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Introducing the NEBNext Direct Cancer HotSpot Panel Using a unique approach, the NEBNext Direct Cancer HotSpot Panel enriches for 190 common cancer targets from 50 genes prior to next generation sequencing. Combining a novel method for hybridization-based target enrichment with library preparation, the NEBNext Direct technology reduces processing time and minimizes sample loss. Ideal for automation, NEBNext Direct enables highly-specific deep sequencing of genomic regions of interest for the discovery and identification of low frequency variants from challenging sample types. Visit NEBNextDirect.com to learn more and to inquire about sampling this product. Target with precision. TARGETS INCLUDE REGIONS FROM THE FOLLOWING CANCER-RELATED GENES, INCLUDING >18,000 COSMIC FEATURES: ABL1 EGFR GNAQ KRAS PTPN11 AKT1 ERBB2 GNAS MET RB1 ALK ERBB4 HNF1A MLH1 RET APC EZH2 HRAS MPL SMAD4 ATM FBXW7 IDH1 NOTCH1 SMARCB1 BRAF FGFR1 IDH2 NPM1 SMO CDH1 FGFR2 JAK2 NRAS SRC CDKN2A FGFR3 JAK3 PDGFRA STK11 CSF1R FLT3 KDR PIK3CA TP53 CTNNB1 GNA11 KIT PTEN VHL For research use only; not intended for diagnostic use. NEW ENGLAND BIOLABS ® and NEB ® are registered trademarks of New England Biolabs, Inc. NEBNEXT DIRECT is a trademark of New England Biolabs, Inc.

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Page 1: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

Introducing theNEBNext Direct™

Cancer HotSpot PanelUsing a unique approach, the NEBNext DirectCancer HotSpot Panel enriches for 190 commoncancer targets from 50 genes prior to next generationsequencing. Combining a novel method forhybridization-based target enrichment with librarypreparation, the NEBNext Direct technology reducesprocessing time and minimizes sample loss. Ideal forautomation, NEBNext Direct enables highly-specificdeep sequencing of genomic regions of interest for thediscovery and identification of low frequency variantsfrom challenging sample types.

Visit NEBNextDirect.com to learn moreand to inquire about sampling this product.

Target withprecision.

TARGETS INCLUDE REGIONS FROM THE FOLLOWING

CANCER-RELATED GENES, INCLUDING >18,000

COSMIC FEATURES:

ABL1 EGFR GNAQ KRAS PTPN11

AKT1 ERBB2 GNAS MET RB1

ALK ERBB4 HNF1A MLH1 RET

APC EZH2 HRAS MPL SMAD4

ATM FBXW7 IDH1 NOTCH1 SMARCB1

BRAF FGFR1 IDH2 NPM1 SMO

CDH1 FGFR2 JAK2 NRAS SRC

CDKN2A FGFR3 JAK3 PDGFRA STK11

CSF1R FLT3 KDR PIK3CA TP53

CTNNB1 GNA11 KIT PTEN VHL

For research use only; not intended for diagnostic use.

NEW ENGLAND BIOLABS® and NEB® are registered trademarks of New England Biolabs, Inc.

NEBNEXT DIRECT™ is a trademark of New England Biolabs, Inc.

Page 2: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

Don’t miss the debut of

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Science is expanding its reach into immunology—now

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Page 3: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

2015 Winner

Shigeki Watanabe, Ph.D.

Johns Hopkins University

For research on synaptic

vesicle endocytosis

Eppendorf & Science Prize for Neurobiology

The annual Eppendorf & Science Prize for Neurobiology

is an international award which honors young scientists

for their outstanding contributions to neurobiological

research based on methods of molecular and cell biology.

The winner and finalists are selected by a committee

of independent scientists, chaired by Science’s Senior

Editor, Dr. Peter Stern. Researchers who are not older

than 35 years are invited to apply.

You could be next to win this prize and to receive

> Prize money of US$25,000

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> Full support to attend the Prize Ceremony held in

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> An invitation to visit Eppendorf in Hamburg, Germany

It’s easy to apply!

Learn more about the Prize and its past winners at:

www.eppendorf.com/prize

Call for EntriesApplication Deadline

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AAAS®andScience®are

registeredtrademarksoftheAmericanAssociationfortheAdvancementofScience,USA.Eppendorf®andtheEppendorflogoare

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Page 4: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

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Page 5: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

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“A compelling read filled with big, bold ideas.”

—Brian J. Enquist, Nature

“Sean Carroll is one of our great science writers. . . . This is a

visionary book.”

—Peter Forbes, The Guardian

“Unbiased, comprehensible information on radiation risk is

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“With stunning illustrations and accessible text, Silent Sparks

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messages and reveals what has been learned about these

beloved beetles through the sleuthing of researchers.”

—Bernd Heinrich, author of The Homing Instinct

Produced by the Science/AAAS Custom Publishing Office

Upcoming Features

Exosomes/MicrovesiclesóJune 10

ProteomicsóJuly 15

GenomicsóOctober 7

LIFE SCIENCE TECHNOLOGIES

SUPERRESOLUTION MICROSCOPY

The latest superresolution

microscopes (GSDIM, PALM, SIM,

STED, STORM) bend a centuries-old

rule in the �eld as well as researchersí

minds on cellular structures they

thought they knew.

See the full story on page 850.

Superresolutionmicroscopy

Page 6: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

850 sciencemag.org/custom-publishing SCIENCE

LIFE SCIENCE TECHNOLOGIES

SUPERRESOLUTION MICROSCOPY

Produced by the Science/AAAS Custom Publishing Office

No, there hasnít been a breakdown in copy editing,

this sentence really does end with two periods..

Your brain caught that apparent mistake because

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power to distinguish objects spaced that close together.

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constrains the microscopeís power, and at some point, two

objects will appear as one.

In 2006, Xiaowei Zhuangís team at Harvard UniversityF���CF

described the superresolution technique known as stochastic

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the technique to make it multicolor, higher resolution, and 3D.

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Zhuang, a Howard Hughes Medical Institute Investigator and

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tion, light arising from a single molecule produces not a circle

but an ellipse, which indicates the moleculeís axial position

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further by positioning objectives above and below the sample,

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campal neurons, they discovered something completely unex�

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copy, actin in the axons of these neurons appears as a uniform,

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beneath the plasma membrane of the axonal shaft they discov�

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cell can precisely position key membrane proteins, such as

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pulses. ìHaving these very important signaling molecules being

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Biology and Biomedical Engineering at the Yale University

School of Medicine, who builds superresolution microscopes

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body had seen these actin rings before, even though people

had looked with the electron microscope for 20 years to under�

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Superresolution explained

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tute for Biophysical Chemistry�F D�F ��FCDEF���CFC�F��E�,FCDEF

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the same fundamental principle.

ìIf you have freely propagating light, that light will always be

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and ground state depletion microscopy with individual

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and Leica�F�E��E�C�E��A��EF�C��D��C���F��������C����

based methods, using photoactivatable or photoswitchable

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at random while the majority remain dark. Under those

conditions, researchers can be relatively certain that

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molecule, thus making it possible to assign their positions

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complete dataset has been collected.

ìBasically we take advantage of what I typically call the

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Exosomes/MicrovesiclesóJune 10 ProteomicsóJuly 15 GenomicsóOctober 7

Upcoming Features

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ability to resolve two objects is constrained by the wavelength

of the light used to view them. But in 2000, researchers showed

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the next decade an alphabet soup of superresolution tech�

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resulting images are both beautiful and revealing, documenting

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Page 7: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

851SCIENCE sciencemag.org/custom-publishing

LIFE SCIENCE TECHNOLOGIES Produced by the Science/AAAS Custom Publishing Office

SUPERRESOLUTION MICROSCOPY

like an optical zoom,î explains Silvio Rizzoli, a professor at the

University of Gˆttingen, Germany, who uses this technique (as

well as STED and STORM) in his neural synapse research.

���������������������������

Stefan Jakobs, head of the Mitochondrial Structure and

Dynamics Research Group at the Max Planck Institute for

Biophysical Chemistry, uses superresolution methodologies to

study the role mitochondria play in cell death or apoptosis. One

protein, called Bax, was known to induce mitochondrial mem-

brane disruption in response to apoptotic signals. What wasnít

clear was how the protein does that.

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Bax molecules assemble into rings in the mitochondrial outer

membrane, like diaphanous green halos crowning the red mem-

brane. That structure explains how Bax translocation during

apoptosis can cause the mitochondrial contents to spill, Jakobs

says. ìIt appears that these rings represent pores,î he explains.

Yet they were impossible to discern via conventional micros-

copy, in which the rings appear as ìa smear.î

To see those rings, Jakobs used anti-Bax antibodies labeled

��������78������������ ����88���78���������8���!8��7���-

resolution microscopy can be hard to come by. ìYou want to

have fast image acquisition and high resolution; you want to

have many colors and low phototoxicity. None of the tech-

niques is currently at the point where all of these parameters

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wavelengths of light. Such proteins are amenable to RESOLFT

and STED live-cell applications, he notes, as they require no

external antibody for labeling. But it is critical not to overex-

press these proteins, he says, as that can lead to artifacts. So,

Jakobsís team has used the gene-editing system known as

CRISPR/Cas9 to couple rsEGFP2 to protein-coding genes in

order to ensure endogenous expression levels. Such work is

ìchallenging,î he admits, ìbut I think it is very important for the

�6�����������8������"�

Another genetic labeling option involves tagging genes with

self-labeling protein tags, such as the SNAP-tag and CLIP-tag

(available from New England BioLabs) or Promegaís

indistinguishable objects are closely positioned in space, she

says, ìwe donít allow them to overlap in time.î

Stimulated emission depletion (STED), reversible saturable

8�����6��78���������������8��$%��+,5�'��������7��7�����6-

lumination microscopy (SIM), conversely, use ìpatterns of light

Ö that determine where the molecules are active and where

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and RESOLFT and cofounded a company called Abberior to

�8�������6�.���78�8��8���!8����8������8�����7�����867-

tion methods. STED, for instance, is usually implemented as a

point-scanning approach that uses a ìdonutî of light, super-

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periphery of the illumination volume to stay dark via a process

called ìstimulated emission,î thereby shrinking the emitting

region to the donutís center. (RESOLFT uses photoswitchable

�78�8��8����8������#����������������'�

According to Jochen Sieber, STED and GSDIM microscopy

product manager at Leica Microsystems, localization systems

������66��8�������������867��8������������8��%��+,5����7��

take far longer as they require the acquisition and merging of

thousands of frames to build a single image. (Indeed, National

Institutes of Health Distinguished Investigator Jennifer Lip-

pincott-Schwartz, in whose lab PALM was invented, notes that

��8�� ����������88���87���8��866���/��8��������������6�!�����

rate is about 5 minutes.)

SIMócommercialized by Nikon, GE Healthcare Life Scienc-

es, and Zeissóis generally regarded as the fastest approach

available and also the easiest, as it requires no special sample

preparation. But at 100 nm, it also yields the poorest resolution.

Recently, Janelia Research Campus Investigator Eric

0��.������8������-�66�����1�66�������28������8!�����!8�����-

versity won the 2014 Nobel Prize in Chemistry for his work

on superresolution, demonstrated that even that limit is not

insurmountable. In late 2015, Betzig and his team documented

two strategies, including ìnonlinear SIM with patterned activa-

tion,î to increase the methodís resolution to about 45 nm. ìMy

personal feeling is that SIM is the superresolution method that

is likely to answer the most biological questions in the next de-

cade,î Betzig says, laughing. ìAnd yet itís the one that wasnít

involved in the Nobel Prize.î

Raman Das, Medical Research Council (MRC) Career De-

velopment Fellow at the University of Manchester, United

Kingdom, answered one such question in 2014 when he used

SIM (among other methods) to document a new form of cell

subdivision called ìapical abscission.î Das was interested in

�8����8�����8����66�������������8��������6��8����������������

���8���7�8�����������������7���0��7���������6������������8��

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����������������66����7�66����7���8��������6676���4����"����8#����

those proteins in one shotóa process that Das likens to closing

a draw-string purse. ìThis results in an acute loss of cell polar-

ity in these cells,î he explains, leading to the formation of func-

tional neuronal architecture.

Perhaps the simplest, and certainly least expensive, super-

resolution option is expansion microscopy. Developed by Ed

Boyden of the Massachusetts Institute of Technology, the

technique improves imaging resolution by encasing samples in

a ìswellable polymerî gel that causes the sample to grow iso-

��8����66����87�� #�!86��6�������57����8��66�������������������

increasing resolution from 200 nm down to 40 nm. ìItís kind of IMA

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When [Zhuangís team] used STORM to zoom in, just beneath

the plasma membrane of the axonal shaft they discovered

a literal cytoskeleton: periodic rings of green actin 180 nm

apart, separated by magenta spectrin spacers.

cont.>

Page 8: Target with precision. · Silent Sparks The Wondrous World of Fireflies Sara Lewis Cloth $29.95 “A compelling read filled with big, bold ideas.” —Brian J. Enquist, Nature “Sean

852 sciencemag.org/custom-publishing SCIENCE

LIFE SCIENCE TECHNOLOGIES

SUPERRESOLUTION MICROSCOPY

Produced by the Science/AAAS Custom Publishing Office

One solution is replacing traditional charge-coupled devices

<==>?������������?��?����� ������������������������

semiconductor (sCMOS) cameras, which can capture thou-

sands of frames per second. In 2013, Bewersdorf and col-

leagues demonstrated noise-handling algorithms that allowed

an sCMOS camera to record superresolved images at up to 32

frames per second, for instance. Today, Leicaís GSDIM instru-

ment includes an sCMOS camera that reduces data acquisition

times to ìwell below a minute,î says product manager Peter

Laskey, thus putting live-cell applications ìwithin reach.î So too

does Nikonís new N-STORM 4.0, which also boasts a brighter

��?�������������������������������?���������������������

ing to general manager Stephen Ross.

Researchers also have devised alternative strategies to

circumvent the speed problem. Melike Lakadamyali, a group

leader at the Institute of Photonic Sciences in Barcelona,

�������������?������?���?�������������?�����������������?��

�� ��������������������������?�����?����������?��?���?�����

������������?������������ ��?����������������������

of a living cell.î

To answer that question, Lakadamyali plays to her systemís

strengths. By recording a live-cell movie of the vesicles at high

temporal (but low spatial) resolution, her team can track vesicle

���������������������?���� �����?�������!"#$���������

microtubule positions.

The researchers found that vesicles tend to pause at mi-

crotubule intersections; the pausing ìcorrelates with tight in-

tersections in which the separation of the two microtubules is

small,î says Lakadamyali. But they also found that motors can

eventually navigate through the intersection. The group is now

using 3D particle tracking to focus on microtubule-associated

����� ������?���������������������������������?����������

skirting roadblocks.

��?���=����#������������%����?����#?�����&��������

Kingís College London, is similarly interested in live-cell local-

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developed an algorithm, called ìBayesian analysis of blinking

and bleaching (3B),î that allows researchers to image more

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tially overlapping, thereby reducing the number of images re-

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looked at systems like cardiac myocytes, [and] been able to

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Still, for all that high resolution can teach, microscopy is

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his samples. Thatís why he has edged away from pure super-

resolution toward ultrahigh-speed imaging with a new (albeit

superresolution-compatible) design called ìlattice light-sheet

microscopy,î soon to be commercialized by Zeiss and 3i. "���?������������������������������������������?(�

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future has never looked brighteróor sharper.

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of interest in live cells by adding cell-permeable dyes linked

to the appropriate substrate. The trick, of course, is that such

dyes must be both bright and cell-permeable, and few current

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at the Institute of Chemical Sciences and Engineering at

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commercialized by Spirochrome.

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rial and microtubules respectively, in live-cell STED and SIM

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in localization superresolution microscopy, the method can be

largely incompatible with live cells.

Featured Participants

Massachusetts Institute of Technologywww.mit.edu

Max Planck Institute for Biophysical Chemistrywww.mpibpc.mpg.de/en

New England BioLabswww.neb.com

National Institutes of Healthwww.nih.gov

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Yale University School of Medicinemedicine.yale.edu

Zeisswww.zeiss.com

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GE Healthcare Life Scienceswww.gelifesciences.com

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