Quantitative interpretation of binding reactions of rapidly diffusing species using fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) measurements offer an important tool for analyzing diffusion and binding processes. Confocal scanning laser microscopes that are used in FRAP experiments bleach regions with a radially Gaussian distributed profile. Previous attempts to derive analyti...

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Bibliographic Details
Published in:Journal of microscopy (Oxford) 2009-03, Vol.233 (3), p.384-390
Main Author: TSIBIDIS, G.D
Format: Article
Language:English
Subjects:
Actins - metabolism
Binding mechanisms
Cell Nucleus - metabolism
Diffusion
Fluorescence Recovery After Photobleaching - methods
FRAP
Green Fluorescent Proteins - metabolism
HeLa Cells - metabolism
HeLa Cells - ultrastructure
Humans
instantaneous processes
intracellular dynamics
Kinetics
mathematical modelling
mathematical models
Models, Biological
Nuclear Proteins - metabolism
Protein Binding
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Summary:Fluorescence recovery after photobleaching (FRAP) measurements offer an important tool for analyzing diffusion and binding processes. Confocal scanning laser microscopes that are used in FRAP experiments bleach regions with a radially Gaussian distributed profile. Previous attempts to derive analytical expressions in the case of processes governed by fast diffusion have overlooked the characteristics of the instruments used to perform FRAP measurements and therefore led to approximating solutions. In the present paper, bleaching laser beam characteristics are incorporated into an improved model to provide a more rigorous and accurate method. The proposed model simulates binding inside bounded regions, and it leads to FRAP curves that depend on the on and off rates that can be employed to determine the rate constants. It can be used in conjunction with experimental data acquired with confocal scanning laser microscopes to investigate the biophysical properties of proteins in living cells. The model aims to improve the accuracy when determining rate constants by taking into account a more realistic scenario of the light-matter interaction.
ISSN:0022-2720
1365-2818
DOI:10.1111/j.1365-2818.2009.03132.x