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WIREs Syst Biol Med
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Subdiffractive microscopy: techniques, applications, and challenges

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Cellular processes rely on the precise orchestration of signaling and effector molecules in space and time, yet it remains challenging to gain a comprehensive picture of the molecular organization underlying most basic biological functions. This organization often takes place at length scales below the resolving power of conventional microscopy. In recent years, several ‘superresolution’ fluorescence microscopic techniques have emerged that can surpass the diffraction limit of conventional microscopy by a factor of 2–20. These methods have been used to reveal previously unknown organization of macromolecular complexes and cytoskeletal structures. The resulting high‐resolution view of molecular organization and dynamics is already changing our understanding of cellular processes at the systems level. However, current subdiffractive microscopic techniques are not without limitations; challenges remain to be overcome before these techniques achieve their full potential. Here, we introduce three primary types of subdiffractive microscopic techniques, consider their current limitations and challenges, and discuss recent biological applications. WIREs Syst Biol Med 2014, 6:151–168. doi: 10.1002/wsbm.1259 This article is categorized under: Laboratory Methods and Technologies > Imaging

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The need for subdiffractive imaging and current commercially available subdiffractive approaches. (a) Cartoon of two‐point sources of light separated by 150 nm and their overlapping point‐spread functions (PSFs) (approximated by Gaussians) typical of confocal microscopy. Approximated PSF in the x–z plane is shown on the right at the same scale. (b) Diffraction‐limited PSF overlaid to scale on an electron microscopy (EM) image of a dendritic spine, illustrating the difficulty of diffraction‐limited microscopy in localizing synaptic proteins. (c) The number of publications per year citing the use of each subdiffractive method. See note on page 14 for search terms used to generate this plot (d) Appearance of two‐point sources separated by 150 nm via current commercially available subdiffractive microscopy methods. The PSF cartoons represent the effective precision associated with each method.Data for panel c were collected from scopus.com and limited to articles in journals using the search terms below. Data are plotted through 2012.
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Subdiffractive imaging of protein complexes in eukaryote cell biology and microbiology. (a) An interferometric photoactivated localization microscopy (iPALM) system was used to map the spatial distribution of focal adhesion‐associated proteins in the z dimension. (Reprinted with permission from Ref . Copyright 2010 Nature Publishing Group). (b) Structured illumination microscopy (SIM) was used to examine multiple centrosomal proteins. The images of each protein were aligned and averaged (left), and mapped to the center of the centriole (right). (Reprinted with permission from Ref . Copyright 2012 Nature Publishing Group). (c) Stimulated emission depletion (STED) microscopy combined with optimized sample preparation and antibody labeling showed that the centriolar protein cep164 makes a nine‐cluster ring that cannot be seen with confocal microscopy. (Reprinted with permission from Ref . Copyright 2012 Elsevier Ltd). (d) SIM was used to map the arrangement of nuclear pore‐associated proteins. (Reprinted with permission from Ref . Copyright 2008 The American Association for the Advancement of Science (AAAS)). (e) SIM was combined with particle averaging analysis to determine the organization of the y‐shaped Nup107‐160 complex (left) at the nuclear pore, which was further mapped onto cryo‐electron microscopy (EM) structure of the pore complex (center, right). (Reprinted with permission from Ref . Copyright 2013 The American Association for the Advancement of Science (AAAS)). (f) Live 3D SIM was used to track bacterial Z‐ring proteins and show that they form dynamic clusters. (Reprinted with permission from Ref . Copyright 2012 PLOS Biology)
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Studies utilizing subdiffractive microscopic methods in neuroscience. (a) The subsynaptic localization of both presynaptic and postsynaptic proteins examined using stochastic optical reconstruction microscopy (STORM). (Reprinted with permission from Ref . Copyright 2010 Elsevier Ltd). (b) Single‐particle tracking photoactivated localization microscopy (sptPALM) was used to track actin and identify discrete regions of actin polymerization in dendritic spines. (Reprinted with permission from Ref . Copyright 2010 Elsevier Ltd). (c) sptPALM measured AMPA receptor dynamics in cultured neurons. (Reprinted with permission from Ref . Copyright 2012 National Academy of Sciences of the United States of America). (d) sptPALM data showed that the change in catalytic subunit of protein kinase A (PKAc) mobility upon increased cAMP concentration can be extracted from single‐molecule trajectories of photoactivatable fluorescent protein (PAFP)‐tagged PKAc (BRL and HZ, unpublished). (e) STORM revealed a previously unobserved periodic ring structure containing actin, adducin, and spectrin in the axons of cultured neurons. (Reprinted with permission from Ref . Copyright 2013 The American Association for the Advancement of Science (AAAS)). (f) In vivo stimulated emission depletion (STED) images showing the dynamic nature of EYFP‐labeled spines at high resolution, 10–15 µm below the surface of the cortex. (Reprinted with permission from Ref . Copyright 2012 The American Association for the Advancement of Science (AAAS))
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The principles and representative images of the subdiffractive microscopic methods. Scale bars are 4 µm. Images in the top panel indicate actin in Hela cell. (Reprinted with permission from Ref . Copyright 2000 John Wiley and Sons Ltd.). Middle panel images (actin in embryonic chick fibroblast) are courtesy of Elise Stanley (Toronto Western Research Institute and University of Toronto). Bottom images (actin tagged with mEos3.2 in fox lung fibroblast) are our unpublished work.
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