Hasil untuk "Microscopy"

J. Goldstein

Marius Pachitariu, C. Stringer, Mario Dipoppa et al.

Two-photon microscopy of calcium-dependent sensors has enabled unprecedented recordings from vast populations of neurons. While the sensors and microscopes have matured over several generations of development, computational methods to process the resulting movies remain inefficient and can give results that are hard to interpret. Here we introduce Suite2p: a fast, accurate and complete pipeline that registers raw movies, detects active cells, extracts their calcium traces and infers their spike times. Suite2p runs on standard workstations, operates faster than real time, and recovers ~2 times more cells than the previous state-of-the-art method. Its low computational load allows routine detection of ~10,000 cells simultaneously with standard two-photon resonant-scanning microscopes. Recordings at this scale promise to reveal the fine structure of activity in large populations of neurons or large populations of subcellular structures such as synaptic boutons.

Bi-Chang Chen, W. Legant, Kai Wang et al.

L. Gremer, Daniel Schölzel, Carla Schenk et al.

M. Liao, E. Cao, D. Julius et al.

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5–6 (S5–S6) and the intervening pore loop, which is flanked by S1–S4 voltage-sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with a short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.

J. Schnitzbauer, Maximilian T. Strauss, Thomas Schlichthaerle et al.

K. Dunn, M. Kamocka, J. McDonald

Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the "colocalization" of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software.

Y. Dufrêne, T. Ando, Ricardo Garcia et al.

Hongda Wang, Y. Rivenson, Yiyin Jin et al.

K. Neuman, A. Nagy

Tony J. Collins

ImageJ is an essential tool for us that fulfills most of our routine image processing and analysis requirements. The near-comprehensive range of import filters that allow easy access to image and meta-data, a broad suite processing and analysis routine, and enthusiastic support from a friendly mailing list are invaluable for all microscopy labs and facilities-not just those on a budget.

M. Krieg, Gotthold Fläschner, D. Alsteens et al.

G. Binning, H. Rohrer, C. Gerber et al.

J. Huisken, J. Swoger, F. Del Bene et al.

R. Henderson, J. Baldwin, T. Ceska et al.

G. Vicidomini, P. Bianchini, A. Diaspro

N. Gostling, JH Cheung, T. Roose et al.

Yaron M Sigal, Ruobo Zhou, X. Zhuang

Super-resolution microscopy has overcome a long-held resolution barrier—the diffraction limit—in light microscopy and enabled visualization of previously invisible molecular details in biological systems. Since their conception, super-resolution imaging methods have continually evolved and can now be used to image cellular structures in three dimensions, multiple colors, and living systems with nanometer-scale resolution. These methods have been applied to answer questions involving the organization, interaction, stoichiometry, and dynamics of individual molecular building blocks and their integration into functional machineries in cells and tissues. In this Review, we provide an overview of super-resolution methods, their state-of-the-art capabilities, and their constantly expanding applications to biology, with a focus on the latter. We will also describe the current technical challenges and future advances anticipated in super-resolution imaging.

Catxere A. Casacio, L. Madsen, Alex Terrasson et al.

C. Peddie, C. Genoud, A. Kreshuk et al.

Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging. Volume electron microscopy techniques are high-resolution imaging approaches that reveal the 3D structure of cells, tissues and small model organisms at nanometre resolution. This Primer introduces the different imaging modalities, specialized sample processing and key applications in biosciences.