Article

Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
(Impact Factor: 32.24). 12/2010; 143(7):1047-58. DOI: 10.1016/j.cell.2010.12.002
Source: PubMed

ABSTRACT Anyone who has used a light microscope has wished that its resolution could be a little better. Now, after centuries of gradual improvements, fluorescence microscopy has made a quantum leap in its resolving power due, in large part, to advancements over the past several years in a new area of research called super-resolution fluorescence microscopy. In this Primer, we explain the principles of various super-resolution approaches, such as STED, (S)SIM, and STORM/(F)PALM. Then, we describe recent applications of super-resolution microscopy in cells, which demonstrate how these approaches are beginning to provide new insights into cell biology, microbiology, and neurobiology.

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• "It should be noted that all results in the present study were obtained by using a conventional confocal microscope. With the help of new super-resolution confocal microscopic techniques (Huang et al., 2010), dRBFC many assist in discovering more exciting details about the localization and trafficking of the viral VRC that carries out active viral replication. "
Article: Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation
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ABSTRACT: Double-stranded RNA (dsRNA) is an important type of RNA that plays essential roles in diverse cellular processes in eukaryotic organisms and a hallmark in infections by positive-sense RNA viruses. Currently, no in vivo technology has been developed for visualizing dsRNA in living cells. Here, we report a dsRNA binding-dependent fluorescence complementation (dRBFC) assay that can be used to efficiently monitor dsRNA distribution and dynamics in vivo. The system consists of two dsRNA-binding proteins, which are fused to the N- and C-terminal halves of the yellow fluorescent protein (YFP). Binding of the two fusion proteins to a common dsRNA brings the split YFP halves in close proximity, leading to the reconstitution of the fluorescence-competent structure and restoration of fluorescence. Using this technique, we were able to visualize the distribution and trafficking of the replicative RNA intermediates of positive-sense RNA viruses in living cells.
Virology 11/2015; 485:439-451. DOI:10.1016/j.virol.2015.08.023 · 3.32 Impact Factor
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• "Among the methods of acquiring super-resolution fluorescence images [1] [2], structured illumination microscopy (SIM) offers a relatively modest, twofold resolution improvement over widefield microscopy [3]. However, as SIM uses only a relatively small number of widefield images to capture the information required to improve resolution, it is in principle more suitable for live sample imaging; SIM offers the advantages of fast acquisition over a large area and weaker irradiation of the sample compared to alternative techniques such as stimulated emission depletion [4] and single-molecule localisation [5] [6] [7], and it is compatible with all fluorophores used in widefield and confocal imaging. "
Article: Construction of an instant structured illumination microscope
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ABSTRACT: A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The "instant structured illumination microscope" (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 μm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x-y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation. Copyright © 2015. Published by Elsevier Inc.
Methods 07/2015; 316. DOI:10.1016/j.ymeth.2015.07.012 · 3.65 Impact Factor
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• "This analysis discards the information from crowded molecules with overlapping images through filtering. Typically, SMLM requires accumulating thousands of frames to generate a super-resolution image, resulting in the long image acquisition time [16] [19] [20]. "
Article: Super-resolved imaging with ultimate time resolution
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ABSTRACT: Precisely and accurately locating point objects is a long-standing common thread in science. Super-resolved imaging of single molecules has revolutionized our view of quasi-static nanostructures $\it{in-vivo}$. A wide-field approach based on localizing individual fluorophores has emerged as a versatile method to surpass the standard resolution limit. In those techniques, the super-resolution is realized by sparse photoactivation and localization together with the statistical analysis based on point spread functions. Nevertheless, the slow temporal resolution of super-resolved imaging severely restricts the utility to the study of live-cell phenomena. Clearly, a major breakthrough to observe fast, nanoscale dynamics needs to be made. Here we present a super-resolved imaging method that achieves the theoretical-limit time resolution. By invoking information theory, we can achieve the robust localization of overlapped light emitters at an order of magnitude faster speed than the conventional super-resolution microscopy. Our method thus provides a general way to uncover hidden structures below the diffraction limit and should have a wide range of applications in all disciplines of science and technology.