PDF - In This Issue

All for one and one for all - p111

doi:10.1038/nmeth0209-111

The development of large-scale centralized biobanks raises the stakes in a familiar conversation on ethics in medical research and poses unique challenges to lawmakers that will require informed discussions between scientists and the public.

Abstract - | Full Text - All for one and one for all | PDF (87 KB) - All for one and one for all

Of active polymerases and antisense transcripts - p113

Nicole Rusk

doi:10.1038/nmeth0209-113

Methods to map active RNA polymerases and to assign transcripts to the sense or antisense strand are valuable additions to the functional analysis of the transcriptome.

Abstract - | Full Text - Of active polymerases and antisense transcripts | PDF (370 KB) - Of active polymerases and antisense transcripts

Sequencing in a flash - pp114 - 115

Michael Eisenstein

doi:10.1038/nmeth0209-114a

A new system capable of simultaneously monitoring thousands of individual polymerase enzymes enables accurate, multiplex DNA sequencing in real time.

Abstract - | Full Text - Sequencing in a flash | PDF (238 KB) - Sequencing in a flash

A measure of floppiness - pp114 - 115

Irene Kaganman

doi:10.1038/nmeth0209-114b

By analyzing the data generated by the Northeast Structural Genomics Consortium (NESG), researchers quantified the physical properties that control protein crystallization.

Abstract - | Full Text - A measure of floppiness | PDF (238 KB) - A measure of floppiness

News in brief - p115

doi:10.1038/nmeth0209-115

Full Text - News in brief | PDF (123 KB) - News in brief

Imaging goes label-free - p116

Allison Doerr

doi:10.1038/nmeth0209-116

Researchers develop a high-sensitivity, label-free imaging technology based on stimulated Raman scattering.

Abstract - | Full Text - Imaging goes label-free | PDF (376 KB) - Imaging goes label-free

When is a stem cell specified? - p118

Natalie de Souza

doi:10.1038/nmeth0209-118

Using time-lapse imaging, researchers study the potency and fate specification of live neural stem cells in culture.

Abstract - | Full Text - When is a stem cell specified? | PDF (128 KB) - When is a stem cell specified?

Photoactive channels obey stoplights - p120

Amy Donner

doi:10.1038/nmeth0209-120

Kinetically bi-stable channelrhodopsin-2 variants enable sensitive, step-like and reversible photoexcitability in neurons.

Abstract - | Full Text - Photoactive channels obey stoplights | PDF (126 KB) - Photoactive channels obey stoplights

Pairing cells to enhance fusion - pp123 - 124

Peter W Zandstra

doi:10.1038/nmeth0209-123

A microfluidic device enhances the efficiency of cell membrane fusion by increasing the efficiency of cell pairing.

Abstract - | Full Text - Pairing cells to enhance fusion | PDF (626 KB) - Pairing cells to enhance fusion

See also: Article by Skelley et al.

Red lights, camera, photoactivation! - pp124 - 125

Samuel T Hess

doi:10.1038/nmeth0209-124

Two groups present new photoactivatable fluorescent proteins that will be useful for super-resolution fluorescence microscopy.

Abstract - | Full Text - Red lights, camera, photoactivation! | PDF (302 KB) - Red lights, camera, photoactivation!

See also: Brief Communication by McKinney et al. | Article by Subach et al.

Genetically timed, activity-sensor and rainbow transsynaptic viral tools - pp127 - 130

Zsolt Boldogki, Kamill Balint, Gautam B Awatramani, David Balya, Volker Busskamp, Tim James Viney, Pamela S Lagali, Jens Duebel, Emese Pásti, Dóra Tombácz, Judit S Tóth, Irma F Takács, Brigitte Gross Scherf & Botond Roska

doi:10.1038/nmeth.1292

Pseudorabies viruses encoding fluorescent proteins are a powerful method for mapping neuronal circuits. Now a series of pseudorabies virus strains encoding fluorescent sensors and time-shifted florescent proteins allow dissection of complex circuits with concurrent activity analysis while defining an analysis period during which the neurons are still healthy.

Abstract - | Full Text - Genetically timed, activity-sensor and rainbow transsynaptic viral tools | PDF (401 KB) - Genetically timed, activity-sensor and rainbow transsynaptic viral tools | Supplementary information

A bright and photostable photoconvertible fluorescent protein - pp131 - 133

Sean A McKinney, Christopher S Murphy, Kristin L Hazelwood, Michael W Davidson & Loren L Looger

doi:10.1038/nmeth.1296

An improved version of the green-to-red photoconverting EosPF is presented. mEos2 is a functional fusion partner for many cellular proteins and retains the high localization precision of EosFP in super-resolution imaging. Also in this issue, Subach et al. present an inducible mCherry variant for super-resolution imaging.

Abstract - | Full Text - A bright and photostable photoconvertible fluorescent protein | PDF (369 KB) - A bright and photostable photoconvertible fluorescent protein | Supplementary information

See also: News and Views by Hess

Large-scale profiling of protein palmitoylation in mammalian cells - pp135 - 138

Brent R Martin & Benjamin F Cravatt

doi:10.1038/nmeth.1293

S-palmitoylation is a protein post-translational modification involved in trafficking, compartmentalization and membrane-tethering of various proteins. A palmitic acid analog, 17-octadecynoic acid, serves as a metabolically incorporated bioorthogonal click chemistry probe for detecting S-palmitoylated human proteins by mass spectrometry or in-gel fluorescence.

Abstract - | Full Text - Large-scale profiling of protein palmitoylation in mammalian cells | PDF (198 KB) - Large-scale profiling of protein palmitoylation in mammalian cells | Supplementary information

miRNA in situ hybridization in formaldehyde and EDC–fixed tissues - pp139 - 141

John T G Pena, Cherin Sohn-Lee, Sara H Rouhanifard, Janos Ludwig, Markus Hafner, Aleksandra Mihailovic, Cindy Lim, Daniel Holoch, Philipp Berninger, Mihaela Zavolan & Thomas Tuschl

doi:10.1038/nmeth.1294

Conventional in situ hybridization protocols lead to loss of microRNAs, which diffuse out of the formaldehyde-fixed sample owing to their small size. Adding a carbodiimide that stably links the microRNA with the protein matrix around it prevents this diffusion and allows detection of miRNAs at very low expression levels.

Abstract - | Full Text - miRNA in situ hybridization in formaldehyde and EDC–fixed tissues | PDF (584 KB) - miRNA in situ hybridization in formaldehyde and EDC–fixed tissues | Supplementary information

In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts - pp143 - 145

Jean Y Perentes, Trevor D McKee, Carsten D Ley, Hannah Mathiew, Michelle Dawson, Timothy P Padera, Lance L Munn, Rakesh K Jain & Yves Boucher

doi:10.1038/nmeth.1295

A combination of multiphoton laser scanning microscopy and second harmonic generation (SHG) imaging is used to track tumor associated fibroblasts and extracellular matrix remodeling in living mice. The SHG signal is used for image registration over several days.

Abstract - | Full Text - In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts | PDF (241 KB) - In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts | Supplementary information

Microfluidic control of cell pairing and fusion - pp147 - 152

Alison M Skelley, Oktay Kirak, Heikyung Suh, Rudolf Jaenisch & Joel Voldman

doi:10.1038/nmeth.1290

A device that traps cells in micrometer-sized capture cups efficiently achieves cell pairing and subsequent chemically or electrically induced fusion. The authors show that fused NIH 3T3 cells continue to be viable and morphologically normal off the chip, and they also show reprogramming of mouse embryonic fibroblasts after fusion with embryonic stem cells.

Abstract - | Full Text - Microfluidic control of cell pairing and fusion | PDF (638 KB) - Microfluidic control of cell pairing and fusion | Supplementary information

See also: News and Views by Zandstra

Photoactivatable mCherry for high-resolution two-color fluorescence microscopy - pp153 - 159

Fedor V Subach, George H Patterson, Suliana Manley, Jennifer M Gillette, Jennifer Lippincott-Schwartz & Vladislav V Verkhusha

doi:10.1038/nmeth.1298

Improved photoactivatable red fluorescent proteins are generated by including properties desirable for photoactivated localization microscopy (PALM) as selection criteria. The PAmCherry proteins are superior tags for one- and two-color PALM in fixed cells, among other applications. Also in this issue, McKinney et al. present an improved version of the green-to-red EosFP protein.

Abstract - | Full Text - Photoactivatable mCherry for high-resolution two-color fluorescence microscopy | PDF (690 KB) - Photoactivatable mCherry for high-resolution two-color fluorescence microscopy | Supplementary information

See also: News and Views by Hess

A genetically encoded fluorescent reporter of ATP:ADP ratio - pp161 - 166

Jim Berg, Yin Pun Hung & Gary Yellen

doi:10.1038/nmeth.1288

A ratiometric fluorescent sensor that reports the ATP/ADP concentration ratio in living cells was created by fusing the bacterial regulatory protein GlnK1 to a circularly permuted fluorescent protein. The sensor detected inhibition of cellular metabolism caused by transient removal of glucose from the cellular medium or administration of a glycolytic inhibitor.

Abstract - | Full Text - A genetically encoded fluorescent reporter of ATP:ADP ratio | PDF (356 KB) - A genetically encoded fluorescent reporter of ATP:ADP ratio | Supplementary information

Probing the mechanical architecture of the vertebrate meiotic spindle - pp167 - 172

Takeshi Itabashi, Jun Takagi, Yuta Shimamoto, Hiroaki Onoe, Kenta Kuwana, Isao Shimoyama, Jedidiah Gaetz, Tarun M Kapoor & Shin'ichi Ishiwata

doi:10.1038/nmeth.1297

A piezo-resistive dual-cantilever system is combined with fluorescence imaging to examine the mechanical features of the in vitro–assembled vertebrate meiotic spindle.

Abstract - | Full Text - Probing the mechanical architecture of the vertebrate meiotic spindle | PDF (1,223 KB) - Probing the mechanical architecture of the vertebrate meiotic spindle | Supplementary information

Biobanking: freezer burn - pp173 - 178

Nathan Blow

doi:10.1038/nmeth0209-173

Biobanking is gaining momentum as more and more patient samples and clinical information are being stored in facilities around the globe. New technology is helping everyone—from national efforts to smaller research laboratories—to process and track their biospecimen collections.

Abstract - | Full Text - Biobanking: freezer burn | PDF (1,430 KB) - Biobanking: freezer burn

High-speed, low-photodamage nonlinear imaging using passive pulse splitters - p179

Na Ji, Jeffrey C Magee & Eric Betzig

doi:10.1038/nmeth0209-179

Full Text - High-speed, low-photodamage nonlinear imaging using passive pulse splitters | PDF (35 KB) - High-speed, low-photodamage nonlinear imaging using passive pulse splitters