Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples.

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 J

journal_title

Biophysical journal

authors

Braeckmans K,Remaut K,Vandenbroucke RE,Lucas B,De Smedt SC,Demeester J

doi

10.1529/biophysj.106.099838

subject

Has Abstract

pub_date

2007-03-15 00:00:00

pages

2172-83

issue

6

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(07)71021-4

journal_volume

92

pub_type

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