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In this chapter, we discuss methods to measure lateral mobility of membrane lipids and proteins using techniques based on the light microscope. These methods typically sample lateral mobility in very small, micron-sized regions of the membrane so that they can be used to measure diffusion in regions of single cells. The methods are based on fluorescence from the molecules of interest or from light scattered from particles attached to single or small groups of membrane lipids or proteins. Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are presented in that order. FRAP and FCS methodologies are described for a dedicated wide field microscope although many confocal microscopes now have software permitting these measurement to be made; nevertheless, the principles of the measurement are the same for a wide field or confocal microscope. SPT can be applied to trace the movements of single fluorescent molecules in membranes but this aspect will not be treated in detail.

Original publication

DOI

10.1016/j.ymeth.2006.05.008

Type

Journal article

Journal

Methods

Publication Date

06/2006

Volume

39

Pages

147 - 153

Keywords

Biochemistry, Diffusion, Fluorescence, Membrane Lipids, Membrane Proteins, Microscopy, Fluorescence, Photobleaching, Spectrometry, Fluorescence