Chapter

Handbook of Single-Molecule Biophysics

pp 95-127

Date:

Fluorescence Imaging at Sub-Diffraction-Limit Resolution with Stochastic Optical Reconstruction Microscopy

  • Graham T. DempseyAffiliated withGraduate Program in Biophysics, Harvard University
  • , Wenqin WangAffiliated withDepartment of Physics, Harvard University
  • , Xiaowei ZhuangAffiliated withDepartments of Physics, Chemistry and Chemical Biology, Howard Hughes Medical Institute, Harvard University

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Abstract

Fluorescence microscopy is an essential tool in biological research. One major drawback of conventional light microscopy, however, is its relatively low resolution, which is limited by the diffraction of light to several hundreds of nanometers. In recent years, a number of fluorescence imaging techniques with sub-diffraction-limit resolution have been developed, achieving a spatial resolution of tens of nanometers in both the lateral and axial dimensions. This chapter focuses on one of these methods, stochastic optical reconstruction microscopy (STORM), which utilizes photoswitchable flourescent probes to separate spatially overlapping images of individual fluorophores in time and construct superresolution images from the precise positions of these fluorophores determined from the single-molecule images. Application of this technique has been extended to imaging fluorophores of different colors simultaneously, in three dimensions, and in living cells. This chapter describes the implementation of multicolor and three-dimensional STORM to imaging cellular structures. It begins by discussing the choice of photoswitchable fluorescent probe and the scheme with which to label a cellular target of interest. The instrumentation and methods for performing a STORM experiment are then described, followed by an outline of the analysis routines used for creating a STORM image. Applications of the technique along with general protocols and troubleshooting are given at the conclusion of the chapter.