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Page 1: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May
Page 2: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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Page 3: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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Page 4: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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Page 5: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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MilliporeSigma in the U.S. and Canada.

MilliporeSigma, the vibrant M, Milli-Q, Millipore, SAFC, BioReliance,Supelco and Sigma-Aldrich are trademarks of Merck KGaA, Darmstadt,Germany or its afliates. All other trademarks are the property of theirrespective owners. Detailed information on trademarks is available viapublicly accessible resources.

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Page 6: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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Page 7: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

LIFE SCIENCE TECHNOLOGIES

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����3��3$�3��3����� �2�3���3���5��3By Amber Dance

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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Page 8: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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.

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Page 9: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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.

Page 10: Revolutionizing your - Science · 2018-07-11 · Produced by the Science/AAAS Custom Publishing Office M I 2345335 33 3 3 2 3 33 2 3 333 ... Molecular biology: PCR anniversary—May

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