colorado Boulder

www.photonics.com/bioconference

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4/29/15 10:03 AM

BIOSCAN
A closer look at the most significant biophotonics research and technology headlines of the month

Chalcogenide fibers mimic brain functions


SOUTHAMPTON, UK Microfibers
produced from sulfur-based glasses,
called chalcogenides, could be used to
replicate the workings of the human
brain, enabling optical computers capable
of learning and evolving.
According to researchers from the
University of Southamptons Optoelectronics Research Centre (ORC) and from
Nanyang Technological University (NTU)
in Singapore, a variety of broadband
photoinduced effects allow the fibers to
be switched on and off. The investigators
indicate that this optical switching can
be exploited for a variety of next-generation computing applications capable
of processing vast amounts of data with
improved energy efficiency.
By going back to biological systems
for inspiration and using mass-manufacturable photonic platforms, such as chalcogenide fibers, we can start to improve
the speed and efficiency of conventional
computing architectures, while introducing adaptability and learning into the
next generation of devices, said ORCs
Dr. Behrad Gholipour.
In the past decade, neuromorphic computing research has advanced software
and electronic hardware that mimic brain
functions and signal protocols, and that
is aimed at improving the efficiency and
adaptability of conventional computers.
But compared to our biological systems, todays computers are more than a
million times less efficient. Simulating
five seconds of brain activity takes 500
seconds and requires 1.4 MW of power,
compared with the small number of calories burned by the human brain.

Scientists demonstrated a range of optical equivalents of brain functions by exploiting the material
properties of chalcogenide fibers. Samples of chalcogenide glass are shown. Courtesy of University of
Southampton.

By exploiting the material properties of


chalcogenide fibers, a team led by NTUs
Dr. Cesare Soci demonstrated a range of
optical equivalents of brain functions.
These include holding a neural resting
state and simulating changes in electrical
activity in a nerve cell as its stimulated.
In the proposed optical version of
this brain function, the changing optical
properties of the fibers act as the varying electrical activity in a nerve cell, and
light provides the stimulus to change
those properties. This enables switching of a light signal the equivalent of a
nerve cell firing.
The research paves the way for scalable, brain-like computing systems that
enable photonic neurons with ultrafast
signal transmission speeds, higher band-

width and lower power consumption than


their biological and electronic counterparts.
This work implies that cognitive
photonic devices and networks can be
effectively used to develop non-Boolean
computing and decision making paradigms that mimic brain functionalities
and signal protocols, to overcome bandwidth and power bottlenecks of traditional data processing, said Soci.
Funding came from the Agency for
Science, Technology and Research
(A*STAR) in Singapore.
The research was published in Advanced Optical Materials (doi: 10.1002/
adom.201400472).

Optogenetics could counteract erectile dysfunction


BASEL, Switzerland A gene construct
that reacts to blue light could offer a
drug-free remedy for men with erectile
dysfunction.
Researchers at ETH Zurichs Department of Biosystems have tested the
construct, which they call an erectile
optogenetic stimulator (EROS), in male
rats. They found that it could be used to

12

515BI_BioScan.indd 12

reliably turn on erections and, in some


cases, cause ejaculations.
When the construct is exposed to the
light, a precursor molecule (guanosine
triphosphate, or GTP) is converted into
the second messenger (cyclic guanosine
monophosphate, or cGMP), which exists
naturally in a number of human organs.
This allows voltage-dependent calcium

channels to close, thereby reducing


calcium levels in the cells. In turn, this
relaxes muscle cells and increases blood
flow to erectile tissue. An enzyme then
slowly breaks down cGMP so that the
erection wears off with time.
In Angewandte Chemie International
Edition (doi: 10.1002/anie.201412204/
full), the researchers wrote that photo-

BioPhotonics May/June 2015

4/28/15 9:05 AM

stimulated short-circuiting of complex


psychological, neural, vascular and
endocrine factors to stimulate penile erection in the absence of sexual arousal may
foster novel advances in the treatment of
erectile dysfunction.
An estimated 30 million American
men are affected by erectile dysfunction,
according to the National Institutes of
Health. About 4 percent of men in their
50s, almost 17 percent of men in their
60s and 47 percent of men older than 75.2
experience the condition.
Drugs used to treat erectile dysfunction have disadvantages. While they serve
to prolong erections, they cannot trigger
them. Additionally, such drugs cannot
be used by people with certain medical
conditions, including heart disease.
Light-based gene therapy, according to
the Swiss researchers, may offer a direct
route to erection without side effects.
Injection of a gene construct should
not be a barrier to potential users, as
injections in the erectile tissue are already
a standard treatment for erectile dysfunction these days, said ETH Zurichs
Dr. Martin Fussenegger.
Erectile tissue is largely insensitive to

pain. For the most part, it is also detached


from normal blood circulation, so the
probability that the gene construct could
reach other parts of the body is very low.
Additionally, cGMP breaks down relatively quickly.

Fussenegger said clinical testing would


be required before the technique could
be used as a treatment for humans. His
team is currently looking for partners to
develop the technology for clinical use.

A male rat is exposed to blue light after injection with a light-activated gene construct that triggers
erections. Courtesy of Martin Fussenegger, ETH Zurich.

Modified genes triggered by blue light

Researchers demonstrate their technique to control


genes by shining light through a Duke D stencil
to turn on fluorescent genes in cells. Courtesy of
Duke University.

BioPhotonics May/June 2015

515BI_BioScan.indd 13

DURHAM, N.C. Crossing a bacteriums


viral defense system with a flowers
response to the sun yields a light-based
trigger for genes.
This type of control could enable deeper
studies of specific gene functions, create
complex systems for growing tissue and
perhaps facilitate healing technologies,
say researchers at Duke University.
This technology should allow a scientist to pick any gene on any chromosome
and turn it on or off with light, which
has the potential to transform what can
be done with genetic engineering, said
doctoral student Lauren Polstein.
The advantage of doing this with light
is we can quickly and easily control when
the gene gets turned on or off and the
level to which it is activated by varying
the lights intensity. We can also target
where the gene gets turned on by shining
the light in specific patterns, for example

by passing the light through a stencil.


The optogenetic technique targets
specific genes using an emerging geneticengineering system called CRISPR/Cas9;
it was discovered as the technique bacteria use to identify viral invaders and to
slice up their DNA. Researchers used the
same technique to precisely target specific
genetic sequences.
The researchers then turned to another
branch of the evolutionary tree to make it
a light-activated system.
In many plants, two proteins lock
together in the presence of light, allowing
these plants to sense the length of day,
which determines biological functions
like flowering. By attaching the CRISPR/
Cas9 system to one of these proteins and
gene-activating proteins to the other, the
team turned several different genes on
or off just by shining blue light on the
cells.

13

4/28/15 9:05 AM

BIOSCAN

The light-sensitive interacting proteins exist independently in plants, said


Dr. Charles Gersbach. What weve done
is attached the CRISPR and the activator
to each of them. This builds on similar
systems developed by us and others, but
because were now using CRISPR to target particular genes, its easier, faster and
cheaper than other technologies.
The technique could allow scientists
to precisely control the level of a genes
activity from its natural position in chromosomal DNA, which would allow them
to get a more accurate interpretation of
the genes role. The light-induced system
also could provide more control over how
stem cell cultures differentiate into various types of tissues.

By creating different patterns of gene


expression, Gersbach hopes the system
can be used in tissue engineering.
One of the limitations of tissue
engineering right now is that typical
methods make a chunk of bone, cartilage
or muscle, but thats not what tissues look
like naturally, Gersbach said. There are
several cell types mixed together, gradients of tissues between interfaces, and
blood vessels and neurons that penetrate
through them.
We want to spatially control where
different tissues get made in a cell population, and that way create multitissue
constructs that potentially better represent
normal physiology, he said.
In the more distant future, the tech-

nique could be used to regenerate


wounded tissue, Gersbach added.
Far, far down the road, you could
envision the type of device youd see on
Star Trek where you wave a flashlight
over a wound and it heals, he said. Obviously thats not currently possible, but
this type of technology that creates much
better control over biological systems
could move us in that direction.
Funding for the work came from the
National Institutes of Health, the National
Science Foundation and the American
Heart Association.
The research was published in Nature
Chemical Biology (doi: 10.1038/nchembio.1753).

Laser glasses aim to counteract low vision


TOKYO A new pair of smart glasses
that shines laser light directly onto the
retinas could be a boon to people with
poor eyesight.
QD Laser Inc. is developing the device
in collaboration with the Institute for

515BI_BioScan.indd 14

Nano Quantum Information Electronics


at the University of Tokyo and expects to
bring it to market in March 2016.
In the meantime, the device has begun
verification testing at medical universities and other educational institutions

in Japan, QD Laser said. A prototype


was presented at the recent International
Technology and Persons with Disabilities
Conference in San Diego.
The device has a camera module in the
center of the frame and uses semicon-

4/28/15 9:05 AM

BIOSCAN

ductor lasers as RGB light sources. The


company said that its eyewear has several
advantages over LCD devices for people
diagnosed with low vision, including a

wide field of view and high brightness.


QD Laser said the technology could
have applications beyond low-vision care.
The company is eyeing a launch into

markets for workplace support tools and


consumer smart glasses by the end of
2017.

Photothermal nanoparticles extend range of optogenetics


CHICAGO Targeted gold nanoparticles
allow light to activate neurons a finding
that could enable the use of optogenetic
techniques without genetic manipulation.
This is effectively optogenetics
without genetics, said Dr. Francisco
Bezanilla of the University of Chicago.
Many optogenetic experimental designs
can now be applied to completely normal
tissues or animals, greatly extending the
scope of these research tools and possibly
allowing for new therapies involving
neuronal photostimulation.
Optogenetics, which is based on genetically engineering neurons to express
a light-responsive protein originally
discovered in algae, allows scientists to
stimulate both individual neurons and
neural networks with precise flashes of
light. However, since optogenetics is

reliant on genetic modification, its use is


limited primarily to relatively few model
organisms.
Bezanilla and colleagues previously
demonstrated that normal, nongenetically
modified neurons can be activated by heat
generated by IR pulses, but this method
lacks specificity and can damage cells.
To improve the technique, they used
20-nm gold particles that, when stimulated with green light, absorbs the energy
and converts it into heat.
The researchers tested two kinds of
nanoparticles. The first type was coupled
with a synthetic molecule based on Ts1, a
scorpion neurotoxin, which binds to sodium channels in neurons without blocking
them. The second type was coupled with
antibodies that bind to ion channels.
Neurons treated with Ts1-coupled

nanoparticles in culture were readily


activated by photothermal stimulation.
Untreated neurons were unresponsive.
Targeted neurons could be stimulated
repeatedly with no evidence of cell damage. Some individual neurons, targeted
with millisecond pulses of light, produced
more than 3000 action potentials over the
span of 30 minutes with no reduction in
efficacy.
In addition to cultured cells, nanoparticles were tested on complex brain tissue
using thin slices of mouse hippocampus.
In these experiments, the researchers activated groups of neurons and observed the
resulting patterns of neural activity.
Treated neurons could still be stimulated
after 30 minutes of continuous washing,
indicating that the nanoparticles were
bound tightly to the cell surface. Excess

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nanoparticles wash away, minimizing potentially harmful elevated temperatures.


With differently shaped nanoparticles,
it can work in near-infrared, as well as
in visible wavelengths, which has many
practical advantages in living animals,
Bezanilla said. Thus far, most optogenetic tools have been limited to visible
wavelengths.
Nanoparticles ability to retain their efficacy while coupled to various antibodies
suggests flexibility for future applications,
including human therapeutic developments.
In some retinal diseases, such as agerelated macular degeneration, photoreceptor cells that absorb light signals are damaged or dead. Yet, the retinal nerve cells
that carry visual information to the brain
often remain intact and healthy. Nanoparticles targeted to these cells potentially
could absorb light and directly stimulate

the neurons, bypassing defective photoreceptors, the researchers said.


While much additional research must
be done to determine the feasibility of
this nanoparticle approach as a vision
restoration therapy, our results encourage further effort aimed at achieving this
critical clinical objective, said Dr. David
Pepperberg of the University of Illinois at
Chicago.
Although no harmful effects were
observed, the researchers note that
toxicity is a possibility. However, many
live-animal tests and human clinical
trials already have been completed using
formulations of gold nanoparticles without serious side effects. The researchers
are now testing the techniques efficacy in
animal models to verify its potential for
therapeutic use.
The research was published in Neuron
(doi: 10.1016/j.neuron.2015.02.033).

Squid skin inspires IR camouflage tape


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IRVINE, Calif. A protein derived from


squid skin could be adapted into a type of
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Researchers from the University of
California, Irvine, have developed an adhesive film that, when stretched, reflects
near-infrared light. Future versions of the
technology that reflect a broader part of
the IR spectrum could be worn by soldiers
hoping to avoid detection by enemy thermal cameras.

Soldiers wear uniforms with the


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patterns to blend into foliage during the
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A squid-inspired adhesive film that reflects near-infrared light when stretched could provide camouflage for
soldiers.

16

515BI_BioScan.indd 16

BioPhotonics May/June 2015

4/28/15 9:05 AM

BIOSCAN

active infrared visualization.


Squid skin features unusual cells
known as iridocytes, which contain layers
or platelets composed of a protein called
reflectin. The animal uses a biochemical
cascade to change the thickness of the
layers and their spacing, altering how the
cells reflect light. Gorodetskys group
used bacteria to produce reflectin.
One way to make the film reflective in
the IR spectrum is to expose it to acetic
acid vapor, but this isnt practical for
soldiers in the field. Instead, Gorodetskys

team applied the protein to flexible polymer substrates essentially, household


sticky tape. The tape adheres to a range
of surfaces, including cloth uniforms, and
its appearance under an IR camera can
be changed by stretching it. A structural
trigger like this might be a more realistic
application for military use.
Were going after something thats
inexpensive and completely disposable,
Gorodetsky said. You take out this
protein-coated tape, you use it quickly to
make an appropriate camouflage pattern

on the fly; then you take it off and throw


it away.
The film may also have uses outside
the military. Examples include clothing
that selectively traps or releases body heat
to keep people comfortable in varying
environments.
The research was presented at the 249th
National Meeting & Exposition of the
American Chemical Society.
For more information, visit: www.uci.
edu.

Fluorescent probe shows promise in osteoarthritis treatment


BOSTON A near-infrared fluorescent
probe may make it easier to diagnose and
monitor osteoarthritis.
Tested in mice, the probe detected
the activity leading to cartilage loss in
joints. As osteoarthritis progressed, the
probes brightness levels increased. This
was the first study to demonstrate that
near-infrared fluorescence can be used to
detect osteoarthritis changes, according
to a research team led by Tufts University
School of Medicine.
The fluorescent probe made it easy
to see the activities that lead to cartilage
breakdown in the initial and moderate stages of osteoarthritis, said Shadi
Esfahani, postdoctoral fellow at Massachusetts General Hospital and Harvard
Medical School.
X-ray imaging tests used for osteoarthritis patients today dont indicate pain
levels or visualize the amount of cartilage
loss, the researchers said.
For the two-month study, the researchers created injury-induced osteoarthritis
in the right knees of 54 mice. The healthy
left knees of the mice served as a control
group. The mice received pain medication.
The fluorescent probes are activated
by matrix metalloproteinases, enzymes
associated with the beginning stages of
osteoarthritis. They were injected into
both knees of each mouse.
Researchers took images of each knee
every two weeks to determine whether
the fluorescent probes emitted a signal. At each examination, the signals
became brighter in the injured right knees
throughout the early to moderate stages of
osteoarthritis. The probes in the healthy
left knees emitted lower signals that did
not increase significantly throughout the
study.

BioPhotonics May/June 2015

515BI_BioScan.indd 17

The next step will be to monitor the


fluorescent probes over a longer period
of time to determine whether the same
results are produced during late stages of
osteoarthritis, the researchers said.
Dr. Li Zeng of Tufts said she hopes to
use the probes to help develop treatments
for animals. Certain breeds of dogs, for
example, are prone to osteoarthritis.
If the technology can be applied to hu-

mans, the investigators said, it may allow


for earlier treatment, including helping to
analyze the effectiveness of osteoarthritis
drugs and treatment methods.
According to the Centers for Disease
Control and Prevention, osteoarthritis
affects 27 million Americans and usually
is detected in its late stages.
The research was published in Arthritis
& Rheumatology (doi: 10.1002/art.38957).

17

4/28/15 9:05 AM

BIOSCAN

Photoelectric dye may restore


some sight to the blind
OKAYAMA, Japan Photoelectric dyes may be used to send
images to the brains of the blind, potentially presenting a simpler
alternative to artificial eyes based on image sensors.
Blind patients with hereditary diseases such as retinitis pigmentosa have dead photoreceptor cells. However, other neurons
involved in sight remain alive and can be stimulated artificially.
Researchers from Okayama University and from Okayama
University of Science tested the dye in genetically blind rats and
found that the rodents could follow the direction of movement of
a spinning drum painted with vertical black-and-white stripes.
The dye used has an absorption spectrum that spans the visible range from 400 to 600 nm. It is stable, readily synthesized,
and has a low molecular weight and no obvious toxic components, the researchers said.
Coupled to a soft, thin polyethylene film at a concentration of
around 106 dye molecules per m2, the dye was inserted into the
subretinal area through a small opening. Large films used to
provide a wide field of view were rolled up before the insertion.
Polyethylene has been used for medical implants for some
time, and its safety and stability already have been proved, the
researchers said. They tested the dye in vitro using cultured
retinal cells and observed no cytotoxicity.
The prototype of the photoelectric dye-coupled retinal prosthesis, OUReP, is unique in using electric potentials to stimulate
retinal neurons, in contrast with the other systems of retinal
prostheses that generate electric currents, the researchers stated.
Next, the researchers plan to test the likelihood of treatment
success by using optical coherence tomography to assess the
level of degeneration in patients retinas.
The research was published in the Journal of Artificial Organs
(doi: 10.1007/s10047-015-0825-1).

Higher-order modes speed up


fiber conveyor belt

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OKINAWA, Japan Evanescent waves bleeding out of optical fibers can be used to trap and move microparticles like a conveyor
belt a phenomenon that could be useful in drug delivery, cell
research and quantum computing.
While the effect has been demonstrated before using light
of the fundamental mode, researchers at the Okinawa Institute
of Science and Technology Graduate University have taken it
a step further by using higher-order modes. They moved 3-m
polystyrene beads through water up to eight times faster with
higher-order modes than with the fundamental mode.
While it was theoretically proposed that higher-order modes
would produce stronger forces, this is the first time, to our
knowledge, that three-dimensional particle manipulation has
been experimentally demonstrated, said Dr. Viet Giang Truong.
The fibers used in the experiments start with a diameter of 80
m and taper down to 2 m at the waist. As light travels through
the fiber, it cannot fit inside the extremely thin waist, and so it
spreads out, creating an evanescent field around the fiber. This
light field can trap particles close to the fiber surface and move

BioPhotonics May/June 2015

515BI_BioScan.indd 18

4/28/15 9:05 AM

BIOSCAN

them in the direction of light propagation. Higher-order modes


create larger evanescent fields in such tapered fibers. Additionally, part of the increase in the speed of the microparticles is likely
due to microfluidic dynamics, said doctoral student Aili Maimaiti. As the particles pick up speed, they lift slightly away from the
fiber, reducing drag and allowing them to move even faster.
This experiment proves the capability of higher-order modes

The shape of light in the fundamental mode (top) versus a higher-order mode
(bottom) is shown in a simulation, left, and in the experiment, right. Courtesy
of the Okinawa Institute of Science and Technology Graduate University.

in microfibers to trap and propel particles, Truong said. The


next step is to control multiple particles in three dimensions
around the microfiber. We are also keen to demonstrate similar
behavior of atoms around the nanofibers.
Researchers used the microfiber in conjunction with optical tweezers, a tool widely used in research to trap and move
individual particles. The higher-order-mode microfiber gives the
tweezers greater control over the particles to be manipulated, the
researchers said. In the future, microfibers also could increase
optical tweezers sensitivity by communicating more precise
information about the trapped particle.
Optical trapping and manipulation of particles using optical
micro- or nanofibers has the potential to help deliver drugs to
specific locations, such as inside a target cell. The effect also
could be used to measure the interaction forces between cell
components, or to study the proteins involved in the DNA and
RNA transcription and translation processes.
The beauty of ultrathin optical fibers is that they are a very
noninvasive tool, allowing us to probe many different physical systems while only affecting specific parameters that we
choose, said Dr. Sle Nic Chormaic. While this work focuses
on trapping micron-sized particles using higher-order light
modes in optical microfibers, we can use similar techniques
at the atomic level for creating some of the building blocks in
quantum networks.
The research was published in Scientific Reports (doi: 10.1038/
srep09077).

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BioPhotonics May/June 2015

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19

4/28/15 9:05 AM

BIOSCAN

Camera-based monitoring of vital signs improved


HOUSTON Refined signal processing
allows video cameras to monitor vital
signs regardless of the ambient lighting

and the skin tone of the subject.


A new algorithm developed by investigators at Rice University detects subtle

The photoplethysmography (PPG) signal is extracted from four regions marked on the face.
A weighted average of the four readings compares well with a reading from a pulse oximeter.
Courtesy of The Optical Society.

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variations in skin tone caused by blood


circulation to determine pulse and breathing rates.
The idea of using a camera to track
vital signs is based on photoplethysmography (PPG), which is a way to measure
physiological processes under the skin by
monitoring subtle changes at the skins
surface. Camera-based PPG has been
studied previously, but its applications
were limited it did not work reliably unless subjects were fair-skinned and sitting
perfectly still in a well-lit room.
The Rice algorithm, called DistancePPG, overcomes these problems by measuring the eyes, nose and mouth separately. A weighted average, based on blood
perfusion and incident light intensity, is
applied to the data to estimate vital signs.
Interestingly, this technique has been
known in other domains of computer
vision but has not been properly applied
to the problem at hand, said graduate
student Mayank Kumar. Once we understood the motion challenge, the tracking
approach became obvious.
To test their new algorithm, the researchers monitored adults engaging in
common activities. The results were compared to readings from pulse oximeters
attached to subjects earlobes.
The algorithm improved the PPG signal
in situations with low levels of motion,
such as when subjects were reading or
watching a video. However, it remained
relatively inaccurate when subjects were
talking or smiling. These larger movements changed the facial light reflectance
more dramatically and made extracting
a reliable signal difficult, the researchers
said.
Noncontact methods for monitoring
vital signs are especially desirable in neonatal intensive care units, where repeatedly attaching and removing monitors can
injure babies born prematurely and leave
them susceptible to infection.
If the motion problems can be overcome, according to researchers, the
technique could find its way into healthtracker applications for smartphones and
computers.
The work was published in Biomedical Optics Express (doi: 10.1364/
BOE.6.001565).

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515BI_BioScan.indd 20

BioPhotonics May/June 2015

4/28/15 9:05 AM

rapidSCAN Business and Markets


Biolases top executive
optimistic after
tumultuous year

Biolase Inc.s President and CEO Jeffrey M. Nugent said he is confident the
dental laser firm will grow this year
after enduring a tumultuous 2014.
The company reported its 2014 fourthquarter and year-end results in March.
The latter half of 2014 marked the
beginning of the transformation of
Biolase, with a new board of directors
and management, said Nugent. In
June 2014, he was chosen to replace
former CEO Federico Pignatelli.
Nugent went on to say, Among the
many problems we faced last June,
revenues had been declining, and there
were substantial legal and administrative expenses caused by litigation that
worsened the negative momentum
even further.
Revenue for the year was
$47.7 million, down 15.4 percent
from $56.4 million in 2013. The
company attributed the downturn to
distractions caused by shareholder
litigation and management changes,
as well as sales department turnover.
Net loss was $18.9 million, compared with a net loss of $11.5 million
in 2013.
Nugent stated that during the last
six months of the year, the company
secured two equity financings and
took other steps to strengthen its balance sheet. He said the company also
addressed customer complaints and
doubled the warranty period of its flagship WaterLase systems.
Lastly, the company implemented
a new product strategy and, in recent
months, introduced two new products.
Nugent added that Biolase continues
to work on resolving various legacy
cultural and operational issues.

Edmund, Luxexcel partner on 3-D-printed optics

Plastic optical components designed by Edmund Optics and printed by Luxexcel. Courtesy of
Luxexcel.

Edmund Optics Inc. of Barrington,


N.J., has partnered with Luxexcel
Group BV of the Netherlands to
develop 3-D-printed plastic optical
components.
Luxexcels patented Printoptical
technology uses additive manufacturing techniques to create smooth, transparent optical components including
microstructures, arrays, prisms and
free-form lenses. The components are
suited for illumination, photovoltaics
and prototyping applications.
Luxexcel received the Prism Award
in the Additive Manufacturing cat-

Headwall partners with


Ocean Spray on spectral
imaging for agriculture

Ocean Spray Cranberries Inc. will try to


improve the quality and yield of its cranberries using spectral imaging systems
from Headwall Photonics Inc.
The two companies will cooperate
to test a new class of machine vision
inspection capabilities for real-time assessment of product quality for a variety
of agricultural processing applications.

BioPhotonics May/June 2015

515_BI RapidScan.indd 21

egory in February at Photonics West,


which took place in San Francisco.
I am excited that this cooperation
will make it easy for designers to prototype, fine-tune and ultimately perfect their designs, enabling the next
generation of optically driven products, said Samuel Sadoulet, Edmund
Optics president and chief operating
officer.
Edmund Optics produces optics,
imaging and photonics technology for
R&D, and electronics, semiconductor, pharmaceutical, biomedical and
military markets.

Headwall has supplied customized


multispectral and hyperspectral technology to the U.S. Department of Defense,
NASA, the Treasury Department and the
Environmental Protection Agency.

LPKF announces
initiatives after a
disappointing 2014

Closing the books on a difficult 2014,


LPKF Laser & Electronics AG of
Garbsen, Germany, has set its sights on

21

4/28/15 9:06 AM

RAPIDSCAN

new laser machining processes and new


markets.
Earnings before interest and taxes
(EBIT) fell 45 percent in 2014 to 13
million (about $14.1 million). Revenue
was 120 million (about $130.4 million),
down 8 percent from 2013. The company
said this was its first drop in revenue in
11 years.
The company attributed the results

to weak demand for its laser directed


structuring (LDS) and printed circuit
board production equipment, as well as to
economic difficulties affecting customers
in South Korea.
This is clearly an unsatisfactory result
for LPKF, said CEO Dr. Ingo Bretthauer.
We had originally planned to achieve a
lot more during 2014, but weak incoming
orders in two of our six product groups

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simply turned these plans upside down.


While the company could not counteract LDS losses, revenues rose 31 percent
in its Other Production Equipment segment and 20 percent in its Electronics
Development Equipment segment, beating
expectations.
In the years ahead, LPKF said it sees
growth potential in the wearable technologies and LED markets. It expects
revenue ranging from 128 million to
136 million (about $139 million to $147.7
million) in 2015.
The company plans to launch three new
laser-based materials processing methods
this year, including a coating process
to enable the application of heavy-duty
metal layers to plastic substrates, a digital
laser printing method for functional
pastes, and a laser-driven ultrafine glass
drilling process for use by chip makers.
LPKF manufactures machines and
laser systems used in electronics fabrication, medical technology, the automotive
sector and in solar cell production.

OLED microdisplay
contracts buoyed
eMagin revenues in 2014
Organic LED (OLED) microdisplays
firm eMagin Corp. of Bellevue, Wash.,
decreased its losses in 2014 as it forged
ahead with new products and contracts.
The company reported a net loss of
$5.3 million, or 22 cents per diluted
share an improvement over the net loss
of $14.1 million, or 60 cents per diluted
share, in 2013.
Revenues for 2014 were $25.7 million, down 8 percent from $28.0 million
in 2013. The company projects revenues
ranging from $26 million to $29 million
in 2015.
The number of displays shipped
decreased compared to 2013, while the

Continued on page 24

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515_BI RapidScan.indd 22

23.7%

The compound annual growth rate of


the ultrafast laser industry from 2014
through 2019 (partly due to extended
applications in the medical sector),
as projected by BCC Research LLC.

BioPhotonics May/June 2015

4/28/15 9:06 AM

RAPIDSCAN

Ten years ago, the cover story of our May 2005


issue highlighted macroscopic fluorescence
imaging, an advanced method for imaging
organisms on both the micro- and macroscale.
Scientists used model organisms including
mice, fruit flies and zebra fish to investigate
human development and disease states relative
to gene expression. The new method allowed
researchers to associate events that occurred at
cellular and molecular levels with events at the
organism level. Previously, multiple imaging
instruments were needed to document large
fluorescence specimens. Macroscopic fluorescence was providing a larger field of view and
the ability to optically zoom into regions of interest. Technology development at
the time was focusing on microscopic imaging, and resolution limits were being
pushed lower and lower. The story notes that, prior to the advance, three groups
of instruments had been used to help piece together the macro- and microscopic
picture of model organisms: stereomicroscopes, classical light (compound) microscopes and fluorescence black boxes/copy stands. The instruments, although
advantageous, were not ideal imaging tools for large-organism research. Leica
Microsystem GmbHs MacroFluo macroscopic fluorescence system, described
in the story, incorporated a 16:1 optical zoom system. This broad range provided
the ability to capture large-field macro pictures of zebra fish and rat brain sections. It also allowed for high-resolution microimaging.

2005

There are numerous advantages to macroscopic fluorescence imaging, but there


are also several limitations of
which researchers should be
aware. Although the system
produces high-depth-of-field
images and digital, extendeddepth composite images, they
are not true 3-D images like
those seen through the eyepieces of a stereomicroscope.
Lon M. Nelson, who authored the
article Getting the Best of Macro- and
Microimaging, was, at the time of
publication, marketing product manager for
stereomicroscopy at Leica Microsystems Inc.

PEOPLE IN THE NEWS

BioPhotonics May/June 2015

515_BI RapidScan.indd 23

Zeiss

Dr. Michael Totzeck, left, and Dr. Michael


Kempe.
Optics and optoelectronics company Carl Zeiss
AG, of Oberkochen, Germany, has designated
two employees as fellows, the highest level of
the companys technical ladder. Both work within
the Corporate Research and Technology unit. Dr.
Michael Kempe, head of the technology area,
received the honor on the basis of his scientific
knowledge regarding microscope development
and biomedical optics; he was a key figure in the
companys development of high-resolution microscopy. Dr. Michael Totzeck, head of industrial
and consumer innovations, was designated a fellow for his expertise in imaging, lithography and
optical metrology. About 500 Zeiss employees
work in the technical ladder, which was created in
2005. Zeiss now has a total of three fellows.

The supervisory
board of Jenoptik
AG of Jena, Germany, has appointed
Hans-Dieter
Schumacher as
chief financial
officer. Schumacher, who has a
background in both
Hans-Dieter Schumacher.
the international
machine engineering and medical engineering industries, most
recently was CFO of Homag Group AG, where he
had been in charge of the finance, information
technology and human resources departments
since 2011. Jenoptik is an integrated optoelectronics group and operates five divisions: Lasers
and Materials Processing, Optical Systems, Industrial Metrology, Traffic Solutions, and Defense
and Civil Systems.

Jenoptik AG

Biolase Inc. of Irvine, Calif., has named David


C. Dreyer its new chief financial officer. Most
recently, he served as CFO at Patient Safety Technologies Inc., which was sold to Stryker Corp. in
2014. Dreyer has hands-on experience in health
care services, biotechnology, pharmaceutical
and medical device research, manufacturing and
marketing. He serves on the boards of directors
for InfuSystem Inc. and Diplomat Pharmacy Inc.
He will report to Jeffrey M. Nugent, president
and CEO of Biolase. The medical device company
supplies lasers for dentistry and medicine,
among other products.

duties will now include responding to technical


application inquiries from sales representatives,
distributors and end users.

Spectronics Corp.

Spectronics Corp.
of Westbury, N.Y.,
a manufacturer of
UV equipment and
fluorescent materials, has announced
promotions and a
new appointment.
The company hired
Daniel Tristan as its
international sales
manager for Latin
America and the
Asia-Pacific region.
Prior to joining
Spectronics, Tristan
served as director
of sales and marketing at Dreyfus
Global Trade LLC.
Limin Chen, who
was promoted to
vice president of
manufacturing and
special projects,
Daniel Tristan. Limin
began his career
Chen. Daniel Chusid.
at Spectronics as a
mechanical product development engineer and
has been the lead engineer for the pipe freezer,
UV EPROM/wafer eraser and grid lamp products
for several years. Daniel Chusid was promoted
to the position of technical sales and regulatory
compliance specialist. His expertise is rooted in
industry best practices, current standards, regulatory requirements and company policies. His

23

4/28/15 9:06 AM

RAPIDSCAN

Continued from page 22

average selling price of displays increased due to changes in product and


customer mix.
President and CEO Andrew G. Sculley
said the company saw a resurgence in
government and nongovernment R&D
contracts toward the end of the year,
yielding $1.7 million in revenue. The
largest new award was the Defense-wide
Manufacturing Science and Technol-

ogy (DMS&T) program, also known as


ManTech.
Meanwhile, development is nearly complete on a new headset prototype featuring
2000 2000-pixel resolution and a field
of view greater than 100.
Recently, eMagin invested in new
direct-patterning equipment for development of ultrahigh-brightness, full-color
OLED microdisplays above 10,000 nits

for avionics and consumer headset applications.


The company integrates its microdisplays with magnifying optics to deliver
near-eye virtual images comparable to
large-screen computer and television
displays in portable, low-power and
lightweight personal displays for military,
industrial, medical and consumer OEM
products.

First Light Imaging


wins EC grant
to develop IR camera

First Light Imaging SAS will use a grant


from the European Commission to finalize development of an ultrafast, low-noise
IR camera for use in astronomy, medical imagery, industry and defense. The
company designs and manufactures hightechnology cameras used in telescopes.
The company is one of six small- and
medium-sized French enterprises to
receive grants totaling 10.79 million
(about $11.43 million) under the Horizon
2020 Instrument SME Phase 2 program.
Previously, the business had been twice
selected for financing by the Ministry of
Higher Education and Research and Public Investment Bank (BPI) of France.
Founded in 2011, First Light Imaging
grew out of the Marseille Astrophysics
Laboratory, Grenoble Institute of Planetology and Astrophysics, and HauteProvence Observatory.

Hamilton Thorne
acquires reproductive
imaging product line

Hamilton Thorne Ltd. has acquired the


Oosight product line from Cambridge
Research & Instrumentation Inc. (CRi)
of Hopkinton, Mass., a subsidiary of
PerkinElmer Inc. Financial terms of the
transaction were not disclosed.
Oosight provides high-contrast, live
images of unfertilized eggs (oocytes),
capturing quantitative data of important
structures using a patented, noninvasive
polarized-light technique developed by
the Marine Biological Laboratory and
commercialized by CRi as LC-PolScope
technology.
David Wolf, Hamilton Thornes president and CEO, said the Oosight system
complements his companys laser products used in fertility and developmental
biology research laboratories.

24

515_BI RapidScan.indd 24

BioPhotonics May/June 2015

4/28/15 9:06 AM

Femtosecond Pulses:
Control Is Key to New Discoveries
From microscopy to micromanipulation, femtosecond pulses are
broadening their reach throughout the photonics research world.
To fully realize their potential, precise pulse control is critical.

BY MARIE FREEBODY
CONTRIBUTING EDITOR

ltrafast lasers have transformed


the worlds of clinical research by
revealing biological mechanisms
in greater detail than previously possible.
Femtosecond lasers are the critically
enabling tools in multiphoton microscopy,
where shorter pulses generally enable
brighter images.
Short pulses deliver high peak intensity
with relatively low pulse energy, making femtosecond pulses the ideal choice
for imaging living tissue while limiting
potential damage. Precise control of the
pulses width, duration, shape, repetition
rate and propagation ensures that desired
results are enhanced and unwanted effects
are minimized.
Beyond imaging, ultrafast pulses are
also finding new applications in microsurgery and micromanipulation, as demonstrated by the growing treatments in eye
surgery, dentistry, and the fabrication
of precise surgical tools and biomedical
equipment, such as stents.
Optogenetics is a particularly hot area
in neuroscience right now where light is
used to activate or silence neurons in

Laser-cut stents embody the precise micromachining made


possible by carefully controlled pulses from femtosecond
lasers. Courtesy of Trumpf GmbH.

BioPhotonics May/June 2015

515BI_Femto Feat.indd 25

25

4/28/15 9:07 AM

Pulse Control

A laser-cut nitinol stent. Courtesy of Trumpf GmbH.

Cold laser material processing of a matchstick


head with the TruMicro Series 5000. Courtesy of
Trumpf GmbH.

the brain of a lab animal. Although many


of these experiments do not need lasers,
multiphoton excitation with femtosecond
lasers is the preferred method when it
comes to investigating the optogenetics
in living animals with in-depth, singleneuron resolution.
While femtosecond lasers and pulse
shapers are relatively advanced and are
most commonly applied in the laboratory,
mastering their control in biological environments proves much more difficult.
Broadband laser systems are develop-

26

515BI_Femto Feat.indd 26

Shaped laser pulses excite molecules to specific excited states. Courtesy of ICFO (The Institute
of Photonic Sciences).

ing rapidly into user-friendly turnkey


system[s]; while also pulse shapers are
moving towards more accessible systems, said Dr. Niek van Hulst, professor of molecular nanophotonics at The
Institute of Photonic Sciences in Barcelona, Spain. Still, the tools are expensive
and specialized, which is an obstacle for
broader applications.
Pulse duration and pulse width
Temporal control is used for obtaining
brighter multiphoton, second-harmonic

and third-harmonic images, which


has improved the imaging of highly
pigmented tissues, such as melanoma
and red blood cells.
A shorter pulse width at the sample
translates directly into brighter images.
This is because the excitation efficiency
in any multiphoton method has a highly
nonlinear dependence on peak intensity
and, hence, peak power.
A technical complication of broadband
short-pulse excitation in microscopy is
dispersion, van Hulst said. The high-

BioPhotonics May/June 2015

4/28/15 9:07 AM

NA [numerical aperture] objective with a


centimeter of glass does disperse the
incident pulse, such that the pulse gets
stretched: The pulse is chirped with
different colors passing at different moments. Correction by pulse compensation
and compression is crucial in multiphoton
microscopy.
Adaptive temporal control is used to
make sure the pulse duration is shortest
at the focal plane, said Marcos Dantus,
founder and CTO of Biophotonic Solutions Inc., and who also is a professor
of chemistry and physics at Michigan
State University. Given that microscope
objectives have very high dispersion
characteristics, temporal shaping can also
make improvements over one order of
magnitude.
The peak intensity of the pulse is
intimately related to the pulse duration.
For ultrashort laser pulses with extremely
high peak intensities, nonlinear effects
come into play that define what kind of
process occurs.
Although shorter pulse widths yield
brighter images, they also increase the

Pulse Control

Two-photon fluorescence, second harmonic and merged images of C. elegans worm muscles; before
(top) and after (bottom) cutting with a laser at position of the arrow. Scale bar is 10 m. Courtesy of
ICFO (The Institute of Photonic Sciences). Reference: S. Santos et al (2013). PLOS ONE, 8(3), p. e58600.
doi: 10.1371/journal.pone.0058600.g006.

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Multimodal microscopy image of an adult Drosophila melanogaster unstained eye. Cross section by
the imaging plane through the eye elements shows their contents with high optical resolution. Excitation
source: sub-50-fs pulses from a Yb fiber laser oscillator with spectrum centered at 1060 nm, built at the
Dantus Research Group using adaptive pulse compression. Scale bar is 20 m. Courtesy of Ilyas Saytashev, Dantus Research Group, Michigan State University.

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515BI_Femto Feat.indd 28

possibility of photobleaching or otherwise


damaging the sample the shortest pulse
widths are not always optimum for every
application.
Given a certain bandwidth and pulse
width produced by the laser, delivered
pulse width can be adjusted by introducing the appropriate level of chirp. In
some applications, those having a lot of
downstream optics, particularly transmissive components, such as acousto-optic
modulators (AOMs), can cause undesirable stretching of the pulse through the
material. This is known as positive group
velocity dispersion (GVD).
So the laser pulses are often prechirped with negative GVD before they
leave the laser head, in order to offset this
downstream stretching and minimize
pulse width at the actual sample or experiment, said Marco Arrigoni, director of
marketing for scientific lasers at Coherent
Inc. in Santa Clara, Calif. Providing adjustable prechirp enables the researcher to
experiment for themselves, and to empirically find the optimum pulse width based
on image brightness and any observed
trade-off in photodamage.
In some of Coherents products, prechirp can be set by a user either by adjusting a pair of gratings in the laser head (as
in the companys Fidelity ytterbium fiber
laser) or via a software program that automatically adjusts spacing and positioning
of a set of prisms, according to the desired
wavelength and prechirp value (as in
Coherents Chameleon product line).
In our Vitara lasers, we use an external chirped mirror-based accessory to
deliver an adjustable amount of prechirp,
said Arrigoni. Here multiple bounces of
the output beam on the mirrors result in
the desired amount of prechirp, adjusted
by selecting an appropriate number of
bounces of the fixed GVD mirrors.
Pulse shape
Advances in pulse shaping and coherent control of broadband pulses have been
key to some of the recent achievements
in ultrafast spectroscopy on biomolecular
complexes.
For nonlinear multiphoton microscopy, peak power is important; thus
pulses need to be Fourier-limited in the
focus, which requires optimized dispersion control, van Hulst said. For STED
[stimulated emission depletion], control
of both spatial and temporal overlap is
mandatory.

BioPhotonics May/June 2015

4/28/15 9:07 AM

Adaptive optics can be used to enhance


peak intensity by shaping the spatial,
temporal and spectral characteristics of
the pulse, revealing never-before-seen
images, as well as achieving ultimate
precision manufacturing.
With femtosecond pulses, we enter the
next order of magnitude in terms of precision at microprocessing, said Dr. Max
Kahmann, product manager of TruMicro
at Trumpf GmbH in Ditzingen, Germany.
Especially processes where any kind of
thermal influence is extremely critical,
femtosecond pulses open the field. Some
materials (e.g., nitinol) are very sensitive
in terms of achieved quality and precision
regarding the pulse duration.
While peak intensity depends on the
pulse shape, results are further complicated by the type of material in which the
beam is incident, as well as by the application. The results may also depend on
how steep the intensity distribution over
time is, especially in the beginning of the
pulse, Kahmann said.
Proper spatial control allows users to
retrieve ultrafast dynamics of biological systems, as well as to enable better
imaging through scattering media a
technique that has, for example, greatly
improved retinal imaging. However,
leaked power unwanted energy applied
outside of the pulses poses a significant
challenge. This can be a problem, and it is
important to suppress.

Two-photon excited fluorescence (TPEF) microscopy image of a mouse kidney section, stained with
Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidins and DAPI. Excitation source: sub-10-fs pulses from a
Ti:sapphire laser oscillator and adaptive pulse compression. TPEF signals were detected in epi-direction by
a spectrally resolved multichannel photomultiplier tube. Scale bar is 20 m. Courtesy of Ilyas Saytashev,
Dantus Research Group, Michigan State University.

Wavelength
While wavelength is not necessarily a
parameter that should be controlled, it is
important to choose the correct wavelength according to the application.
The wavelength gives the Rayleigh
length, as well as the smallest possible
beam diameter size. Further, the absorption behavior might depend on the wavelength for different materials, Kahmann
said.
While continuous-wave sources lase at
a single wavelength, modern pulsed lasers
are generally mode-locked, meaning they
lase over a broader band of colors with
a range of wavelengths simultaneously
circling the laser cavity. The modes of
all these colors are in phase (i.e., modelocked) and, as a result, in the time
domain the laser is pulsed.
For example, the titanium-sapphire
laser operates within the 10- to 20-nm
bandwidth with around 100-fs pulses.
Modern broadband lasers operate within

BioPhotonics May/June 2015

515BI_Femto Feat.indd 29

29

4/28/15 9:07 AM

Multimodal microscopy image of an unstained, undissected Drosophila melanogaster RFP mutant larva.
TPEF from fat cells pseudocolored in yellow; second-harmonic generation (SHG) from fibrous tissue signal
shown in blue. Excitation source: sub-10-fs pulses from a Ti:sapphire laser oscillator and adaptive pulse
compression. Scale bar is 20 m. Courtesy of Ilyas Saytashev, Dantus Research Group, Michigan State
University.

30

515BI_Femto Feat.indd 30

the 100- to 200-nm bandwidth with


around 10-fs pulses.
First, such pulsed lasers typically
have ~105 times higher peak power for the
same average (CW) [continuous wave]
power, which is important for nonlinear
applications: multiphoton microscopy,
optical sectioning, etc., van Hulst said.
Second, the short pulses allow [users]
to study dynamic processes on [the] femtosecond-nanosecond timescale: energy
transfer, quenching, lifetime. Finally, the
broad bandwidth allows coherent control
of the excitation and thus certain energy
pathways.
When it comes to imaging, spectral
control is used for functional imaging and
enhancing contrast; it currently is being
used to determine the margins of cancerous tumors.
Adaptive spectral control is used with
broadband femtosecond laser pulses to
selectively excite some chromophores but
not others, Dantus said. Spectral control
is used to gain chemical selectivity via
multiphoton transitions or via vibrational
excitation.

BioPhotonics May/June 2015

4/28/15 9:07 AM

Pulse Control

pulse energy (without an external modulator), Kahmann said.

Other controllable laser


parameters
Beam propagation Using diffractive
optical elements (DOEs), the intensity
distribution in the focus plane, as well as
along the beam axis, can be fitted to a specific application (e.g., creating multispot
in the focus plane).
Repetition rate The possibility of
using only every second or third pulse
can be important for cutting materials.
Once the cutting edge radius gets smaller,
the scanning speed of the laser has to be
reduced to avoid overlap from one pulse
to the next, which could cause adverse
thermal heating effects.
While repetition rate and pulse energy
can be controlled from pulse to pulse by
external modulators, pulse duration and
pulse shape can only stay constant if the
right laser technology is selected (e.g.,
fiber-amplified lasers, disk-amplified
lasers).
Other solid-state laser techniques
(e.g., Q-switched slab lasers) allow [users]
to adjust the repetition rate without an
external modulator, but a change of the

An example of the benefits of next-generation


dual-wavelength excitation lasers. Pancreas tissue
was imaged using SHG at 1040 nm to show
collagen surfaces and a Raichu-Rac biosensor
expressed as a FRET probe, revealing cell crypts
at 830 nm. Courtesy of Ewan McGhee and Kurt
Anderson of Beatson Institute, Glasgow, Scotland.

repetition rate changes the pulse duration


and the pulse shape and thus the process
parameters, like the intensity, and it
further does not enable you to control the

Sophisticated pulse shaping


of the future
The number of adjustable parameters,
combined with the huge number of
possible materials and applications for
which ultrafast lasers may be used, leaves
researchers with a mind-blowing number
of options to explore. Currently, they
are only at the beginning stages of this
pursuit.
Highly sophisticated forms of pulse
control, such as spectral phase and amplitude shaping, are being reported in a number of scientific journals. According to
Coherents Arrigoni, though, this makes
more sense in conjunction with very
short, broadband pulses (i.e., <20 fs).
At this stage, most biological applications of femtosecond lasers still rely on
pulses longer than 50 femtoseconds, so
sophisticated pulse shaping is not widely
adopted as of now, he said.
marie.freebody@photonics.com

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BioPhotonics May/June 2015

515BI_Femto Feat.indd 31

31

4/28/15 9:07 AM

Nanodiamonds
Shine New Light on Bio Applications
Defect complexes in nanodiamond particles, which consist
of vacancies trapped by nitrogen atoms, form different color
centers depending on the nitrogen state present in the diamond.
New research into these color centers indicates that nanodiamond particles are ideal for molecular-imaging and cell-labeling
bioprobe applications.

BY DR. OLGA SHENDEROVA


ADMAS NANOTECHNOLOGIES INC.

anodiamond (ND) particles


have emerged recently as a key
platform for many developments
in nanoscience and nanotechnology due
to their outstanding mechanical performance, biocompatibility and unique optical properties a combination of assets
not often met in the nanoworld. Their
superhardness and exceptional chemical
resistance motivate the application of
nanodiamonds in novel, wear-resistant
polymer and metal coatings and as additives in lubricants, where they dramatically decrease friction and wear. The high

refractive index and biocompatibility of


NDs also make them appealing for use in
health care products, such as sunscreens.1
The most exciting area of research has
been the unique photoluminescent properties of crystallographic defects in the
diamond lattice, which are often referred
to as color centers.
Most color centers are based on complexes of vacancies in the diamond lattice
in combination with impurity atoms,
which can include nitrogen (N), silicon
(Si) and certain types of transition metals.
The unprecedented photostability of the

color centers, biocompatibility and ease


of conjugation with biomolecules have encouraged applications using NDs containing color centers as fluorescent biolabels
or biomarkers in life sciences.2 Additionally, optically detectable spin properties
of selected color centers particularly the
nitrogen vacancy center have stimulated
developments in high-resolution magnetometers, atomic-sized sensors for electric
fields, and quantum measurements of temperature gradients within living cells.3, 4
Nanodiamonds are synthesized by
detonation of carbon-containing explo-

Figure 1. (a) A high-resolution transmission electron microscope image of a ~6-nm detonation nanodiamond (ND) particle, demonstrating the perfect
crystallographic structure of the diamond nanoparticle core.5 Image courtesy of O. Shenderova et al, reprinted with permission. (b) A schematic model
illustrating the structure of a single ~5-nm ND particle after oxidative purification. Structural features of the ND surface include a layer of functional groups
(oxygen atoms and hydrocarbon chains are shown in red and green, correspondingly) and patches of graphitic sp2 carbon (shown in black). Image courtesy
of Vadym Mochalin, Drexel University.

32

515BI_Nanodiamonds Feat.indd 32

BioPhotonics May/June 2015

4/28/15 9:09 AM

Figure 2. (a) Image of fluorescent diamond particles containing nitrogen vacancy (NV) centers captured in an inverted fluorescence microscope. Inset shows the
crystallographic structure of the NV center. (b) Photoluminescence emission spectra of 100-nm ND particles containing NV centers dispersed in deionized water at
a concentration of 1 mg/ml. Excitation wavelength is 532 nm. Image courtesy of O. Shenderova.

sives (called detonation nanodiamonds,


as shown in Fig. 1)5 or by fragmentation
of micron-sized diamond particles produced by phase transformation of carbon
precursors under high pressure and high
temperature.1 Nitrogen is the most common impurity in diamond and typically is
incorporated into the crystal lattice as isolated, substitutional nitrogen atoms or two
nearest-neighbor substitutional nitrogen
atoms, among numerous other nitrogencontaining defects. Complexes consisting

of vacancies trapped by nitrogen atoms


form different color centers, depending
on the type of nitrogen state present in
the diamond. The nitrogen vacancy (NV)
defect (responsible for diamonds red and
near-infrared fluorescence) and nitrogen
vacancy nitrogen (NVN) color centers
(H3 centers) with bright-green fluorescence are the optically active defects that
have received the most consideration.24, 6
The NV center is a defect formed within
the diamond by one substitutional nitro-

gen atom and a vacancy (Fig. 2), while an


NVN center consists of a pair of nitrogen
atoms adjacent to a vacancy (Fig. 3).
Vacancies can be produced by irradiating
the diamond with high-energy particles.
High-temperature annealing (greater than
about 700 C) causes diffusion of vacancies and the formation of complexes with
nitrogen atoms.6
The optical properties of the NV center
are well suited for bioimaging applications, with optical excitation ranging

Figure 3. (a) Image of fluorescent diamond particles containing NVN centers captured in an inverted fluorescence microscope. Inset shows the crystallographic
structure of the NVN center. (b) Photoluminescence emission spectra of 70-nm ND particles containing NVN centers dispersed in deionized water at a concentration of 1 mg/ml. Excitation wavelength is 442 nm. Peak at 488 nm corresponds to Raman shift of diamond. Image courtesy of O. Shenderova.

BioPhotonics May/June 2015

515BI_Nanodiamonds Feat.indd 33

33

4/28/15 9:09 AM

Nanodiamonds for Biology

from 490 to 560 nm and emission in the


red/near-infrared ranging from 635 to
800 nm (Fig. 2a), away from most autofluorescent cell components. Emission
occurs in a spectral window of low absorption, which is attractive for biological labeling due to greater penetration of
light in surrounding tissue. The spectra
of NV centers exhibit a zero-phonon line
at 638 nm for the negatively charged
NV defect and a zero-phonon line at 575
nm for the neutral state (Fig. 2b). The
intensity of the luminescence from NDs
containing NV centers depends on the
quantity of NV centers within a particle.
For example, when compared side by side
under identical measurement conditions
using total internal reflection fluorescence microscopy, 100-nm NDs may
have a photoluminescence brightness
exceeding the brightness of the ATTO
532 dye by more than an order of magnitude. The H3 center emits green fluorescence with a maximum intensity around
530 nm when excited by blue light
(Fig. 3). A remarkable feature of NV and
NVN centers is that they do not photo-

bleach or blink, even when subjected to


continuous, high-energy excitation conditions.4 Their unprecedented photostability makes them superior to conventional
chromophores.
The negatively charged NV center in
nanodiamond particles also has emerged
as an important element within quantum
sensing.3, 4 Since the spin state of the
negatively charged NV center can be
optically detected and the state of the spin
is sensitive to the surrounding magnetic
field, an ND particle containing a single
negatively charged NV center can be
used as an ultrasensitive magnetometer
working at ambient conditions. Besides
magnetic fields, negatively charged NV
centers demonstrate high sensitivity to
electric fields, temperature and strain.3, 4
Depending on the intended applications,
ND particles containing either a large
number of color centers per particle, or
just one negatively charged NV center
(one electron spin) per particle, are
required. Using ND particles containing
a large number of color centers is suitable
for applications like biolabels with bright

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fluorescence. Using ND particles containing only one negatively charged NV


center best suits nanosensor applications.
Currently, commercially available NDs,
ranging from 50 to 100 nm in size, provide bright fluorescence, while particles
ranging from 10 to 20 nm in size typically
contain one negatively charged NV center
per particle (Fig. 4).
Since color centers are embedded within the diamond matrix, their fluorescence
properties are not affected by surface
modification when the centers are located
at a distance more than a few nanometers
from the surface. Although the surface of
bulk diamond is considered chemically
inert, nanodiamonds typically contain
nu-merous oxygen-containing surfacefunctional groups that are introduced
during their purification based upon the
use of strong oxidizers.7 Nanodiamonds
also can be purified using hydrogen to
etch sp2 carbon. Due to multiple methods
of surface functionality and, consequently, different surface moieties, nanodiamonds can be readily incorporated into
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BioPhotonics May/June 2015

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Nanodiamonds for Biology

biological entities. Such examples include


proteins, enzymes, hormones, antigens,
DNA or drugs attached via both electrostatic and covalent interactions. A
nanodiamonds surface is amenable to
derivatization with a variety of organic
functional groups for subsequent linkage with bioactive molecules, making
it suitable for use within diagnostic and
therapeutic applications.2, 7
Nanodiamond particles serve as a platform for several imaging capabilities, as
is summarized in Fig. 5. Besides the wellknown fluorescence properties of NDs,
characteristic Raman signals of pristine
NDs were used for the detection and
imaging of NDs in biological specimens.
Additionally, NDs can be externally
labeled with molecular contrast agents
for MRI or positron emission tomography.2 The first reported use of fluorescent
nanodiamonds (FNDs) containing color
centers for in vitro biological labeling
appeared in 2005,8 demonstrating that
FNDs containing NV centers can be
spontaneously internalized by cells while
having very low toxicity. The brightness

and stability of the fluorescence of these


diamonds make them suitable for singleparticle tracking within cells. Improved
image contrast of nanodiamonds in cells
also can be achieved through time-gated
detection of the NV center fluorescence,
taking advantage of the NDs long-lived
fluorescence lifetime up to ~20 ns
which is substantially longer than the
~3-ns lifetime for cell and tissue autofluorescence.2
The perfect photostability of FNDs
enabled superresolution imaging by stimulated emission depletion (STED).9 Highresolution 3-D imaging can be readily
achieved in real time. Using STED microscopy, single FND particles (~30 nm) were
distinguished in cells with subdiffraction
resolution of approximately 40 nm.9
Multiphoton excitation imaging is another powerful tool that allows imaging
of living-organism tissues with deeper
penetration depths. It excites a fluorophore at the focal spot and thereby reduces
cell autofluorescence. Additionally, the
multiphoton microscopy provides better
image contrast and lower photodamage

to cells. The presence of single FND particles (~40 nm) in cells was detected using
a femtosecond infrared laser.10
The optically detected magnetic resonance capability of negatively charged
NV centers was used to improve the
image contrast of FNDs in vitro and
in vivo and to overcome the problem of
autofluorescence caused by endogenous
molecules. In wide-field fluorescence
imaging in vivo, alternating microwave
irradiation modulated only the fluorescence intensity of the negatively charged
NV center, while background fluorescence
remained constant. Image processing
effectively removed background autofluorescence signals and significantly
improved image contrast.11 In an alternative approach, a modulated magnetic field
was used to achieve background-free,
wide-field imaging of negatively charged
NV centers in NDs, which were located
in sentinel lymph nodes.12 The image
contrast improved by nearly two orders
of magnitude.
Certain color centers in NDs possess
cathodoluminescence and remain stable

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4/28/15 9:09 AM

Nanodiamonds for Biology

Figure 4. (a) Volumetric particle size distribution for NDs with an average particle size of 10 nm and containing an average of one negatively charged NV
center per particle and (b) NDs with an average particle size of 100 nm and containing ~900 NV centers per particle. Image courtesy of O. Shenderova.

during prolonged electron beam exposure,


providing valuable labels for bioimaging
based on correlative light and electron
microscopy (CLEM). Color images of
green and red FNDs within living HeLa
cells were obtained using a CLEM microscope,13 revealing structural details with
excellent spatial resolution. This demon-

Bioimaging with Nanodiamonds


Light Scattering
Rayleigh Scattering
Raman Mapping
External Labels
Fluorescence Imaging
Magnetic Resonance Imaging
Positron Emission Imaging
Crystallographic Defects
Photoluminescence Imaging
PL Lifetime Gated Imaging
Cathodoluminescence Imaging
Photoacoustic Imaging
Imaging Based on Optically
Detected Magnetic Resonance
Figure 5. Nanodiamond-based bioimaging
capabilities. Image courtesy of O. Shenderova.

36

515BI_Nanodiamonds Feat.indd 36

strates that FNDs may advance clinical


diagnostic sensitivity.
In summary, NDs containing color
centers possess bright fluorescence without photobleaching and blinking which,
in combination with their biocompatibility and facile biofunctionalization,
make them ideal bioprobes for molecular
imaging and cell labeling. The unique
combination of spin and photoluminescent properties present in certain types of
color centers in NDs opens new perspectives for advancing the discovery of new
bioimaging and sensing methods.
Meet the author

Dr. Olga Shenderova is president of Admas


Nanotechnologies Inc. in Raleigh, N.C.; email:
oshenderova@adamasnano.com.

References

1. V.N. Mochalin et al (2012). The properties


and applications of nanodiamond. Nat
Nanotechnol, Vol. 7, pp. 11-23.
2. V. Vaijayanthimala et al (2014). Nanodiamond-mediated drug delivery and imaging:
challenges and opportunities. Expert Opin
Drug Del, Vol. 12, pp. 1-15.
3. R. Schirhagl et al (2014). Nitrogen-vacancy
centers in diamond: Nanoscale sensors for
physics and biology. Annu Rev Phys Chem,
Vol. 65, pp. 83-105.
4. M. Doherty et al (2013). The nitrogenvacancy colour centre in diamond. Phys
Rep, Vol. 528, pp. 1-45.
5. O. Shenderova et al (2011). Surface
chemistry and properties of ozone-purified
nanodiamonds. J Phys Chem C, Vol. 115,
pp. 9827-9837.

6. Y.R. Chang et al (2008). Mass production


and dynamic imaging of fluorescent nanodiamonds. Nat Nanotechnol, Vol. 3, p. 284.
7. A. Krueger and D. Lang (2012). Functionality is key: Recent progress in the surface
modification of nanodiamond. Adv Funct
Mater, Vol. 22, pp. 890-906.
8. S.J. Yu et al (2005). Bright fluorescent
nanodiamond: No photobleaching and low
cytotoxicity. J Am Chem Soc, Vol. 127, pp.
17604-17605.
9. B.M. Chang et al (2013). Highly fluorescent nanodiamonds protein-functionalized
for cell labeling and targeting. Adv Funct
Mater, Vol. 23, pp. 5737-5745.
10. Y. Hui et al (2010). Two-photon fluorescence correlation spectroscopy of lipidencapsulated fluorescent nanodiamonds in
living cells. Opt Express, Vol. 18, p. 589.
11. R. Igarashi et al (2012). Real-time background-free selective imaging of fluorescent
nanodiamonds in vivo. Nano Lett, Vol. 12,
pp. 5726-5732.
12. S.K. Sarkar et al (2014). Wide-field in vivo
background-free imaging by selective
magnetic modulation of nanodiamond
fluorescence. Biomed Opt Express, Vol. 5,
pp. 1190-1202.
13. Y. Nawa et al (2014). Multicolor imaging
of fluorescent nanodiamonds in living HeLa
cells using direct electron-beam excitation.
Chem Phys Phys Chem, Vol. 15, pp. 721-726.

Acknowledgment

The author wishes to acknowledge support


from the National Institutes of Health under
SBIR Contract HHSN268201300030C, NHLBI
COAC Services Branch, RFP No. PHS 2013-1,
Topic 80, Fluorescent Nanodiamonds for In
Vitro and In Vivo Biological Imaging.

BioPhotonics May/June 2015

4/28/15 9:09 AM

Spectroscopy Aids Disease Detection,


Faces Obstacles
Spectroscopy advances are steadily reducing disease-detection
time frames. These new methods also hone the accuracy of
disease identification and provide for more timely treatment
protocols. As with anything groundbreaking, though, roadblocks
inevitably arise. As with the diseases themselves, time is precious
when resolving these hurdles. Sometimes, though, theres just no
time to spare.
BY RODD M. PEDROTTI
EDITOR

uring a disease is no easy task.


Virtually every disease requires its
own unique treatment. However,
many diseases have no existing panacea
despite the time, money and manpower
invested in researching them. And, for
those that do have treatment options
available either to destroy or to curb
the disease, success may come down to
timing. How long has the disease been advancing, at what stage does it exist when
its first detected and identified by medical experts, and in what stage of approval
are the possible treatment protocols?
Spectroscopy advances most notably
those that have come to light in the last
few years are helping to reduce the
amount of time needed to detect diseases
and thereby allow for more timely and
viable treatment options. Additionally,
these new methods are increasing the
accuracy of disease identification.
As with anything new, growing pains
exist as novel developments inch their
respective ways into new applications and
procedures. In some cases, theyre halted
from accessing a clinical setting altogether
due to stringent FDA regulations and big
business concerns.
Some lifesaving technologies and
those who invented them are still
awaiting FDA approval and the financial
investments needed to transition to clinical use. Others, though, are being used in
clinical settings right now.
Ahead of the curve; hampered
by corporate concerns
In November 2014, BioPhotonics
contributing editor Gary Boas checked in
with Boston Universitys Dr. Irving Bigio,

BioPhotonics May/June 2015

515_SpectroscopyFeat.indd 37

who has been developing a technique


called elastic scattering spectroscopy
(ESS) since the 1990s (UV-VIS Spectroscopy in the Clinic Whats Stopping
It? www.photonics.com/A56917). ESS
provides real-time, semiquantitative
point-source measurements of microstructural changes and, because it can be
miniaturized, can be easily incorporated
within endoscopic biopsy tools.
Bigio and his colleagues already have
successfully demonstrated ESS for use
in a range of applications suitable to the
upper gastrointestinal (GI) tract and the
colon. Examples include determining
the presence of dysplasia in Barretts

esophagus, distinguishing benign from


adenomatous polyps in the colon, and
helping to differentiate Crohns disease
from ulcerative colitis. ESS also showed
its potential for detecting cancer in thyroid nodules and for real-time pathology
to guide breast surgery.
In the June 2014 issue of Inflammatory
Bowel Diseases, Bigios team reported
a study demonstrating the use of ESS
as an optical marker of inflammatory
bowel disease activity. This established
its potential ability to discriminate
between ulcerative colitis and Crohns
disease.
As part of the study, they devised a
diagnostic algorithm based on a pattern
recognition scheme to classify measured
spectra. The algorithm allowed them to
determine whether any specific diagnosis
obtained from an optical biopsy has a
high probability of being correct. This
increases the reliability of those measurements that the instrument does not determine. And, for the 10 to 15 percent of
cases where the algorithm indicates that
some uncertainty still exists, the doctor
can collect additional measurements or
perform a physical biopsy.

To facilitate clinical adoption of the technology, Bigios team developed elastic scattering spectroscopy
(ESS) devices that easily integrate into endoscopic tools. An example is the incorporation of optical
fibers into the biopsy forceps that doctors use to remove growths. Courtesy of Irving Bigio, Boston
University.

37

4/29/15 9:26 AM

Spectroscopy Advances

As it stands right now, this technology


has been fully mature for more than half
a year. Certainly, it could benefit a range
of clinical applications. To date, though, it
has yet to be brought into the clinic.
One of the two primary obstacles originally faced by Bigio and his team was the
reticence of clinicians, many of whom
are wary of adopting new technologies
especially those that might present a steep
learning curve. To address this, Bigio and
colleagues designed devices that integrated seamlessly into the clinicians work
flows. One example was incorporating
optical fibers into the biopsy forceps used
to remove polyps during endoscopies.
Clinicians tested the new design and were
very enthusiastic about it.
Problem solved? Not quite. The team
now faced a more formidable obstacle: Attracting funding from an investment community that is increasingly averse to risk.
Bigios group already had performed
large-scale clinical studies with ESS. Furthermore, these studies met performance
standards established by the American
Gastroenterological Association for opti-

The 3-in-1 spectroscopy probe. Courtesy of James Tunnell and Austin May, University of Texas at Austin.

cal biopsy tools for colonoscopy or upper


GI endoscopy: 90 percent sensitivity and
90 percent negative predictive value.
Regardless, investors were (and still
are) reluctant to embrace it. Theres
no venture in venture capital anymore,
Bigio said. He indicated that groups will

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38

515_SpectroscopyFeat.indd 38

say they are interested in a new technology but only after it has received FDA
approval. Simply put, they dont want to
be involved in funding FDA trials.
Bigio confirmed that the latter impediment still exits. The one (and only
obstacle that is impeding commercial
development of our technology is the lack
of investors willing to risk investing
in a medical technology that may take
a couple of years to generate profits.
They seem to want technologies that are
already FDA-approved, he stated. Bigio
went on to say that some potential investors currently are studying the business
opportunity, but, as of this point in time,
no decisions have yet been made.
Investor restraint has become a
problem throughout the medical instrumentation field. The current trend is that
when larger companies want new products, they simply buy existing companies.
But, as Bigio indicated in June 2014, this
sort of development strategy is shortsighted. Companies will buy other, smaller
companies for hundreds of millions of
dollars, but they wont spend $5 million
to fund a new idea that will open a whole
new product area.
In short, more than half a year later,
Bigio, his research colleagues and the
technology he helped spearhead all are
in the same anticipatory situation. The
technology exists. In fact, it performs to
standards set by the American Gastroenterological Association. However, in the
clinical world where its needed the
device is only as real as its existence in
each respective place of treatment: It does
not exist.

BioPhotonics May/June 2015

4/28/15 9:16 AM

Spectroscopy Advances

Cancer detection so good,


biopsies are less prevalent
Not all lifesaving technologies face the
quandary detailed above. One noninvasive
skin cancer detection device is a prime
example.
Skin cancer is the most common form
of cancer and, according to the American
Cancer Society (ACS), it accounts for
almost 50 percent of all cancers in the
U.S. In fact, the most recent ACS data indicates that 3.5 million cases of basal and
squamous cell skin cancer are diagnosed
yearly in this country, with melanoma
expected to account for more than 73,000
cases of skin cancer this year alone.
About eight months ago, a new device
hit the medical world that offers doctors
the potential to see a clearer picture of
cancerous skin lesions and thereby reduce
unnecessary biopsies. A team at the
University of Texas at Austin developed
a probe that combines three distinct
spectroscopy methods to measure the
properties of skin tissue: Raman spectroscopy, diffuse reflectance spectroscopy and
laser-induced fluorescence spectroscopy.
The 3-in-1 device offers a fast, comprehensive and noninvasive examination of
melanoma and other skin cancer lesions.
According to the researchers, the
probe reveals information invisible to the
human eye, offering a better screening
tool for cancer that can eliminate many
negative biopsies. They noted that as normal skin becomes cancerous, cell nuclei
enlarge, top layers of skin thicken, and
skin cells increase their consumption of
oxygen and become disorganized. These
changes alter the way light interacts with
the tissue.
Diffuse optical spectroscopy can be
sensitive to absorption by proteins such
as hemoglobin, while Raman spectroscopy is sensitive to vibrational modes of
chemical bonds, including those found in
connective tissues, lipids and cell nuclei.
Currently, as per the researchers, the
only definitive way to diagnose skin
cancer is to perform a biopsy. However,
determining which lesions to biopsy can
be an imprecise process. For every case
of skin cancer detected, approximately
25 negative biopsies are performed.
The researchers noted that this can be
expensive and sometimes unnecessarily
time-consuming.
Skin is a natural organ to apply imaging and spectroscopy devices to because
of its easy access, said Dr. James Tun-

BioPhotonics May/June 2015

515_SpectroscopyFeat.indd 39

device took to the winners podium at the


annual South by Southwest Interactive Innovation Awards, claiming victory in the
SciFi No Longer category. The event
took place in Austin, Texas, on March 17,
2015.

nell, an associate professor of biomedical


engineering at the University of Texas at
Austin. He went on to say, This probe
that is able to combine all three spectral
modalities is the next critical step to
translating spectroscopic technology.
Dr. Tunnell told BioPhotonics about
progress updates, stating, We are
planning clinical studies starting this
summer. It should also be noted that this

Meet the author

Rodd M. Pedrotti is editor of BioPhotonics;


email: rodd.pedrotti@photonics.com

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Single-Molecule Localization
Microscopy with sCMOS Cameras
EMCCD-based cameras with high quantum efficiencies and
low readout noise characteristics traditionally have been the preferred technology for SMLM superresolution imaging. However,
with tailored localization algorithms, sCMOS cameras have
become a notable alternative for superresolution microscopy.
BY RUISHENG LIN, ALEX CLOWSLEY,
ISURU JAYASINGHE AND CHRISTIAN SOELLER,
BIOMEDICAL PHYSICS, UNIVERSITY OF EXETER

he ability of optical superresolution


microscopy to circumvent the
so-called diffraction limit has
revolutionized the use of fluorescence
microscopy to study subcellular and
molecular-scale biological processes.
Single-molecule localization microscopy
(SMLM) also commonly referred to as
PALM, STORM and dSTORM, among
other acronyms overcomes the resolution limit by measuring the position of
large numbers of marker molecules and
can achieve effective lateral detail resolution in excess of 20 nm14 in addition to
similar improvements in axial resolution.
Typically, to reconstruct a single 2-D
or 3-D image, several thousand camera
frames have to be acquired and analyzed.5

Preferred camera technologies combine


the ability to sustain high frame rates with
the capability to detect the comparatively
low light levels associated with singlemolecule fluorescence emission. Such
cameras should enable the rapid image
generation that often is required for livecell recordings.
Traditionally, electron multiplying
CCDs (EMCCDs) have served as the
dominant technology within this realm.
EMCCDs are widely used in superresolution imaging because the best backthinned EMCCDs combine high quantum
efficiency with very low readout noise.
In the consumer market for digital
cameras, CMOS-based sensors effectively
have replaced CCD sensors due to lower

manufacturing costs and considerable improvements in CMOS sensor technology.


For example, CMOS-based DSLR cameras are now widely used by hobbyists
for light-sensitive tasks like astrophotography. Research-grade CMOS cameras,
sometimes branded as scientific CMOS
or sCMOS cameras, have become available from several manufacturers. These
cameras are capable of recording at high
frame rates (approaching 1000 frames
per second in reasonably sized regions
of interest) and have low readout noise,
peak quantum efficiencies up to about
70 percent, and provide megapixel densities and high dynamic ranges.610 In addition, the cost of CMOS cameras typically
is a fraction of that of EMCCDs, which

Figure 1. (a) Simplified schematic of a custom-built STORM microscope system. Beams from several laser modules are coupled and propagate through a
neutral-density filter wheel. A field stop is used to adjust the area of illumination in the sample. A 4F optical system is inserted with its back focal plane coplanar
with the conjugate plane of the sample plane, and the two mirrors in the system are mounted in kinematic holders, allowing the illumination position and beam
angle to be adjusted. Images are recorded using either an EMCCD or a sCMOS camera. (b) Maps and histograms of the pixel-dependent noise variance
in the full region (2048 2048) of an Andor Zyla 4.2 sCMOS camera. The variance ranges from <1 to 2500 ADU2 (analog-to-digital units squared), indicating
nonuniform noise characteristics of the sensor chip.

40

515BI_SMLM Feat.indd 40

BioPhotonics May/June 2015

4/28/15 9:10 AM

makes CMOS cameras an attractive


alternative for single-molecule-based
superresolution imaging.
One potential drawback of CMOS
cameras is their pixel-dependent sensor
characteristics. Unlike EMCCDs, where
all photoelectrons are converted and
amplified through a common readout
structure, CMOS cameras pixels each
have their own readout and processing
electronics. Therefore key properties like
readout noise, offset and sensitivity of
CMOS cameras are not uniform throughout the entire sensor area.11,12
In SMLM, each raw data frame
contains images of a subset of the whole
population of marker molecules, and a
series of frames is used to collect the positions of a large percentage of the marker
population. In the simplest implementation, the fluorescence-emitting molecules
in a single frame are sparse enough so
that their images do not overlap, allowing
their positions to be measured with high
precision. Localizing marker positions is
typically achieved by fitting a model to
the data to determine the central coordinates of each blink.13,14 An efficient and
comparatively simple approach uses a
Gaussian model for rapid 2-D localization, and more complex models can be
used for full 3-D localization in conjunction with point spread function engineering. Unfortunately, the pixel-dependent
characteristic in CMOS cameras makes
using the conventional algorithms
which assume uniform pixel properties
and work well with EMCCD cameras
problematic, because local pixel property
variations introduce bias and therefore
may not reliably determine the position of
each molecule.1517
The bias resulting from pixel nonuniformities can be eliminated by employing
localization algorithms that explicitly
take into account the camera-specific
pixel properties. These algorithms use
information on the nonuniform noise
characteristics, offsets and sensitivity to
determine an unbiased position estimate.
It has been shown that if CMOS cameras
are provided with suitable algorithms,
these cameras can achieve highly precise
localization and achieve performance
that rivals that of EMCCDs all while
allowing for a larger field of view and fast
frame rates.7
We sought to implement a simple, unbiased algorithm for 2-D position determination in SMLM, the goal of which was to

BioPhotonics May/June 2015

515BI_SMLM Feat.indd 41

Figure 2. Comparison of the localization results from the conventional and new algorithms.
(a) Temporal variance of an image series of fluorescent beads (0.2-m orange-red fluospheres) reveals
the noisy pixels and their presence relative to bead locations, which are seen clearly due to the shot noise
properties of bead signals (see inset). (b) and (c) Localization results from the conventional and new
algorithm. Bias is introduced by the presence of noisy pixels. Note the correlation between the spread
direction in b and the high-variance pixels in the inset in a. The bias is essentially eliminated by our
algorithm c. (d) and (e) Reconstructed images generated from the localization results in b and c. (f)
and (g) The profiles of the green lines in d and e illustrate a 22 percent (measured as full width at half
maximum of 17.0 nm versus 21.7 nm) improvement of the localization uncertainty, resulting in better
localization precision and the absence of bias.

evaluate whether current CMOS cameras


are a viable alternative to EMCCDs,
while still preserving the simplicity of the
software interface that we currently use.
Setup and data acquisition
A simplified schematic of an SMLM
system, such as the one in our laboratory
that uses both an EMCCD and a CMOS
camera, is shown in Figure 1a. Acquisition is controlled using our Pythonbased PYthon Microscopy Environment
(PYME), which can be obtained at the
Bitbucket code repository. PYME offers a
data acquisition module that performs microscope and camera control and is optimized for PALM-/STORM-type superresolution imaging. Customizable acquisition
protocols allow users to predefine a series
of hardware setting changes (e.g., beam
intensity or camera settings) at defined
times during the acquisition process while

providing CPU-parallel, real-time analysis. In short, PYME seamlessly integrates


the Python programming language with a
robust combination of powerful features
for microscopy.
We evaluated the performance of a Zyla
4.2 CMOS camera (10 Tap connection).
An Andor Ixon Ultra EMCCD served as
the reference. Both cameras were controlled via manufacturer software development kits (SDKs), which were used in
custom back-end implementations within
our PYME environment.
Determination
of camera properties
The Zyla CMOS camera offers two
real-time filters that ensure more visually
appealing images by filtering out spurious noise and applying static blemish
correction. During data acquisition for
superresolution imaging, these filters

41

4/28/15 9:10 AM

SMLM Imaging

should be disabled. Offset, dark current


and read noise can be measured by
recording a series of dark frames. Offset
and total temporal noise (i.e., read noise
plus dark current contributions) are measured as the mean and standard deviation
of the frame series, respectively, and are
determined on a pixel-by-pixel basis. The
dark current is proportional to the integration time and can be obtained by employing a linear regression on data obtained
with different camera integration times.
In practice, given the short integration
times used for SMLM, determining the
total temporal noise was sufficient. The
extent of the variability in read noise
between pixels is illustrated in the colorcoded map (Figure 1a), and the frequency
histogram of the measured temporal noise
variance is shown in Figure 1b.
To determine sensitivity variation,
the gain can be measured via a photon
transfer curve on a pixel-by-pixel basis.7
Alternatively, we estimated the photon
response uniformity by imaging a smooth
input signal (e.g., a heavily defocused
fluorescein droplet) followed by 2-D
Gaussian filtering of the offset subtracted
mean signal. In response, the smoothing
filter locally flattens the variation; the
variation then can be obtained by comparing raw offset corrected mean with
smoothed mean. In our experiments, the
variations were relatively small and did
not have a strong influence on the model
fitting process.
Most important for the user, the
determination of pixel-specific camera
maps essentially is a one-off procedure,
and processed camera maps can be stored
for all future SMLM analyses. Some
care may have to be taken during the
characterization procedure to match the
gain settings and other adjustable camera
parameters to those typically used for
SMLM acquisition.
CMOS localization algorithm
In our implementation, all pixel properties are predetermined and available as
camera maps that indicate offset, read
noise and pixel sensitivity, all of which
are handled by a user-defined class in
Python that is provided to the algorithm
during real-time analysis. The raw data
from the camera first is corrected by
removing local pixel offsets and applying
pixel sensitivity corrections; photoelectron conversion also is implemented at
this stage. We fit a 2-D Gaussian model

42

515BI_SMLM Feat.indd 42

Figure 3. Superresolution image of microtubules


in COS-7 cells labeled with Alexa 680. (a) The
entire 30 40 m2 region of interest. The corresponding (b) diffraction-limited and (c) superresolution images of the boxed region. A significant improvement of resolution reveals more detail
of the microtubule structure.

with a background using a weighted


least squares (WLS) algorithm. The
weight given to each pixel is inversely
proportional to the total noise variance
in each pixel, which is estimated as the
combination of read noise (Figure 1b) and
Poisson shot noise, the variance of which
is estimated from the pixel photoelectron
count. As a result, high-noise pixels have
a smaller weight and contribute less to the
localization process. Thresholds can be
chosen to exclude pixels from fitting completely, which is achieved by assigning an
artificially high read noise to them (e.g.,
106), thereby making their contribution
negligible. The parameters that minimize
the WLS fit are recorded and include
center locations, amplitude, image width
and background. The covariance matrix
returned from the least squares fit routine
is used to estimate confidence intervals
for all parameters.
It has been noted that the WLS problem
can become unstable under certain conditions but is well-behaved in the presence
of non-negligible backgrounds, which
we typically find is the case in SMLM
experiments with biomedical samples
and exceptions are detected by our
implementation. Overall, the simplicity
of the algorithm, its rapid convergence
using a Levenberg-Marquardt routine and
its well-understood behavior make it very
suitable for SMLM.

Validation with
subresolution beads
We validated the algorithm using an
image series (~4000 frames) of 100-nm
beads recorded with low-intensity illumination and high frame rate (100 fps).
The average number of photons in each
event was approximately 900 close
to the photon yield of commonly used
fluorochromes. Both the conventional as
well as the new CMOS algorithm were
used to determine bead locations; the
same drift correction was applied in the
data analysis process. The localization
results were rendered into a 2-D position
map by a quad-tree-based adaptive histogram method (Figure 2).18 The comparison clearly shows the distortion in the
cloud of localizations resulting from the
uncorrected bias using the conventional
algorithm. It also shows an estimated
22 percent improvement to the localization precision with the use of the cameraspecific maps.
Qualitative comparison
with EMCCD data
Although the quantum efficiency
of EMCCDs is higher than that of our
CMOS camera (90 percent vs. 5070
percent, respectively), the additional
noise that is generated during the electron
amplification process in the EMCCD effectively reduces the signal-to-noise ratio
by a factor of 2, equivalent to reducing
the effective photon count by half. In
essence, this halves EMCCDs effective
quantum efficiency and makes it comparable to that of the CMOS camera. We
qualitatively compared both the signals
and the locations of the beads by switching between cameras and concluded that
the CMOS data was at least comparable
to data obtained from EMCCDs for localizations. In most cases, though, the data
indicated slight improvements of CMOSbased localizations versus those of our
EMCCD-based localizations.
Imaging biological samples
with a CMOS camera
We now regularly use the Zyla CMOS
camera for SMLM and show some typical
results as illustration. Figure 3 shows a
superresolution image of microtubules
in COS-7 cells labeled with secondary
antibodies conjugated to Alexa 680. Compared with the blurred, diffraction-limited
image, the superresolution image clearly
shows the details of the microtubular

BioPhotonics May/June 2015

4/28/15 9:10 AM

SMLM Imaging

network. Additionally, although the 30


40-m2 region of interest uses only about
5 percent of the cameras active pixel
area (equivalent to 148 148 m2 in the
sample, typically large enough to image
a multitude of cells), it would almost
cover the entire pixel area of an EMCCD
camera. We rarely use the full size of the
detector for SMLM, as we can cover sufficiently large sample areas using a subset
of pixels. In addition, the relatively high
laser intensities required for STORM
limit the size of the illuminated field, and
large ROIs impose high CPU demands
on real-time analysis. Accordingly, most
users will be able to choose a more affordable, USB-based connection option
for a CMOS camera (if available) that is
able to support maximal frame rates for
reasonably sized ROIs. In our experience,
the large pixel count of CMOS sensors
is useful when identifying areas of the
sample for SMLM imaging, as the chip
size allows for covering a comparatively
large field of view typically much larger
than that covered by EMCCDs.
Another example is the superresolu-

tion image of small membrane structures


in equine heart cells, called transverse
tubules, which are shown in Figure 4
and are stained by the membrane marker
WGA Alexa 680. A significant improvement in resolution is achieved and details
of the complex membrane topologies are
revealed in the superresolution image.
In conclusion, modern sCMOS cameras
provide a formidable option for superresolution imaging. The advantages of
fast data acquisition, large field of view
and reasonably high effective quantum efficiency make it an attractive choice. With
tailored algorithms, the apparent disadvantages (compared to those of EMCCDs)
can be fully compensated. In the near

future, we aim to fully integrate cameraspecific algorithms into the PYME suite
so that the microscope operator can
seamlessly benefit from algorithmic

Figure 4. A 10-m-thick section of equine


ventricular cardiac muscle labeled using wheat
germ agglutinin (WGA), conjugated to Alexa
Fluor 680, reveals the cell membrane and T-tubule
network. (a) Superresolution image of a small
area in the tissue sample; note the improved
resolution as compared to the conventional widefield region. (b) The corresponding wide-field and
(c) superresolution images of the boxed region.
The improvement in resolution reveals finer tubules
and the detailed shape of wider tubules.

Lambda DG-4/DG-5 PLUS


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Wavelength Switcher with improved digital servo

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oth-No. 32

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BioPhotonics May/June 2015

515BI_SMLM Feat.indd 43

www.lasos.com

43

4/28/15 9:10 AM

SMLM Imaging

improvements. The combination of ongoing advances in hardware and software


technologies should help to further pave
the way for routine and straightforward
superresolution imaging.
Meet the authors

Dr. Ruisheng Lin is an associate research


fellow at the University of Exeter, UK; email:
r.lin@exeter.ac.uk. Alex Clowsley is a PhD
student at the University of Exeter, UK; email:
ac353@exeter.ac.uk. Dr. Isuru Jayasinghe is
an associate research fellow at the University
of Exeter, UK; email: i.d.jayasinghe@exeter.
ac.uk. Professor Christian Soeller heads the
Laboratory for Biophysics and Biophotonics in
the Biomedical Physics Group at the University of Exeter, UK; email: c.soeller@exeter.
ac.uk.

References

1. E. Betzig et al (2006). Imaging intracellular


fluorescent proteins at nanometer resolution.
Science, Vol. 313, pp. 1642-1645.
2. T.J. Gould et al (2012). Optical nanoscopy:
from acquisition to analysis. Annu Rev
Biomed Eng, Vol. 14, pp. 231-254.
3. S.W. Hell (2009). Microscopy and its focal
switch. Nat Methods, Vol. 6, pp. 24-32.

4. S. van de Linde et al (2012). Live-cell


superresolution imaging with synthetic
fluorophores. Annu Rev Phys Chem,
Vol. 63, pp. 519-540.
5. M.J. Rust et al (2006). Subdiffraction-limit
imaging by stochastic optical reconstruction
microscopy (STORM). Nat Methods,
Vol. 3, pp. 793-795.
6. H.T. Beier and B.L. Ibey (2014). Experimental comparison of the high-speed
imaging performance of an EM-CCD and
sCMOS camera in a dynamic live-cell
imaging test case. PLOS ONE, Vol. 9,
p. e84614.
7. F. Huang et al (2013). Video-rate nanoscopy
using sCMOS camera-specific single-molecule localization algorithms. Nat Methods,
Vol. 10, pp. 653-658.
8. Z.L. Huang et al (2011). Localization-based
superresolution microscopy with an sCMOS
camera. Opt Express, Vol. 19, pp. 1915619168.
9. F. Long et al (2012). Localization-based
superresolution microscopy with an sCMOS
camera part II: experimental methodology
for comparing sCMOS with EMCCD
cameras. Opt Express, Vol. 20, pp. 1774117759.
10. S. Saurabh et al (2012). Evaluation of
sCMOS cameras for detection and localiza-

tion of single Cy5 molecules. Opt Express,


Vol. 20, pp. 7338-7349.
11. H. Deschout et al (2014). Precisely and
accurately localizing single emitters in
fluorescence microscopy. Nat Methods, Vol.
11, pp. 253-266.
12. A. Small and S. Stahlheber (2014). Fluorophore localization algorithms for superresolution microscopy. Nat Methods,
Vol. 11, pp. 267-279.
13. D. Baddeley et al (2011). 4-D superresolution microscopy with conventional fluorophores and single wavelength excitation in
optically thick cells and tissues. PLOS ONE,
Vol. 6, p. e20645.
14. D. Baddeley et al (2009). Light-induced
dark states of organic fluochromes enable
30-nm resolution imaging in standard
media. Biophys J, Vol. 96, pp. L22-24.
15. K.I. Mortensen et al (2010). Optimized
localization analysis for single-molecule
tracking and superresolution microscopy.
Nat Methods, Vol. 7, pp. 377-381.
16. R.J. Ober et al (2004). Localization
accuracy in single-molecule microscopy.
Biophys J, Vol. 86, pp. 1185-1200.
17. C.S. Smith et al (2010). Fast, single-molecule localization that achieves theoretically
minimum uncertainty. Nat Methods, Vol. 7,
pp. 373-375.

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BioPhotonics May/June 2015

4/28/15 9:10 AM

BREAKTHROUGHPRODUCTS
Dynamic-Focus Lenses

The new Dynamic Focus VZM lenses from Edmund Optics Inc. use an internal liquid lens to
seamlessly adjust focus over a 7 range, from 0.65 to 4.6. The lens maintains the zoom
capabilities of standard VZM zoom imaging lenses. Dynamic Focus VZM lenses are suited for
a range of applications, including microscopy. They feature a lockable iris, zoom control and a
rotatable mount for optimal camera orientation. The internal liquid lens is controlled via a 6-pin
Hirose connector and USB port. The lenses include software, allowing them to be programmed
to cycle through focus with a square, sinusoidal or sawtooth waveform, or adjustable frequency
and focus-shift range.
sales@edmundoptics.com

Portable Raman Microscopes

A family of portable Raman microscopes from BioTools


Inc. bridges the gap between microscopy and spectroscopy. The Mobile Raman (pictured) is designed for
examining particles, liquids, contaminants and fibers,
while the -BioRaman is intended for protein analysis.
When coupled with surface-enhanced Raman scatter
(SERS) substrates or capillaries, the instruments can
collect a spectrum in 10 s, requiring only four to eight
spectra to generate a well-defined Raman signature. Both
systems offer multiple sample-handling modalities and a
fast-scanning piezoelectric stage for Raman mapping. The
instruments are suited for the pharmaceutical industry,
research and forensics, doctors offices and infusion clinics, and for food and water safety testing.
rkdukor@btools.com

Linear Focus Actuator

For optical applications requiring both


high precision and high-speed positioning
over a short-to-medium stroke, Equipment
Solutions Inc. provides the LFA-2010 Linear
Focus Actuator. This compact motion control
positioner has a 10-mm range of motion with
<50-nm positioning repeatability and a step
response time of <3 ms. Its high-force, lownoise voice coil motor typically is configured
with an analog position feedback sensor and
can be optionally equipped with a 1-m resolution digital quadrature feedback element.
A tubular architecture allows the 20-mm clear
optical path to go directly through the middle
of the motor. The actuator has applications in
confocal microscopy and biotechnology.
info@equipsolutions.com

Microscope Illuminator

The Lumen 100-LED by Prior Scientific Inc. provides


illumination for a range of fluorescence microscopy applications, with no vibration and minimal
heat production. The system covers wavelengths
throughout the visible spectrum and allows for
precise intensity control in 1 percent increments.
Adapters are available to connect the illuminator to
most upright and inverted microscopes currently
on the market. A combiner allows two LEDs to be
used simultaneously. The unit can be enhanced by
the addition of excitation filters to further optimize
the bandwidth for specific applications. It is suitable for the majority of fluorophores used in
fluorescence applications, and it may also be used in electrophysiology and optogenetics.
jcommesso@prior.com

Microscopy Camera

A digital CCD color camera by Leica Microsystems GmbH is


suited to fluorescence and bright-field microscopy applications. The DFC7000 T has a highly sensitive quad-tab sensor
and is cooled. Users can acquire bright-field images due to
the high camera resolution and color fidelity. Meanwhile, the
cameras high signal-to-noise ratio and sensitivity allow for
crisp, multicolor fluorescence imaging even in the near-IR.
This dual functionality means users do not need to switch
cameras during experiments. The camera can be integrated
into the microscope environment using the companys
imaging software. The Leica DFC7000 T is based on the Sony
ICX674AQG CCD sensor with Exview HAD II technology,
featuring 2.8 megapixels.
news@leica-microsystems.com

Cryogenic Optical Measurement

The updated Cryostation C2 from Montana Instruments Corp. is a 3.2 to 350 K optical measurement platform. A redesigned sample-support structure improves stiffness for better positional
stability and increased thermal performance. The system has evolved to ensure low vibration
(<5 nm) across various experimental setups and in demanding applications. Other design
improvements reduce energy transfer to the table. The new shape improves access to the
experiment from all directions, and an integrated light makes it easier to set up the experiment
and work with the sample. Updates in firmware and electronics result in more intuitive temperature control and optimized wide-range temperature stability of 5 mK at base temperature.
info@montanainstruments.com

BioPhotonics May/June 2015

515_NewProdLeads.indd 45

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4/28/15 9:11 AM

BREAKTHROUGHPRODUCTS

Visual Field Analyzer


The HFA3, a new version of the Humphrey
Field Analyzer (HFA) for glaucoma diagnostics, is offered by Carl Zeiss Meditec AG. In
perimetry, a trial lens provides the refractive
correction needed so each patient can see
clearly during the test. The HFA3 replaces the
old manual process with the patented Liquid
Trial Lens, which automatically delivers the
appropriate refractive correction using measurement information entered into the instrument. Its RelEYE feature allows doctors to
instantly review the patients eye position at
any stimulus point to reveal common testing
problems, such as droopy lids (ptosis). Faster
gaze tracking provides information that helps
doctors assess the reliability of test results.
william.taggart@zeiss.com

electric systems. The ASI and ASC functions


allow for automatic sensor identification and
automatic system calibration, respectively.
ASI capabilities are available for digital
amplifiers in the NV40/3 (pictured), NV120/1
and NV40/1 series. ASI functions allow users
to exchange the same type of actuator and
use it with the same amplifier. Additionally,
an actuator for ASI-compatible amplifiers is
equipped with an external preamplifier. Valid
only for standard calibration, new calibration
no longer is necessary. ASC provides an additional level of functionality. The integrated
circuit, built into a closed-loop actuator,
saves calibration parameters, such as name,
serial number, motion axis, PID values and
filter settings.
sales@piezojena.com

Intelligent Piezo Controllers

Fluorescence Spectrometer

Piezosystem Jena GmbH of Jena, Germany,


now offers two new functionalities for piezo-

Edinburgh Instruments Ltd. has combined


its FS5 single-photon-counting spectrofluorometer with a liquid nitrogen cryostat. This
enables users to measure spectral properties of a sample with a temperature range of
77 to 500 K, such as in phosphorescence or
delayed fluorescence measurements where
samples are sometimes frozen at liquid
nitrogen temperatures in order to preserve
the fragile triplet state. The system enables
automated 3-D measurements in order to
produce temperature-dependent maps of
excitation, emission, synchronous scans,

and phosphorescence and fluorescence


decays. Using a variety of sample holders,
it can measure samples of different states,
including solid crystals, thin films, powders
and liquids.
enquiries@edinst.com

OCT Fiber Probes

OFS Fitel LLC has announced new optical fiber assembly capabilities to build high-quality probes with flexible tip lensing designs for
OCT applications. The technology allows the
devices to meet critical OCT imaging specifications, such as insertion loss, internal back
reflection, beam size and working distance.
ofs@ofsoptics.com

FLIM Attachment
A fluorescence lifetime imaging microscopy
(FLIM) system from Lambert Instruments
BV is compatible with widefield fluorescence
microscopes. The Lambert Instruments FLIM
attachment for time-domain imaging (LIFATD) includes a camera with a built-in image
intensifier, which can reduce the exposure
time to 3 ns and alter timing of the exposure
in the subnanosecond range. A pulsed laser

oth-No. 32

Hall B3, Bo

Come into our


world!

Gas lasers

Solid state lasers

Optical systems
Fiber technologies

Microscopy Raman Spectroscopy


Metrology Flow Cytometry Holography
LASOS Lasertechnik GmbH Franz-Loewen-Str. 2 07745 Jena Germany

www.lasos.com

46

515_BI New Prods.indd 46

BioPhotonics May/June 2015

4/28/15 11:20 AM

BREAKTHROUGHPRODUCTS

excites the fluorophores in the sample. By using a laser with a small pulse width, the accuracy of lifetime measurements is optimized.
Images are recorded with custom software,
which also processes the data to determine
fluorescence lifetime. The fluorescence lifetimes are then shown as a colorized overlay
on the original image.
ria@lambert-instruments.com

has a stable housing and wavelength tuning


for easier signal optimization. Diffraction
gratings with aberration correction and good
efficiency from 190 nm UV to the IR are available. Applications include Raman filtering,
UV edge rejection, photoluminescence, and
light source and detector characterization.
mcp@mcphersoninc.com

Tunable UV Raman Filter

An intravascular imaging system by Infraredx


Inc. allows in vivo chemical analysis to help
manage coronary artery disease. The
Advanced TVC Imaging System combines
near-infrared spectroscopy with intravascular ultrasound. The FDA-approved device is
intended to help cardiologists predict the risk
of periprocedural heart attacks by assessing
not only the degree of stenosis but also the
presence and extent of lipid-core plaques,
which are known to complicate stenting procedures and are suspected to be the cause of
most heart attacks. The companys extended
bandwidth intravascular ultrasound technology harmonizes multiple signal frequencies
to produce an image of the complete vessel,
which allows for easy identification of the
lumen, plaque and vessel structure.
gfrazier@infraredx.com

Intravascular Imaging System

McPherson Inc.s compact double-monochromator Model 275D is suited for deployment


in UV Raman spectroscopy systems to detect
chemical, biological and explosive activity.
It works as a tunable filter and can work with
wavelengths as short as 190 nm with no special preparation. The double-monochromator

Digital Videoscope
The minnieScope-XS from Enable Inc. is the
worlds smallest 400 400-pixel videoscope

and is designed to provide embedded, realtime optical imaging for medical devices. It
has an outside diameter (OD) of ~1.5 mm at
the tip and a shaft OD <1 mm. The system
includes a CMOS image sensor, imaging
optics, highly flexible cable, illumination and
a video processing unit. Steering conduits
are also available to provide full 360 steerability with single-handed operation.
sales@enableimaging.com

Biomechanical Camera System

A portable, four-camera, high-speed recording system by NorPix Inc. is suited for biomechanical applications. Based on the companys StreamPix software, the recording
system can record from four synchronized
cameras at up to 330 fps and a resolution
of 2048 1088 in 8-bit format. Applications
include motion capture, sports biomechanics, animal science, ergonomics, kinematics
and equine biomechanics.
ln@norpix.com

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BioPhotonics May/June 2015

515_BI New Prods.indd 47

47

4/28/15 10:24 AM

APPOINTMENTS
CALL FOR PAPERS
SPIE/OSJ Biophotonics Japan October 2728
Deadline: Abstracts, June 22
Tokyo. SPIE and the Optical Society of Japan (OSJ) invite submissions
for oral and poster presentations at SPIE/OSJ Biophotonics Japan.
Papers will be accepted in a variety of areas including advanced biophotonics and microscopy; tissue characterization; diffuse optical
imaging; light sources for biomedicine; light/laser interaction with
biological tissue; and clinical and biomedical spectroscopy and
imaging. Among other topics to be considered are optical coherence
imaging techniques, optoacoustic methods and applications, lasers
and nonlinear optics for medicine, optical fibers and sensors, and
molecular-scale medicine and imaging. Manuscripts will be published
in Proceedings of SPIE.
Contact: SPIE
+1 (360) 676-3290
customerservice@spie.org http://spie.org
ACP 2015 November 1923
Deadline: Paper submissions, July 20
Hong Kong. Researchers are encouraged to submit original work for
presentation at Asia Communications and Photonics Conference 2015

JUNE

The Big M Manufacturing Convergence


(June 24) Detroit. Contact SME Headquarters, +1 (313) 425-3000; service@sme.com;
http://bigmevent.com.
Photonex Scotland Roadshow 2015 (June 3)
Glasgow, U.K. Contact Laurence Devereux,
Xmark Media Ltd., +44 1372 750555; ld@
xmarkmedia.com; http://photonex.org/
strathclyde.
Imaging and Applied Optics (June 711)
Arlington, Va. Includes Adaptive Optics (AO);
Applications of Lasers for Sensing & FreeSpace Communication (LS&C); Applied
Industrial Optics: Spectroscopy, Imaging
and Metrology (AIO); Computational Optical
Sensing and Imaging (COSI); Freeform
Optics (Freeform); Imaging Systems and
Applications (IS); and Propagation through
and Characterization of Distributed Volume
Turbulence and Atmospheric Phenomena
(pcDVT). Contact The Optical Society, +1
(202) 223-8130; info@osa.org; www.osa.org.
Inter/Micro 2015 (June 812) Chicago. Contact McCrone Research Institute, +1 (312) 8427100; intermicro@mcri.org; www.mcri.org.

l Sensors Expo & Conference (June 911)

Long Beach, Calif. Contact Wendy Loew, group


show director, +1 (617) 219-8343; wloew@
questex.com; www.sensorsexpo.com.
Photonics North 2015 (June 911) Ottawa,
Ontario. Contact Conferium, +1 (418)
522-8182; conference@conferium.com;
www.photonicsnorth.com.
World Preclinical Congress 2015 (June 1012)
Boston. Contact Cambridge Healthtech Institute, +1 (781) 972-5400; chi@healthtech.com;
www.worldpharmacongress.com.
2015 BIO International Convention (June
1518) Philadelphia. Contact Biotechnology
Industry Organization, +1 (202) 962-6655;

48

515_Appointments.indd 48

(ACP 2015). Technical areas to be addressed include biophotonics and


optical sensors; novel fibers and fiber-based devices; and optical materials, devices and integration. Other areas considered include optical signal processing and microwave optics; network architectures,
management and applications; and optical transmission systems,
subsystems and technologies.
Contact: Hong Kong Polytechnic University acp2015@polyu.edu.hk
www.acp-conf.org
SPIE Photonics West February 1318, 2016
Deadline: Abstracts, August 3
San Francisco. Authors are encouraged to submit abstracts for oral or
poster presentations at SPIE Photonics West, which encompasses the
conferences BiOS, LASE and OPTO. The BiOS conference is organized
into five program tracks: Photonic Therapeutics and Diagnostics;
Clinical Technologies and Systems; Tissue Optics, Laser-Tissue Interaction and Tissue Engineering; Biomedical Spectroscopy, Microscopy
and Imaging; and Nano/Biophotonics.
Contact: SPIE
+1 (360) 676-3290
customerservice@spie.org http://spie.org

convention@bio.org; http://convention.bio.
org/2015.
AOIM X, International Workshop on Adaptive Optics for Industry and Medicine (June
1519) Padova, Italy. Contact Stefano Bonora,
stefano.bonora@dei.unipd.it; http://aoim.pd.
ifn.cnr.it.
World of Photonics Congress (June 2125)
Munich. Contact Ellen Richter-Maierhofer,
congress manager, +49 89 949 20370; info@
photonics-congress.com; www.photonicscongress.com.
CLEO/Europe-EQEC 2015 (June 2125)
Munich. The European Conference on Lasers
and Electro-Optics and the European Quantum Electronics Conference. Part of World
of Photonics Congress. Contact European
Physical Society, conferences@eps.org;
www.cleoeurope.org.

try (June 2225) Munich. Held in conjunction


with World of Photonics Congress. Contact
Visitor Service, +49 89 949 11468; info@
world-of-photonics.com; www.world-ofphotonics.com.
SPIE Optical Metrology 2015 (June 2225)
Munich. Part of World of Photonics Congress.
Contact SPIE, +1 (360) 676-3290; customer
service@spie.org; http://spie.org.
International Symposium on Molecular
Spectroscopy (June 2226) Urbana, Ill.
Contact Birgit McCall, coordinator, +1 (217)
244-0984; birgit@isms.illinois.edu; http://
isms.illinois.edu.
Infrared Spectroscopy I Course: Interpretation of IR and Raman Spectra (June 2226)
Brunswick, Maine. Contact James de Haseth,
IR Courses Inc., +1 (706) 248-6386; dehaseth
@ircourses.org; www.ircourses.org.

ECBO 2015, European Conferences on


Biomedical Optics (June 2125) Munich.
Part of World of Photonics Congress. Contact
SPIE +1 (360) 676-3290; customerservice@
spie.org; http://spie.org.

Biodefense World Summit 2015 (June 2226)


Bethesda, Md. Contact The Knowledge
Foundation, +1 (617) 232-7400; custserv@
knowledgefoundation.com; www.biodefense
worldsummit.com.

TRVS 2015, Time-Resolved Vibrational


Spectroscopy Meeting (June 2125)
Madison, Wis. Contact Conference Services,
+1 (608) 890-1077; trvs@union.wisc.edu;
www.union.wisc.edu/trvs2015.

Advanced Photonics (June 27July 1) Boston.


Includes Integrated Photonics Research, Silicon and Nano-Photonics (IPR); Novel Optical
Materials and Applications (NOMA); Optical
Sensors (Sensors); Photonic Networks and
Devices (Networks); and Signal Processing in
Photonics Communications (SPPCom). Contact The Optical Society, +1 (202) 223-8130;
info@osa.org; www.osa.org.

EOS WPC2015 at the World of Photonics Congress (June 2224) Munich. Third EOS Conference on Optofluidics (EOSOF); Fourth EOS
Conference on Manufacturing and Testing of
Optical Components (EOSMTOC); EOS Conference on Optomechanical Engineering (EOSOME); EOS Conference on Light Engineering
(EOSLE). Contact Oili Kohonen, European
Optical Society, +358 40 564 0480; kohonen@
myeos.org; www.myeos.org/events.

lLaser World of Photonics, the International


Trade Fair for the Laser and Photonics Indus-

OECC 2015, Optoelectronics and Communications Conference (June 28July 2)


Shanghai. Contact Kan Wu, kanwu@sjtu.edu.
cn; www.oecc2015.sjtu.edu.cn.

l Indicates shows Photonics Media will be attending.


Complete listings at www.photonics.com/calendar.

BioPhotonics May/June 2015

4/28/15 9:13 AM

ADVERTISERINDEX
Photonics Media Advertising Contacts
Please visit our website
Photonics.com/mediakit for all
our marketing opportunities.
New England & FL
Rebecca L. Pontier
Associate Director of Sales
Voice: +1 (413) 499-0514, Ext. 112
Fax: +1 (413) 443-0472
becky.pontier@photonics.com
NY, NJ & PA
Timothy A. Dupree
Regional Account Manager
Voice: +1 (413) 499-0514, Ext. 111
Fax: +1 (413) 443-0472
tim.dupree@photonics.com
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Europe & Israel
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Voice: +1 (413) 499-0514, Ext. 103
Fax: +1 (413) 443-0472
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& Western Canada
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peggy.dysard@photonics.com
Asia (except Japan)
Advertising Sales Department
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Japan
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advertising@photonics.com

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Quantel .................................................... 47
www.quantel-laser.com

Edinburgh Instruments Ltd. .................. 19


www.edinst.com

SCHOTT North America Inc.


Lighting and Imaging .......................... 14
www.us.schott.com/lightingimaging

Edmund Optics ....................................... 22


www.edmundoptics.com/life-sciences

Semrock Inc. ........................................... 35


www.semrock.com

SUTTER INSTRUMENT ......................... 43


www.sutter.com

Forth Dimension Displays Ltd. ............. 28


www.forthdd.com

Gould Fiber Optics ................................... 6


www.gouldfo.com

LASOS Lasertechnik GmbH ............ 43, 46


www.lasos.com
Lumencor Inc. ...................................... CV3
www.lumencor.com

TechnoSpex Pte Ltd. .............................. 30


www.technospex.com

Xintu Photonics Co., Ltd. ....................... 46


www.tucsen.com

Zemax Optical Design ............................. 3


www.zemax.com/demo

Mad City Labs Inc. ................................... 44


www.madcitylabs.com
Melles Griot ............................................ 15
www.mellesgriot.com

Photonics Media has


launched a new Online
Reader Service tool that
allows you to instantly request
information about ads and
product announcements that
appear in our magazines.

Request info from the advertisers in this issue at Photonics.com/rsbp


BioPhotonics May/June 2015

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4/28/15 9:13 AM

POSTSCRIPTS

An ongoing investigation on the effects of street lighting on artificial


grassland communities has shown that amber lighting, simulating
a commonly used type of sodium street lamp, affects the food source
of pea aphids, causing a decline in the population of the insect.
Courtesy of Jon Bennie, University of Exeter.

Lighting stunts aphids edibles

s drivers or pedestrians at night,


we are happy for roadside lighting
to safely guide us on our way. The
byproduct of this illumination, however,
is an extended stay in the spotlight for
humble but important roadside attractions like grass, plants, flowers, insects
and other creatures.
A long-term, ongoing study reveals that
artificial nighttime light sources such
as street lamps can have an impact on
the natural environment in ways that
are complicated and difficult to predict.
Researchers at the University of Exeters
Environmental and Sustainability Institute (ESI) in Penryn, England, published
their work in the journal Philosophical
Transactions of the Royal Society B (doi:
10.1098/rstb.2014.0131).
Our results suggest that by lighting
up our nighttime environment, we trigger
complex effects on natural food webs,
said Dr. Jonathan Bennie, part of the ESI
team. While we are all aware that street
lights often attract insects at night, we
show that they have more permanent,
widespread impacts on wildlife and
ecosystems.
In July 2012, Bennie and his colleagues set up 54 experimental mesocosms outdoors: a diversity of artificial

50

515BI_Postscripts.indd 50

grassland communities housed in transparent box-like structures. The team


used the artificial ecosystems to study
the population density of the pea aphid,
a grassland insect, under two types of
low-level lighting and various other conditions, such as the presence or absence
of predatory ladybugs. The flowering
shoots of a wild-growing legume, known
as Greater Birds-foot Trefoil, is the
aphids main food source. The legume
was also transplanted to the man-made
communities.
The researchers selectively applied a
cool white light similar to commercial
LED street lighting to the mesocosms.
They also applied amber lighting to
simulate low-pressure sodium lighting,
another common form of street lighting.
Downward-facing LEDs were mounted
across the top of the mesocosms to
supply the light treatments, which were
switched on at sunset and off at sunrise
by light-detecting photocells. Unlit
mesocosms served as control treatments.
Over time, the investigators found that
the low-intensity amber light inhibited the
growth of the legumes flowering shoots.
The researchers observed that the number
of aphids was significantly lower under
the amber light treatment during mid-

August the team attributed this to the


decrease in the food source.
This is the first time that the effects
of artificial nighttime lighting on plants
have been shown in turn to influence the
organisms that feed on them, said Kevin
Gaston, director of ESI, who has studied
the ecological impacts of urbanization for
many years. These kinds of cascading
effects are probably very widespread but
are challenging to disentangle.
The team also found that the effects
from the white light treatments were
intermediate between those of the amber
and the control treatments.
As for the challenges involved, Gaston
noted that the experiments were very
demanding, requiring a lot of time and
effort to sustain and collect data. These
are the first findings from major, longterm experiments funded by the European
Research Council, he said.
We are particularly interested to
determine how the impacts of nighttime
lighting might change as the experiment
runs for longer. It seems likely that initial
impacts will cause further cascades of
subsequent effects.
Caren B. Les
caren.les@photonic.com

BioPhotonics May/June 2015

4/28/15 9:14 AM

LUMENCOR

CUSTOMER FOCUS

Aurelie Snyder
OHSU

SEEING MORE with LESS


Your job is to assist and advise
researchers in the use of advanced
microscopy techniques, correct?
Yes, I am an imaging specialist at the
Advanced Light Microscopy Core at The
Jungers Center at Oregon Health Sciences
University. It is a shared resource of high-end
microscopy systems. Im responsible for 10
microscope systems and support a user base
of over 350 researchers and clinicians.

How have Lumencors light engines


impacted your work?
We installed a Lumencor light engine as
part of our wide-eld deconvolution imaging
system. It replaced a metal halide light
source. There was so much more intensity
with the light engine that the speed of
analysis was dramatically improved. Our
data acquisition rates (frames per second)
increased by a factor of 2 for xed samples
and by a factor of 4 for live cell samples.
Illumination necessary to achieve a good
image was reduced from hundreds of
milliseconds to tens of milliseconds.

Anybody who has struggled


with phototoxicity problems in
live cell imaging experiments
will readily appreciate the
benets of being able to limit
light exposure.
What are some other benets that you and
users of your facility have noticed?
The illumination is even across the eld of
view through the eyepieces, which is larger
than that of the camera detection area. An
additional attraction is the rapid electronic
switching between excitation channels,
providing a further decrease in acquisition
speed compared to microscopes equipped
with mechanical lter wheels.

www.lumencor.com

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EN E S T EDT. S E

More coHAIRence

When you cant afford a bad laser day


www.cobolt.se

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2014-11-03
14:29
4/28/15
9:15 AM

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