NDLI: Simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy.

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Year: 2013 Issue: 82

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Simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy.

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Simultaneous multicolor imaging of biological structures with fluorescence photoactivation localization microscopy.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Curthoys, Nikki M. Mlodzianoski, Michael J. Kim, Dahan Hess, Samuel T.
Description	Author Affiliation: Curthoys NM ( Department of Physics and Astronomy, University of Maine.)
Abstract	Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/50680
Journal	Journal of Visualized Experiments
Issue Number	82
Language	English
Publisher	MyJove Corp.
Publisher Date	2013-12-09
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Fluorescence Polarization Instrumentation Microscopy, Fluorescence Animals Fluorescent Dyes Chemistry Mice Microscopy, Video Nih 3t3 Cells Photobleaching Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Research Support, U.s. Gov't, Non-p.h.s. Video-audio Media
Content Type	Text
Resource Type	Article

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