Interpretation of fluorescence correlation spectroscopy and photobleaching recovery in terms of molecular...

The theoretical basis and experimental implementation of FCS and FPR measurements are now well established. Because of the requirements for system stability and long data acquisition times FCS is relatively rarely used. But FCS can provide unique information, especially about extents of aggregation or polymerization and therefore is a useful supplement to FPR for certain applications. FPR measurements are now carried out routinely in many laboratories in a variety of formats using different beam profiles, optical systems, and analytical schemes. A particular version may be better adapted to a specific application. The spot photobleaching approach, however, seems simplest and most versatile for cellular studies and is now most often used. Important experimental considerations in setting up a spot photobleaching instrument are discussed in detail in Chapter 10 by Wolf (this volume) and elsewhere (Petersen et al., 1986a). In interpreting FPR measurements it is also important to take into account the possibility of systematic errors from a number of sources. In Chapter 10 in this volume, Wolf discusses many factors that must be properly controlled in carrying out FPR measurements. Additional consideration of some of these points is presented by Petersen et al. (1986a). One potentially troublesome type of error arises from the possibility that chemical reactions initiated by the photobleaching pulse or during the measurement of recovery could significantly perturb the system. Evidence from a variety of sources [summarized, for example, in Petersen et al. (1986a)] indicates that photobleaching fluorophores can induce chemical cross-linking of cellular proteins under some conditions. But measurements in a number of different systems have demonstrated that, even if these types of reactions occur in FPR measurements, nevertheless they do not perturb the measured mobilities. If possible, however, this point should be checked for each new system because variations in structure or environmental conditions could enhance the chemical cross-linking reactions mediated by photogenerated free radicals. In practice, the principal difficulty in carrying out FPR measurements on cells is frequently the low intensity of the fluorescent signal which can be obtained from specifically labeled cell surface ligands or microinjected components. This low intensity results from the typically low capacity of an individual cell for the specifically labeled macromolecule. Even in the absence of systematic errors, low emission intensity will reduce the accuracy of measurements due to shot noise. This is an important practical limitation on measuring accuracy. Low measurement accuracy severely limits the extent to which the data can be interpreted mechanistically. Precision can be improved by averaging many recovery experiments.(ABSTRACT TRUNCATED AT 400 WORDS)