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Volume 21 Issue 4
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NEWS
7 BIOSCAN
14 RAPIDSCAN
FEATURES
30 SUPERRESOLUTION IMAGING
ADDS ANOTHER DIMENSION
5 EDITORIAL
DEPARTMENTS
THE COVER
A surgical laser can score
a cataract in a cross, pie,
bulls-eye or grid formation.
Image courtesy of Lensar.
Design by Art Director
Suzanne L. Schmidt.
32 BREAKTHROUGHPRODUCTS
35 APPOINTMENTS
37 ADVERTISER INDEX
38 POST SCRIPTS
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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.
www.photonics.com
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EDITORIAL
his month, we congratulate the winners of the 2014 SPIE Awards. Among them are
the following three, who have had an impact on the world of biophotonics. (For a list
of all the winners, visit www.photonics.com/a55977.)
The SPIE Britton Chance Biomedical Award was awarded to Dr. Brian Wilson, head
of the Division of Biophysics and Bioimaging at the Ontario Cancer Institute, in honor
of Wilsons many contributions to biomedical optics over the past 30 years, including
his pioneering work in photodynamic therapy (PDT). His work included the concepts of
PDT beacons, metronomic PDT and nanotechnology-enabled PDT, as well as technology
development and clinical trials in the brain, the prostate and metastatic bone lesions.
The recipient of the SPIE Biophotonics Technology Innovator Award is Dr. Naomi
Halas, director of the Laboratory for Nanophotonics and the Stanley C. Moore Professor
in Electrical and Computer Engineering at Rice University in Houston. Halas is recognized for her invention of biocompatible nanoparticles and their innovative applications in imaging, diagnostics and photothermal cancer therapy. She invented a class of
nanoparticles with optical resonances that can be designed in to a nanostructure. Her
use of gold nanoshells in living systems is in clinical trials.
Also on the list is Dr. Jeff Squier, a professor in the department of physics at the Colorado School of Mines in Golden, recipient of the Harold E. Edgerton Award. His achievements include the demonstration of the first Ti:sapphire-based regenerative amplifier for
high-energy ultrashort pulse generation as part of a group at the University of Rochester
in New York. Most recently, he introduced a method to simultaneously capture nonlinear
optical images from multiple depths. He is recognized for his seminal contributions to,
among others, femtosecond lasers and amplifiers, laser filamentation, ultrafast spectroscopy and ophthalmic procedures with ultrafast lasers.
Speaking of ophthalmic lasers, in our cover story, science writer Valerie Coffey takes
an unblinking look at cutting-edge laser technologies advancing ophthalmological
treatments. Refractive laser surgery is used to treat eye conditions including near- and
farsightedness, astigmatism and one day soon the scourge of middle age: presbyopia.
Look into the latest on lasers in eye treatment in Seeing the Light: How Photonics Continues to Improve Eyesight, beginning on page 19.
Also in this issue, contributing editor Marie Freebody examines the versatile FRET
spectroscopy technique in FRET Pursues Affordable, Robust, User-Friendly Instruments, starting on page 23; Dr. Edward Freniere and Michael Gauvin of Lambda Research Corp. explain the role of optomechanical design software in developing efficient
and effective biomedical optical products in Software Enhances Life Sciences Applications, Biomedical Simulations, beginning on page 27; and, in Superresolution Imaging
Adds Another Dimension, news editor Gary Boas outlines the potential impact of 3-D
capabilities added to the technique, beginning on page 30.
Enjoy the issue. Send comments to me at karen.newman@photonics.com.
Karen A. Newman
karen.newman@photonics.com
CONTRIBUTORS
News editor Gary Boas has
extensive experience as
a writer and editor in the
research community; he is
also a contributing editor to
Photonics Spectra. Page 30.
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BIOSCAN
A closer look at the most significant biophotonics research and technology headlines of the month
The 3-D atomic force microscope developed by researchers at the University of Missouri could help
researchers understand proteins better. Images courtesy of the University of Missouris Department
of Physics and Astronomy and Department of Biochemistry.
BIOSCAN
Researchers scattered a focused laser directly off an atomic force microscope tip apex to rapidly and precisely measure the tapping tip trajectory in a 3-D space. F = Force; pN = pico Newton; = angstrom.
the drinking behaviors of animals, and helps explain the underlying neurochemical basis of drug addiction.
By stimulating certain dopamine neurons in a precise pattern, resulting in low but prolonged levels of dopamine release,
we could prevent the rats from bingeing. The rats just flat-out
stopped drinking, said Dr. Caroline E. Bass, assistant professor
of pharmacology and toxicology in the UB School of Medicine
and Biomedical Sciences.
The rodents continued to avoid alcohol even after the stimulation of neurons ended.
BIOSCAN
Dr. Caroline E. Bass holds two fiber optic implants used in the experiments to
deliver light to the brain. The light induces dopamine release, which changes
the behavior of the animals. Courtesy of Douglas Levere, University of Buffalo.
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Surgical laser systems such as the Lensar Laser System break up the cloudy cataract via laser-scored
patterns (a-d: cross, pie, bulls-eye and grid). The use of a laser reduces the amount of ultrasound
required, which reduces postoperative inflammation. Courtesy of Lensar.
Cataracts
Cataracts, a cloudiness of the eyes
clear crystalline lens, are the leading
cause of vision loss in the world, affecting
half of all individuals over the age of 60.
Nearly 22 million cataract surgeries were
performed globally in 2013, according to
estimates from MarketScope. The global
market for femtosecond lasers for cataract
surgery is growing exponentially and will
reach $2.4 billion by 2019, according to a
market report from RnR Market Research
of Dallas.
Cataract surgery involves fragmentation of the cloudy cornea, and its emulsification and removal (aspiration of the fragments), followed by the transplantation of
an artificial intraocular lens (IOL) made
of silicone or acrylic. Since the use of
femtosecond excimer lasers became common in 2009, the surgery has evolved into
an outpatient technique that takes only
a few minutes. Use of lasers reduces or
eliminates the use of ultrasound, typically
administered manually to break up and
emulsify the cataract. This helps reduce
damage to epithelial cells in the eye. The
results are more consistent, accurate and
predictable.
Recent advances in cataract treatment
include the development of better IOLs
that can compensate for the natural ability
of the cornea. One type of IOL, the multifocal IOL, mimics accommodation the
ability to focus from near to far distances
by providing multiple focal points at
near, intermediate and far distances.
Pseudoaccommodative IOLs flex to help
restore near vision by using the muscles
and natural physiological mechanisms of
the eye to bring a focus from far to near.2
What is remarkable about the latest versions of IOLs is that they can be used not
only to reclaim vision lost to cataracts,
but also to address myopia (nearsightedness), astigmatism (blurred vision),
hyperopia (farsightedness) or presbyopia
(the need for reading glasses) at the same
19
Human donor lenses from the Bristol Eye Bank reveal spectral responses similar to those obtained
with pigs lenses in an experiment to explore early cataract diagnosis via tryptophan fluorescence:
80-year-old female (27552A); 79-year-old male (27553B); 60-year-old female (27554A);
and 66-year-old male (27557B). Courtesy of B. Dhillon/Edinburgh Instruments.
The Lensar Laser System prepares the patient for a surgical procedure using a liquid interface of a
balanced salt solution to irrigate the eye and reduce laser incision pressure. Courtesy of Lensar.
20
Vision correction
Refractive laser surgery is the treatment of the cornea to correct myopia,
hyperopia, astigmatism and presbyopia.
One recent development in surgical vision
correction is the use of wavefront aberrometry to measure the eye front to back.
The technique maps in 3-D the subtle
optical aberrations that affect vision.
Called adaptive optics by astronomers
who use it to measure and correct aberration introduced by the atmosphere in real
time wavefront mapping enables custom
lasik treatment. The map is transferred to
the laser system and matched to the eyes
position. Surgeons then use the systems
cool blue excimer laser to resurface the
cornea and correct for aberrations.
In all-laser refractive surgery, advanced
femtosecond lasers replace the mechanical instruments called microkeratomes,
which use a steel blade to create the thin
circular flap on the outermost layer of the
cornea. Also called IntraLasik, the technique has been in commercial practice for
several years to treat myopia, hyperopia
and presbyopia.
Late last year, laser system developer
Lensar Inc. of Orlando, Fla., announced
a clinical trial to study the use of a technique called lenticular softening to restore
accommodation of the crystalline lens in
presbyopic patients. Presbyopia is caused
by a loss of elasticity in the lens, hardening it so that it has less accommodating
(zoom) power to focus on nearby objects.
The condition typically occurs in middle
and old age and affects a billion people
worldwide, according to the Archives of
Ophthalmology.
Presbyopia is the holy grail in ophthalmology, said Lensar CEO Nick Curtis.
We look forward to studying and expanding the body of data on the use of a
femtosecond laser for lenticular softening
with the eventual goal of accommodation
restoration.
An initial advanced feasibility study
of 80 subjects demonstrated the Lensar
laser systems ability to soften the lens
The Dangers
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Courtesy of Shinp.
The fundus of the eye shows a pattern of grids placed by the PASCAL laser. The Endpoint Management
software enables physicians to choose whether the burns are invisible or barely visible to provide a visual
reference. Courtesy of Topcon Medical Laser Systems.
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References
Excitation/Emission spectra
Fluorescence decays
Anisotropy and kinetics
Time-resolved emission spectra
Photochemistry
Materials research
Singlet oxygen
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time
The excitation path of a home-built microscope capable of three-color single-molecule FRET and alternating-laser excitation (ALEX) microscopy. The microscope
uses several visible lasers (405, 473, 532, 561 and 640 nm); the modulation and intensity of the lasers are controlled using an acousto-optical tunable filter.
The same microscope can be used for localization-based superresolution imaging techniques. Courtesy of Justin Pinkney.
BY MARIE FREEBODY
CONTRIBUTING EDITOR
lthough FRET (Frster resonance energy transfer) is now a mature and established
technique, it can always be faster, brighter, more sensitive, more multiplexed
and more selective. So research never ceases to extend FRETs capability and to
expand the range of applications for which it can be used.
Improvements to the technique would not only make it easier to use in existing applications, but also open it up to novel applications such as single-molecule analysis inside
living cells. Visualizing interactions, conformations and dynamics in their natural
biological contexts is still in its infancy, but new developments in internalization proce-
23
FRET Spectroscopy
Combining FRET with tethered fluorophore motion offers insight into chromosome segregation. (a)
Schematic of the recombination reaction with a DNA substrate for recombination. The DNA substrate
has the fluorophores attached to DNA adjacent to each recombination site (dif site); the positions of the
acceptor and the donor fluorophores are indicated with red and green circles, respectively. Progression
through the recombination was monitored using two observables: Image width (referred to as PSF width)
of the acceptor and FRET. Recombination between two dif sites leads to a formation of product DNA
molecules; one of them remains attached to the slide and has fluorophores flanking dif on both sides.
(b) Nonproductive synaptic complexes (top). Representative time trace of the intensities of donor (green)
and acceptor (red) under donor excitation; and acceptor-under-acceptor excitation (black). FRET
efficiency (middle) and PSF width (bottom) are shown. The nonproductive events are defined by the
ultimate broadening of the PSF coincident with the disappearance of the FRET signal. All data were
acquired at a frame rate of 10 Hz, unless otherwise stated. Histogram of FRET efficiency (right) and
dwell time (inset) of XerCD-dif synaptic complexes (n = 380). Dwell times were fit to single exponentials.
Courtesy of Pawel Zawadzki and Peter May.
Highly sensitive photon-counting detectors are the centerpiece of most multiparameter FRET spectroscopy
techniques. They permit detecting the arrival of single photons with picosecond time resolution. Shown is a
custom-built setup featuring eight photon-counting detectors that permit simultaneous detection of various
colors and polarization directions. Courtesy of the Lemke Group.
24
FRET probes
The design of FRET probes is still
considered an art rather than a science.
Crucial to the efficacy of FRET is the
selection of the right probes and developing new ones tailored to the biological
system under scrutiny.
Already using all the FRET tools that
exist offers incredible possibilities, said
professor Dr. Niko Hildebrandt of the
Nano BioPhotonics Group at the Institute of Fundamental Electronics at the
University of Paris-Sud in Orsay, France.
The challenge is always to find the good
FRET pair (or pairs) for the desired application and to make it work (or to find that
it does not). So the main challenge is to
use existing FRET pairs and to integrate
them into applications which can benefit
from the versatility of FRET.
When it comes to fluorescent proteins,
we are approaching an optimum in terms
of spectroscopic properties, according
to Raik Grnberg of the Institute for Research in Immunology and Cancer at the
University of Montreal. Several recent
variants (such as mTurquoise2) have more
than 90 percent quantum efficiency [and]
mono-exponential decay behavior, and
are fairly photostable, he said.
In recent years due to the work of
groups led by, for example, Markus Sauer
and Thorsten Seidel of Bielefeld University in North Rhine-Westphalia, Germany; Philip Tinnefeld of the University
of Brunswick Institute of Technology in
Lower Saxony, Germany; and Xiaowei
Zhuang of Harvard University in Cambridge, Mass. we have gained a better
understanding of how popular additives
modulate the bleaching and blinking
behavior of organic fluorophores. Along
FRET Spectroscopy
This illustration shows a SWIFT device to which, for example, nucleosomes (also shown) can be
loaded via the sample inlet. Courtesy of the Lemke Group.
25
FRET Spectroscopy
for biosensing.
The largest distance over which FRET
can be measured is with quantum dots
and lanthanides, with a Frster radius of
over 10 nm, Suhling said. The brighter
the donor, the better but as the quantum
yield of many donors is high already, improvements in brightness will be limited,
as the quantum yield can only ever be a
maximum of 100 percent. More photostable uorophores will, however, be very
useful for prolonged studying of protein
interactions or conformational changes.
FRET challenges
One of the biggest headaches for FRET
users is the sample preparation, which
requires routine molecular biology/biochemistry and cell biological work that is
both slow and labor-intensive. But lab-ona-chip technologies could alleviate some
of the drudgery.
Lemke and colleagues recently published a possible way to overcome this
in their latest paper, which appeared in
Nature Methods (doi:10.1038/nmeth.2809)
in January. Their automated microuidic
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LED emission into the brain to determine oxygenation in tissue. Images courtesy of Lambda Research Corp.
27
28
b
LED emission into human tissue with bulk scatter shown: (a) is the same as (b), except that the
setup in (b) has a paddle to hold the LED and detector.
Tissue propagation
Glucose monitoring
Heart rate monitoring
Fluorescence, Raman, UV, VIS, NIR and IR spectroscopy
Flow cytometry
Microarrays and plate readers
Nucleic acid amplification
Assay, cell and tissue-based imaging
Confocal laser scanning and fluorescence microscopy
Medical imaging and endoscopy
In vitro and in vivo diagnostics
Biosensors
Molecular detection: quantum dots, nanocrystals and luminescent reporters
Laser-induced fluorescence (LIF) detection
Frster resonance energy transfer (FRET)
Flux absorbed by surfaces and bulk media
Light scattering in biological tissue
TracePro can also be used in complement with another simulation and design
program by Lambda Research called
OSLO; the two work in tandem for the
development of advanced medical systems. OSLO enables a user to design and
analyze simple to complex optical models,
especially for lenses, mirrors, diffraction
gratings, Fresnel lenses and holographic
optical elements. The two programs have
been successfully used for multiple biomedical applications, including:
Developing pulsed light systems
like the StarLux and Medilux systems
by Palomar Medical Technologies Inc.
to reduce varicose veins, to treat acne,
wrinkles and vascular lesions, and to
remove scars and tattoos.
Locating and eliminating early cancer
in the lungs, colon, cervix and other tissues using fluorescence, laser ablation,
photodynamic therapy and other techniques.
Measuring glucose through optical
measurements of skin and blood samples
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systems.
Developing oximeters to assess blood
oxygenation in the brain and other tissue.
Detecting an object within a tissue
29
Superresolution Imaging
Adds Another Dimension
3-D techniques advance a range of biological research studies.
BY GARY BOAS, NEWS EDITOR
able that Nature Methods named superresolution imaging the 2008 Method of
the Year. As electron microscopy did
in the past, superresolution microscopy,
or nanoscopy, provides the ability to see
details of cellular and even macromolecular structure that were not possible to see
before, the editors wrote when explaining their selection. Notably, however,
nanoscopy is compatible with live cells
and has the capability for multiplex labeling with high molecular specificity.
Zeiss has introduced a module that enables photoactivated localization microscopy, or PALM, in 3-D and
with a lateral resolution of 20 to 30 nm and an axial resolution of 50 to 80 nm. Shown is a PALM nuclear
pore image of epithelial kidney cells. Courtesy of Anna Lschberger and Markus Sauer, University of
Wrzburg, Germany.
30
the lateral and axial directions is desirable. But they anticipate that the technique could be applied to a wide range of
additional biological samples, including
interacting cells and 3-D cell cultures.
They continue to refine the technique
with this in mind. For example, they are
further developing the adaptive optics
schemes used to correct for sample aberrations. More advanced methods may be
required in the presence of higher-order
aberrations stemming from complex
refractive index structure in samples,
Sinclair said. They also are working to incorporate fluorescence lifetime imaging to
enable multilabel 3-D STED microscopy.
gary.boas@photonics.com
31
BREAKTHROUGHPRODUCTS
CCD Cameras
CCD cameras from Raptor Photonics Ltd., developed using Sony ExView HAD II sensor technology, offer a choice of 2.8-, 6- or 9-MP resolution and are available in both mono and RGB. Pixel
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Two new vibration-isolation series platforms
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Raman Microscope
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molecular structure, chemical composition
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BREAKTHROUGHPRODUCTS
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APPOINTMENTS
CALL FOR PAPERS
Neuroscience 2014 November 15-19
Deadline: Abstracts, May 8, 5 p.m. EDT
Washington. The Society for Neuroscience seeks papers for oral
and poster presentation at its 44th annual meeting, Neuroscience
2014. Among the topics to be considered are development, including
human imaging; Alzheimers disease and other dementia, including imaging and biomarkers; and brain blood flow, metabolism and
homeostasis, including functional imaging. Staining, tracing and
imaging techniques also will be discussed within sessions on novel
methods and technology development.
Contact: Society for Neuroscience
+1 (202) 962-4000
program@sfn.org www.sfn.org
SPIE/COS Photonics Asia October 9-11
Deadline: Abstracts, May 12
Beijing. Researchers are invited to submit their work to Photonics
Asia, a conference sponsored by SPIE and COS (Chinese Optical Society). The event will encompass the meeting Optics in Health Care and
Biomedical Optics VI, which will address areas including tissue optics
MAY
ARVO 2014 Annual Meeting (May 4-8) Orlando, Fla. Contact Association for Research in
Vision and Ophthalmology, +1 (240) 221-2900;
arvo@arvo.org; www.arvo.org.
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Andor Technology plc .......................................................................................................... 13
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Hamamatsu Corporation ...................................................................................................... 11
www.hamamatsu.com
www.lumencor.com
m
www.madcitylabs.com
n
Newport Corporation ........................................................................................................... 10
www.newport.com
p
PCO AG ................................................................................................................................ CV4
www.pco.de
Photonics Media ......................................................................................CV2, 26, 33, 34, CV3
www.photonics.com
PI (Physik Instrumente) L.P. .................................................................................................. 18
www.pi-usa.us
PicoQuant GmbH .................................................................................................................. 22
www.picoquant.com
s
SCANLAB AG .......................................................................................................................... 9
www.scanlab.de
SUTTER INSTRUMENT ........................................................................................................ 33
www.sutter.com
37
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