Abstract:
:We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Braeckmans K,Remaut K,Vandenbroucke RE,Lucas B,De Smedt SC,Demeester Jdoi
10.1529/biophysj.106.099838subject
Has Abstractpub_date
2007-03-15 00:00:00pages
2172-83issue
6eissn
0006-3495issn
1542-0086pii
S0006-3495(07)71021-4journal_volume
92pub_type
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