Localization and mobility of bacterial proteins by confocal microscopy and fluorescence recovery after photobleaching.

This chapter describes the use of laser-scanning confocal fluorescence microscopy for determining the localization of fluorescently tagged proteins within bacterial cells, discussing the problems caused by the limited resolution of an optical microscope. It also explains a relatively simple method for using fluorescence recovery after photobleaching (FRAP) to observe and quantify the diffusion of fluorescently tagged proteins in bacterial cells. The techniques are illustrated with reference to measurements on green fluorescent protein (GFP)-tagged proteins in Escherichia coli.