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Molecular biology: PCR anniversary—May 11 Proteomics: Big data sharing—June 15 Genomics: Pharmacogenomics—September 28
Live-cell imaging: Deeper, faster, wider
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LIFE SCIENCE TECHNOLOGIES
1547SCIENCE sciencemag.org/custom-publishing
microscopy
Produced by the Science/AAAS Custom Publishing Office
At the National Institute of Biomedical Imaging and
Bioengineering in Bethesda, Maryland, microscopist Hari
Shroff is also interested in developmental neuroscience. In
collaboration with scientists at Yale University in New Haven,
Connecticut, and the Sloan-Kettering Institute in New York,
heís imaging how the brain of a nematode develops.
ìThe worm embryo: Itís hard to image, and itís very
sensitive to light,î says Shroff. ìYou really have to be fast.î
He and his colleagues wanted to avoid the classic light-
sheet arrangement in which the sample is embedded in a
tube of agarose and surrounded by lasers and cameras.
Instead, they preferred to put the worm embryos on standard
glass coverslips.
To get the incoming light sheet and observing objectives
at right angles to each other, Shroff ís solution was to tilt each
by 45 degrees from vertical. The apparatus looks much like
a standard microscope, except for the two askew objectives
aimed at one stage. The sample stays stationary, and the light
sheet from one objective and the other detection objective
move in tandem. The system is called inverted selective plane
illumination microscopy (iSPIM).
Like Keller, Shroff also dealt with the anisotropy issue, but
he didnít add any more objectives to do it. He simply set
each to either produce a light sheet or collect an image, and
to alternate between the two. First, objective A produces
the sheet and objective B the image, then they swap. The
researchers called this arrangement symmetrical dual-view
iSPIM (diSPIM).
'()*������*���*����(�* ���)*�������*��*���)��*
version they call triple-view SPIM, they added an extra
objective below the coverslip, to collect extra output and
improve spatial resolution at no cost to speed, and with no
added dose of light. Finally, the researchers experimented
with performing diSPIM with a mirrored coverslip. This
creates a ìvirtual imageî beyond the looking glass, as if the
sample and the light sheets were doubled. With the right
algorithms to make sense of it all, Shroff and his team can
�(*�������)���*����*)���*���)�����*��*��()*��*���*
sensitive imaging at twice the speed, all for the cost of an
aluminum-coated coverslip.
Wider: Living, moving landSCAPEsElizabeth Hillman, a biomedical engineer at Columbia
University in New York, has developed a light-sheet
technique that uses only a single objective to both produce
the light sheet and to collect all the signals from the sampleó
which could be an entire, freely moving organism. The system
uses a mirror to sweep the lightóand the focal point of the
cameraóthrough a sample. She refers to her system as ìswept
confocally-aligned planar excitationî (SCAPE) microscopy.
As with other new light-sheet techniques, SCAPE has
the advantage of tremendous speed. With newer cameras,
Hillman is imaging more than 100 volumes of sample per
second. That allows her to image the cells of awake, moving
���)(���*�(��*��*)�*�������*�(����*��*��� ���*��*����*
��*)�*��)���*���)�*��*) �)�����*��������* �)��()*)�*
problem of blurring when the animal moves. ìWeíre going
so fast that we can see timing information no oneís ever seen
before.î
Faster: Sheets of light
Multiphoton imaging still relies on slow point-scanning,
though. ìSpeed and depth is always the trade-off,î says Xu. And
��* ��)�*���*�)��*�������*�����*������*��*��� �*
seconds, so light-sheet microscopy was the answer.
While light-sheet microscopy is an old ideaóscientists at ZEISS
!���������*���*�������)���*���)*���*(�* �)�*�)*��*" �#$���*
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work to process image volumes combined to make light-sheet
mainstream. The ZEISS Lightsheet Z.1 microscope includes a
��%�(����)*������*)��)*���*���*�)��*���������$�*��(�)*��*
or even a small octopus, for exampleódangling in front of the
objective, and permits easy rotation for a better point of view.
A 2008 version of Kellerís light-sheet microscope, called
����)��*������*���*���)%��)*�(������*����������*
(DSLM), gave him the speed he craved: 1.5 billion voxels
per minute. The design differs from that of a conventional
commercial microscope: Keller placed the laser and objective
lens at right angles to each other atop a table; then he
�����*�������*������*��*������*��*�*����*)(�*
between the two. He translocated the sample back and forth
through the light sheet to hit all planes. He could observe
���%����%��*���)����*��*�������*�����*������)�*
and the formation of germ layers.
Since then, Keller has updated the technology. One issue was
that if a sample is large or opaque, the light sheet might not
reach all the way through it. To deal with this, Keller developed
SiMView: He doubled the light sheets and cameras to collect
four images within 20 milliseconds or less, because each
camera can focus on the two sheets in rapid succession.
Another issue he tackled was anisotropy, that is, the fact that
a single objective will typically give better resolution in the
lateral xy direction than in the axial z*����)����*��*���)*�*#&*
image with equal resolution at any viewing angleóa technology
called IsoViewóKeller doubled the number of cameras and
sheets again to four, and digitally combined those images.
Live imaging and computational tracking of cells in an entire developing Drosophila embryo undergoing gastrulation (shown are views of the dorsal and ventral sides of the embryo). The nuclei-labeled embryo was imaged with SiMView light-sheet microscopy and computationally reconstructed with the TGMM cell-lineaging framework.
cont.>
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LIFE SCIENCE TECHNOLOGIES
1548 SCIENCE sciencemag.org/custom-publishing
microscopy
Produced by the Science/AAAS Custom Publishing Office
Amber Dance is a freelance writer living in Los Angeles.
California Institute of Technologymr.caltech.edu
Columbia University
www.columbia.edu
Cornell University
www.cornell.edu
Hunter College, City University of New Yorkwww.hunter.cuny.edu
Imperial College London
www.imperial.ac.uk
Janelia Research Campus
www.janelia.org
Featured participants
Leica Microsystems
www.leica-microsystems.com
National Institute of Biomedical Imaging and Bioengineeringwww.nibib.nih.gov
Osaka University
www.osaka-u.ac.jp/en
University of Burgundyen.u-bourgogne.fr
ZEISS Microscopy
www.zeiss.com/microscopy/us/
home.html
Itís easy to adapt the SCAPE objective to a variety of sample
types, says Hillman. The cells or organisms could be in a plate
or dish, or in the head of a mouse; it doesnít matter.
Columbia has licensed the SCAPE technology to Leica,
though Burke is not certain when Leicaís SCAPE system will
be available. Leica has also licensed another sheet-sweeping
technology, the oblique plane microscopy (OPM) developed
by Chris Dunsby, a biomedical optics researcher at Imperial
College London.
Dunsbyís goal was to perform light-sheet microscopy with
a single objective, while maintaining good resolution. His
technique uses three microscopes in succession to magnify the
sample, capture light from the tilted plane, and sweep the light
sheet.
The complete setup offers high-speed, 3D imaging with
subcellular resolution, says Dunsby. For example, heís used it
along with a calcium-tracking dye in cardiac muscle cells from
a rat, to image the sparks and waves of calcium ions that can
trigger deadly arrhythmias during heart failure. ìWe were able
�������������������������������������� ������� ��������������
in cardiac myocytes,î he explains.
OPM also works in multiwell plates. Dunsby and colleagues
������������� ���������������� ��������� ������� ������������
biosensor, in multicellular kidney cell spheroids. Imaging 42
wells took 9 minutes.
Whatís next?SCAPE and OPM complement each other nicely, says Burke,
and Leica plans to combine the two in an upcoming product.
Whatís next for live-cell imaging? ìI donít think we have
explored the full capabilities of light sheet yet,î says Hellriegel.
For example, he suggests that superresolution techniques
might be incorporated into light-sheet imaging.
���������������������������������������� ���������� ��
indicate cellular events (e.g., neurotransmission) will make
real-time, rapid imaging appealing to many scientists. He also
anticipates that faster, more sensitive cameras will be neededó
and developedófor speedy imaging.
Labels and the lack thereof
�!�� ���#�"#��� �# #"�!�� "�� " !" #�"� !��#�� " ��#�"For one thing, it can be tricky to introduce them into #�����#�"� �#"��#!"����" !"��"�� ���" �� " ��"� !�!��!��"� �� ���"���� #" ��"�!� �!�"���"� ��� �"!�" ��" ���� "��! ����"���"�����"���� "�! "���" ��"��! ���"������ ���"!�"might photobleach over time.
Biophysicist Hyungsik Lim at Hunter College, City University of New York, uses third harmonic genera-tion (THG) microscopy to image myelinóthe ìinsulationî around nerve ìwiresîóin live cultures and tissues with-out adding any labels. THG generates a signal when the energy from three incoming photons is combined into one outgoing photon. The technique is particularly sen-sitive to boundaries where the refractive index of a tis-sue changes, such as those between aqueous solutions and lipid- or protein-rich structures, such as myelin.
Another option is Raman microscopy, a scanning ver-sion of Raman spectroscopy, says Katsumasa Fujita, an applied scientist at Osaka University in Japan, who is introducing Raman to the microscopy world. Raman spectroscopy relies on the incoming light of a single wavelength to excite the molecules in a sample. The photons that make up this light bounce, or scatter, off the molecules in the sample, mostly at the same wavelength they had coming in. But every so often (about one in 100 million photons), a photon will bounce off with a different wavelength, shifted to a lower, more reddish frequency. The frequency of the shifted light depends on the mol-ecule it scattered from. In this way, Raman spectroscopy can identify the components of a sample.
Under the life-science microscope, Raman scattering !��#"��"�"#������"���"��!������"�"��!���"!�" ��"!��!-nent molecules in a specimenówhether it is DNA, pro-tein, or lipid. However, it canít distinguish much beyond that; for example, it canít tell one kinase from another.
Meanwhile, researchers at Columbia University in New York are working on a way to add dozens of color labels !"������������"���#�"����� �!���"� !��#���"�������"����#"! "� "��! ��"���"!�!���"�����#�"��� #�" ��"��!��"�����#"!�"������� �#"��� ��"��" ��"� !�!��!��#"overlap, points out biophysical chemist Wei Min. But the wavelengths emitted in Raman spectroscopy fall into a much tighter range, so it ought to be possible to use many more colors without that overlap.
A former postdoc of Minís, Lu Wei, now a chemistry professor at the California Institute of Technology in Pasadena, took on the challenge. She and her colleagues ��#�����"��"�������� "� !��#�� "���#"��"�������" ��"triple carbonñcarbon bonds, triple carbonñnitrogen bonds, and isotope content to create different colors.
Now, Wei and Min are working on more colors and �� �!�#" !"����" ��"���#" !"#����"��!�!�� ��#"!�"organelles of interestóMin thinks 50 or more colors should be possible.
LIFE SCIENCE TECHNOLOGIES
new products: microscopy
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35-mm Imaging Dishibidiís µ-Dish 35-mm Quad
is a four-compartment
cell culture dish that
guarantees brilliant optical
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enable up to four parallel,
individual experiments in
one dish, where applications
such as transfection,
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performed. Quad is the ideal solution for scientists who conduct
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experimental conditions. The subdivisions not only save time, but also
decrease experimental costs by reducing the cell numbers and reagents
needed. Its unique ibidi Polymer Coverslip Bottom guarantees superior
optics for high-end microscopy. Excellent phase contrast is provided
by the centered plate construction. This Ph+ (Phase Contrast +) feature
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ibidi
For info: 844-276-6363
www.ibidi.com
Electronically submit your new product description or product literature information! Go to www.sciencemag.org/about/new-products-section for more information.
Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizations are featured in this
space. Emphasis is given to purpose, chief characteristics, and availability of products and materials. Endorsement by Science or AAAS of any products or materials mentioned is not
implied. Additional information may be obtained from the manufacturer or supplier.
Apoptosis/Necrosis Detection KitEnzo Life Scienceís GFP-CERTIFIED Apoptosis/Necrosis Detection Kit
detects four distinct cell states: viable cells, early apoptotic cells, late
apoptotic cells, and necrotic cells. Plasma membrane integrity and the
display of phosphatidylserine on the plasma membraneís extracellular
face are both hallmarks of apoptosis, and are used in this kit to
distinguish apoptosis from necrosis. The kit features true multiplexing
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understanding the development, homeostasis, and pathogenesis of
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neuroscience research, drug discovery, and more.
Enzo Life Sciences
For info: 800-942-0430
www.enzolifesciences.com
Spinning Disk Confocal Superresolution MicroscopeWith resolving power that surpasses the limits of conventional optical
microscopes, the Olympus IXplore SpinSR10 imaging system balances
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frame rate and 120-nm XY resolution enable researchers to observe the
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into regions that can be hard to access using traditional superresolution
methods. A suite of features helps minimize phototoxicity and
photobleaching when capturing 3D images, prolonging the viability
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so live-cell, superresolution images can be captured using conventional
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Olympus
For info: 781-419-3900
www.olympus-lifescience.com/advanced-imaging-solutions/spinsr10
Plate Reader
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well-imaging along with label-free and labeled detection technologies.
Its imaging module and easy-to-use Kaleido 2.0 data acquisition and
analysis software allow you to image a 384-well plate in less than 5
min., a two-color image in around 6 min., and a three-color image in
around 7 min. With its image cytometry capabilities, you can quickly
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cells, performing end-point assays, or taking kinetic measurements over
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speed makes it ideal for assay optimization and for assessing cell-based
assay quality to reveal cell-seeding errors, improper liquid handling, and
bacterial contamination. You can use ready-made, click-and-go protocols
or build your own from the toolbox, and the plate stacker gives you the
convenience of walkaway operation.
PerkinElmer
For info: 800-762-4000
www.perkinelmer.com/ensight/index.html
Live-Cell Incubator ImagerEtalumaís high-resolution, versatile, and compact inverted LS
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you stable temperatures and CO2 levels throughout your assays. They
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an automated XY stage with autofocus in Z (LS720) or a manual stage
(LS620) and get images, time-lapse series, and videos recorded directly
to your computer. These fully functioning microscopes empower users
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chips, or custom labware. Their quality is comparable to that of
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without the big-ticket price.
Etaluma
For info: 760-298-2355
www.etaluma.com/live-cell
Digital Imaging SystemThe CELENA S is a small, powerful digital imaging system that
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semiconductor (CMOS) camera, and a computer with user-friendly
software, it allows researchers to capture vivid, publication-quality
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accommodate a wide range of imaging needs. Researchers can
use the CELENA S for multiple applications, such as capturing and
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automated cell counting. The new onstage incubation system features
an environmental chamber, temperature controller, and gas mixer.
Researchers can control temperature, humidity, and gas content with
precision. Live cells can be monitored with the time-lapse function or the
growth monitor.
Logos Biosystems
For info: +82-(31)-478-4185
logosbio.com/digital_microscope/CELENA_S/features.php
1549SCIENCE sciencemag.org/custom-publishing
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