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Fluorescence microscopy is particularly well suited to the study of cell biology, due to its noninvasive nature, high sensitivity detection of specific molecules, and high spatial and temporal resolution. In recent years, there has been an important transition from imaging the static distributions of molecules as a snapshot in time in fixed material to live-cell imaging of the dynamics of molecules in cells: in essence visualizing biochemical processes in living cells. Furthermore, in the last 5 years, there have been important advances in so-called "super-resolution" imaging methods that have overcome the resolution limits imposed by the diffraction of light in optical systems. Live-cell imaging is now beginning to deliver in unprecedented detail, bridging the resolution gap between electron microscopy and light microscopy. We discuss the various factors that limit the spatial and temporal resolution of microscopy and how to overcome them, how to best prepare specimens for high resolution imaging, and the choice of fluorochromes. We also summarize the pros and cons of the different super-resolution techniques and introduce some of the key data analysis tasks that a cell biologist employing high resolution microscopy is typically interested in.

Original publication

DOI

10.1016/B978-0-12-391857-4.00002-1

Type

Journal article

Journal

Methods Enzymol

Publication Date

2012

Volume

504

Pages

29 - 55

Keywords

Cell Tracking, Fluorescent Dyes, Humans, Image Processing, Computer-Assisted, Microscopy, Electron, Microscopy, Fluorescence