Results 1 - 10 of 95

Do-it-yourself guide: How to use the modern single-molecule toolkit.

by Nils G Walter , Cheng-Yen Huang , Anthony J Manzo , Mohamed A Sobhy - Nat. Methods , 2008
"... Single-molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state of the art in single-molecule tools including fluorescence spectroscopy, ..."
Abstract - Cited by 23 (3 self) - Add to MetaCart
Single-molecule microscopy has evolved into the ultimate-sensitivity toolkit to study systems from small molecules to living cells, with the prospect of revolutionizing the modern biosciences. Here we survey the current state of the art in single-molecule tools including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy. We also provide guidelines for choosing the right approach from the available single-molecule toolkit for applications as diverse as structural biology, enzymology, nanotechnology and systems biology.

High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy. Chromosome Res

by Jürgen Reymann , David Baddeley , Manuel Gunkel , Paul Lemmer , Werner Stadter , Thibaud Jegou , Karsten Rippe , Christoph Cremer , Udo Birk
"... Abstract Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved i ..."
Abstract - Cited by 18 (8 self) - Add to MetaCart
Abstract Spatially modulated illumination (SMI) microscopy is a method of wide field fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale architecture in fluorescently labelled cells. The first prototype of the SMI microscope proved its applicability to a wide range of biological questions. For the SMI live cell imaging this system was enhanced in terms of the development of a completely new upright configuration. This so called Vertico-SMI transfers the advantages of SMI nanoscaling to vital biological systems, and is shown to work consistently at different temperatures using both oil-and waterimmersion objective lenses. Furthermore, we increased the speed of data acquisition to minimize errors in the detection signal resulting from cellular or object movement. By performing accurate characterization, the present Vertico-SMI now offers a fully-fledged microscope enabling a complete three-dimensional (3D) SMI data stack to be acquired in less than 2 seconds. We have performed live cell measurements of a tet-operator repeat insert in U2OS cells, which provided the first in vivo signatures of subnuclear complexes. Furthermore, we have successfully implemented an optional optical configuration allowing the generation of high-resolution localization microscopy images of a nuclear pore complex distribution. Abbreviations
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Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

by Graham T. Dempsey, Joshua C. Vaughan, Kok Hao Chen, Mark Bates, Xiaowei Zhuang - Nat. Methods , 2011
"... One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes — the properties of the probes, including photons per switching event, on/o ..."
Abstract - Cited by 16 (0 self) - Add to MetaCart
One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes — the properties of the probes, including photons per switching event, on/off duty cycle, photostability, and number of switching cycles, largely dictate the quality of super-resolution images. While many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here, we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides a set of guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low crosstalk, four-color super-resolution imaging.

High Accuracy 3D Quantum Dot Tracking with Multifocal Plane Microscopy for the Study of Fast Intracellular Dynamics in Live Cells

by Sripad Ram, Prashant Prabhat, Y Jerry Chao, Y E. Sally Ward, Raimund J. Ober*y - Biophys. J , 2008
"... ABSTRACT Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Prev ..."
Abstract - Cited by 11 (0 self) - Add to MetaCart
ABSTRACT Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.
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Q&A: Single-molecule localization microscopy for biological imaging

by A L McEvoy, D Greenfield, M Bates, J Liphardt - BMC Biol , 2010
"... ..."
Abstract - Cited by 4 (0 self) - Add to MetaCart
Abstract not found
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Fast three-dimensional super-resolution imaging of live cells

by Sara A. Jones, Sang-hee Shim, Jiang He, Xiaowei Zhuang
"... We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolutions using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and three-dimensional (3D) ..."
Abstract - Cited by 4 (0 self) - Add to MetaCart
We report super-resolution fluorescence imaging of live cells with high spatiotemporal resolutions using stochastic optical reconstruction microscopy (STORM). By labeling proteins either directly or via SNAP tags with photoswitchable dyes, we obtained two-dimensional (2D) and three-dimensional (3D) super-resolution images of living cells, using clathrin-coated pits and the transferrin cargo as model systems. Bright, fast switching probes enabled us to achieve 2D imaging at spatial resolutions of ~25 nm and temporal resolutions as fast as 0.5 sec. We also demonstrated live-cell 3D volumetric super-resolution imaging. A 3D spatial resolution of ~30 nm in the lateral directions and ~50 nm in the axial direction was obtained at time resolutions down to 1 – 2 sec with several independent snapshots. Using photoswitchable dyes with distinct emission wavelengths, we further demonstrated two-color 3D super-resolution imaging in live cells. These imaging capabilities open a new window for characterizing cellular structures in living cells at the ultrastructural level. The development of super-resolution fluorescence microscopy has allowed the diffraction-limited resolution to be surpassed1–2. This advancement has been achieved either by

Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophysical journal

by Ilaria Testa, Christian A. Wurm, Rebecca Medda, Ellen Ro, L Ch , 1016
"... de tur r s da ple g o str me egl state by transferring them in a dark or a nonexcitable fluo- in very sparsely labeled samples (19–22). Fortunately, process, these approaches have been widely used becauseSubmitted May 24, 2010, and accepted for publication August 10, 2010. ..."
Abstract - Cited by 4 (0 self) - Add to MetaCart
de tur r s da ple g o str me egl state by transferring them in a dark or a nonexcitable fluo- in very sparsely labeled samples (19–22). Fortunately, process, these approaches have been widely used becauseSubmitted May 24, 2010, and accepted for publication August 10, 2010.
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Electrons, Photons, and Force: Quantitative Single-Molecule Measurements from Physics to Biology

by Shelley A. Claridge, Jeffrey J. Schwartz, Paul S. Weiss
"... The ability to measure the structure and behavior of single molecules has evolved in parallel in fields ranging from physics to biology. Molecules studied vary widely: from small molecules less than 1 nm in diameter to DNA molecules with lengths of several thousand nanometers. Some measurements are ..."
Abstract - Cited by 2 (0 self) - Add to MetaCart
The ability to measure the structure and behavior of single molecules has evolved in parallel in fields ranging from physics to biology. Molecules studied vary widely: from small molecules less than 1 nm in diameter to DNA molecules with lengths of several thousand nanometers. Some measurements are performed at cryogenic temperatures (often near 1 K) and ultrahigh vacuum (10-9 Torr), others in liquid at room temperature. Some experiments measure the molecule directly, while others require attachment of a high-contrast label, which may be orders of magnitude

Single-Molecule and Superresolution Imaging in Live Bacteria Cells

by Julie S. Biteen, W. E. Moerner , 2010
"... Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobact ..."
Abstract - Cited by 2 (0 self) - Add to MetaCart
Single-molecule imaging enables biophysical measurements devoid of ensemble averaging, gives enhanced spatial resolution beyond the diffraction limit, and permits superresolution reconstructions. Here, single-molecule and superresolution imaging are applied to the study of proteins in live Caulobacter crescentus cells to illustrate the power of these methods in bacterial imaging. Based on these techniques, the diffusion coefficient and dynamics of the histidine protein kinase PleC, the localization behavior of the polar protein PopZ, and the treadmilling behavior and protein superstructure of the structural protein MreB are investigated with sub-40-nm spatial resolution, all in live cells. Since its advent 20 years ago, single-moleculefluorescence imaging has given rise to a host of exciting experiments (Ambrose andMoerner 1991). Beyond enabling fundamental investi-gations of the physics of emissive molecules, one main advantage of this technique is its use in biologically relevant, live-cell experiments. Optical fluorescence microscopy is an impor-
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Structural analysis of herpes simplex virus by optical super-resolution imaging

by Romain F. Laine, Anna Albecka, Sebastian Van De Linde, Eric J. Rees, Colin M. Crump, Clemens F. Kaminski - Nat. Commun. 2015
"... Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-reso-lution imaging by direct stochastic optical ..."
Abstract - Cited by 2 (1 self) - Add to MetaCart
Herpes simplex virus type-1 (HSV-1) is one of the most widespread pathogens among humans. Although the structure of HSV-1 has been extensively investigated, the precise organization of tegument and envelope proteins remains elusive. Here we use super-reso-lution imaging by direct stochastic optical reconstruction microscopy (dSTORM) in combi-nation with a model-based analysis of single-molecule localization data, to determine the position of protein layers within virus particles. We resolve different protein layers within individual HSV-1 particles using multi-colour dSTORM imaging and discriminate envelope-anchored glycoproteins from tegument proteins, both in purified virions and in virions present in infected cells. Precise characterization of HSV-1 structure was achieved by particle aver-aging of purified viruses and model-based analysis of the radial distribution of the tegument proteins VP16, VP1/2 and pUL37, and envelope protein gD. From this data, we propose a model of the protein organization inside the tegument.