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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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Volume 22 Issue 4
www.photonics.com
NEWS
12 BIOSCAN
21 RAPIDSCAN
FEATURES
25 FEMTOSECOND PULSES: CONTROL IS KEY
TO NEW DISCOVERIES
DEPARTMENTS
7 EDITORIAL
45 BREAKTHROUGHPRODUCTS
48 APPOINTMENTS
49 ADVERTISER INDEX
50 POST SCRIPTS
Now available as a
FREE mobile app
for subscribers:
www.photonics.com/apps
515BI_Contents.indd 4
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.
4/29/15 9:28 AM
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www.photonics.com
Group Publisher
Karen A. Newman
Editorial Staff
Editor
Web Managing Editor
Associate Editor
Contributing Editors
Copy Editors
Rodd M. Pedrotti
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www.GOULDFO.com
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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,
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and opinions expressed in BioPhotonics are those of the contributors the publisher assumes no
responsibility for them.
4/28/15 8:53 AM
EDITORIAL
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.
David Benaron, MD
Professor, Medicine (consulting)
Founder, Stanford Biomedical Optics program
Stanford University School of Medicine
CEO, Spectros Corp.
Karen A. Newman
karen.newman@photonics.com
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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.
Welcome to
Whats Online:
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.
REGISTE
R
NOW
4/28/15 9:01 AM
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.
515BI_IYL.indd 9
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:
www.ascb.org/2015meeting
/ascbiology
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@ascbiology
ascb
4/28/15 9:02 AM
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
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
A Novel Photoacoustic
Camera for Medical Imaging
Navalgund Rao,
Rochester Institute
of Technology
Photoacoustic Imaging
of Breast Cancer
Srirang Manohar,
University of Twente
Quantitative Analysis
of Multimode Fiber
Endoscopes
Antonio M. Caravaca Aguirre,
University of Colorado Boulder
www.photonics.com/bioconference
4/29/15 10:03 AM
BIOSCAN
A closer look at the most significant biophotonics research and technology headlines of the month
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.
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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.
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13
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BIOSCAN
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BIOSCAN
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A squid-inspired adhesive film that reflects near-infrared light when stretched could provide camouflage for
soldiers.
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BIOSCAN
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BIOSCAN
/ofsoptics
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/company/ofs
/OFSoptics
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
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BIOSCAN
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.
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BIOSCAN
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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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.
Plastic optical components designed by Edmund Optics and printed by Luxexcel. Courtesy of
Luxexcel.
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LPKF announces
initiatives after a
disappointing 2014
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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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Zeiss
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
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
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RAPIDSCAN
Hamilton Thorne
acquires reproductive
imaging product line
24
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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
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Pulse Control
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Shaped laser pulses excite molecules to specific excited states. Courtesy of ICFO (The Institute
of Photonic Sciences).
4/28/15 9:07 AM
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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Flexure Stages
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Fiber Alignment
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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.
Applications:
Super Resolution Microscopy
Confocal Microscopy
Structured Illumination Microscopy
Light Sheet Microscopy
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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
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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
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Pulse Control
Endoscopes
Computed Tomography
Fluorescence and tissue scatter
Opthalmetrics
Confocal imagers
Microscopes
Hyper-spectral systems
Beam shapers
Polarization
Fiber optic delivery systems
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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.
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
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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.
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.
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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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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.
36
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References
Acknowledgment
4/28/15 9:09 AM
515_SpectroscopyFeat.indd 37
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
The 3-in-1 spectroscopy probe. Courtesy of James Tunnell and Austin May, University of Texas at Austin.
Webinars
ON DEMAND
Available 24/7
In-Depth
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featuring
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www.photonics.com/webinars
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.
4/28/15 9:16 AM
Spectroscopy Advances
515_SpectroscopyFeat.indd 39
Super-resolution
Microscopy
STED Add-on for the
MicroTime 200
FLIM and FCS Platform
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per-reso
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ELMI me copy at the
eting (M
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MicroTime 200: An all-in-one solution for timeresolved microscopy with unmatched sensitivity
Features
PicoQuant GmbH
Berlin, Germany
info@picoquant.com
www.picoquant.com
Further extensions
4/28/15 9:16 AM
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
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
4/28/15 9:10 AM
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.
41
4/28/15 9:10 AM
SMLM Imaging
42
515BI_SMLM Feat.indd 42
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
4/28/15 9:10 AM
SMLM Imaging
future, we aim to fully integrate cameraspecific algorithms into the PYME suite
so that the microscope operator can
seamlessly benefit from algorithmic
oth-No. 32
Hall B3, Bo
Gas lasers
FEATURES
Optical systems
Fiber technologies
515BI_SMLM Feat.indd 43
www.lasos.com
43
4/28/15 9:10 AM
SMLM Imaging
References
Know
More
Now
Try it now!
44
515BI_SMLM Feat.indd 44
Visit Photonics.com/rsbp
to access Reader Service for
the latest issue.
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
Microscope Illuminator
Microscopy Camera
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
515_NewProdLeads.indd 45
45
4/28/15 9:11 AM
BREAKTHROUGHPRODUCTS
Fluorescence Spectrometer
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
Gas lasers
Optical systems
Fiber technologies
www.lasos.com
46
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
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
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
September
October
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
48
515_Appointments.indd 48
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.
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.
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
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Associate Director of Sales
Voice: +1 (413) 499-0514, Ext. 112
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advertising@photonics.com
OFS .......................................................... 18
www.ofsoptics.com
Alluxa ....................................................... 34
www.alluxa.com
Applied Scientific
Instrumentation Inc. ........................... 16
www.asiimaging.com/bp
Cobolt AB ..............................................CV4
www.cobolt.se
Coherent Inc. ....................................... CV2
www.coherent.com
Quantel .................................................... 47
www.quantel-laser.com
515BI_AdIndex.indd 49
49
4/28/15 9:13 AM
POSTSCRIPTS
50
515BI_Postscripts.indd 50
4/28/15 9:14 AM
LUMENCOR
CUSTOMER FOCUS
Aurelie Snyder
OHSU
www.lumencor.com
515_Lumencor_Pg51.indd 51
4/28/15 2:57 PM
EN E S T EDT. S E
More coHAIRence
Cobolt-annons-213x281.indd
515_Cobolt_Pg52.indd 52 1
2014-11-03
14:29
4/28/15
9:15 AM