Taking advantage of the ability to track dynamic behavior in living cells using fluorescent protein fusions to intracellular targets, the technique of fluorescence recovery after photobleaching (FRAP) and associated methods (loss in photobleaching; FLIP, and inverse FRAP; iFRAP) are proving highly useful for studying the kinetic behavior of proteins. All of these experiments rely on selectively photobleaching the fluorescence within a region of interest with a high-intensity laser, followed by monitoring the diffusion of new fluorescent molecules into the bleached area over a period of time with low-intensity laser light. Photobleaching techniques are ideal for determining kinetic properties, including the diffusion coefficient, mobile fraction, and transport rate of proteins in live-cell imaging.
Photobleaching GFP reveals protein dynamics inside live cells. Trends in Cell Biology 9: 61-65 (1999). An excellent review of FRAP and FLIP techniques as applied to fluorescent proteins in cultured cells. The article discusses practical aspects, examines mobility calculations, and provides dramatic, well-behaved examples of both techniques in live cells.
Studying protein dynamics in living cells. Nature Reviews Molecular Cell Biology 2: 444-456 (2001). A comprehensive and well-written review with a thorough bibliography of techniques used in monitoring cellular dynamics using photobleaching techniques. Included are a discussion of diffusion theory, an introduction to the analysis of protein mobility, and numerous comparative examples of protein diffusion rates.
From fixed to FRAP: Measuring protein mobility and activity in living cells. Nature Cell Biology 3: E145-E147 (2001). The authors describe photobleaching kinetics measurements using fluorescent proteins in single cells and provide several examples. Also discussed are the basic aspects of FRAP calculations and data analysis.
Systematic evaluation of FRAP experiments performed in a confocal laser scanning microscope Journal of Microscopy 220: 20-30 (2005). A systematic analysis of FRAP measurements using highly defined model systems in confocal microscopy. Discussed are the theoretical basis of FRAP, evaluation of spatially resolved data, diffusion coefficient estimates, and experimental protocols.
FRAP analysis of binding: Proper and fitting. Trends in Cell Biology 15: 84-91 (2005). A comprehensive review of photobleaching techniques that includes suggestions for quantitative interpretation of recovery curves. This paper is also an excellent source of information about the basic aspects of FRAP and contains numerous references.
Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29: 14-28 (2003). The authors examine the experimental design, mathematical modeling, and analysis of FRAP experiments to extract information about the distribution of a protein between mobile and immobilized pools with a confined compartment (the nucleus).
Diffusional mobility of Golgi proteins in membranes of living cells Science 273: 797-801 (1996). A classic paper and one of the first to report FRAP data using fusions of EGFP to Golgi and endoplasmic reticulum membrane proteins. The experimental approach in this report has set the baseline for the examination of kinetics in living cells with fluorescent proteins.
Fluorescence photobleaching analysis for the study of cellular dynamics. European Biophysics Journal 31: 36-51 (2002). In this review, the authors examine the theory and practice of performing photobleaching investigations using confocal microscopy. Included are several applications using fluorescent proteins as well as a comprehensive guide to the literature in this field.
Nucleocytoplasmic shuttling revealed by FRAP and FLIP technologies. Current Opinion in Biotechnology 16: 28-34 (2005). A comprehensive discussion of basic FRAP and FLIP techniques with applications to shuttling of proteins between cellular compartments. The paper contains several nice examples of collecting and analyzing experimental data.
Kinetic modeling approaches to in vivo imaging. Nature Reviews Molecular Cell Biology 2: 898-907 (2001). A general review of protein dynamics focusing on fluorescent protein fusions. The authors discuss practical aspects and the derivation of computational models for FRAP, FLIM, and correlation spectroscopy with several application examples.