Breaking the Diffraction Barrier: Super-Resolution Imaging of Cells

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


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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    • "However, the requirement of sample embedding for high-quality serial sectioning poses extra challenges for super-resolution imaging. Since STORM imaging relies on switching and localization of individual fluorophores to reconstruct super-resolution images (Huang et al., 2010; Rust et al., 2006), the resolution of a STORM image depends not only on the localization precision of individual fluorophores determined by their photon output but also on the localization density determined by the labeling density. Achieving optimal STORM resolution thus requires the labeling and embedding conditions to simultaneously retain optimal fluorophore properties and high-density labeling in resin-embedded samples. "
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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. "
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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.
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