Fluorescence perturbation techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, photoactivation, photoconversion, and FLIP.

The technique of fluorescence recovery after photobleaching (FRAP) was introduced in the mid-1970s to study the diffusion of biomolecules in living cells. For several years, it was used mainly by a small number of biophysicists who had developed their own photobleaching systems. Since the mid-1990s, FRAP has gained increasing popularity because of the conjunction of two factors: First, photobleaching techniques are easily implemented on confocal laser-scanning microscopes (CLSMs), and so FRAP has become available to anyone who has access to such equipment. Second, the advent of green fluorescent protein (GFP) has allowed easy fluorescent tagging of proteins and their observation in living cells. Thanks both to the versatility of modern CLSMs, which allow control of laser intensity at any point of the image, and to the development of new fluorescent probes, additional photoperturbation techniques have emerged during the last few years. After the photoperturbation event, one observes and then analyzes how the fluorescence distribution relaxes toward the steady state. Because the photochemical perturbation of suitable fluorophores is essentially irreversible, changes of fluorescence intensity in the perturbed and unperturbed regions are due to the exchange of tagged molecules between those regions. This article first discusses the materials required for performing FRAP experiments on a CLSM and the software for data analysis. It then describes general considerations on how to perform FRAP experiments as well as the necessary controls. Finally, different possible ways to analyze the data are presented.