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PMID: 17208970 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Biophysical journal ·Vol. 92 ·No. 6 ·2007-03-15 ·Pages 2172-83

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

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.

MeSH Terms
Algorithms Diffusion Fluorescence Recovery After Photobleaching/instrumentation,methods Image Interpretation, Computer-Assisted/methods Imaging, Three-Dimensional/instrumentation,methods Microscopy, Confocal/methods Reproducibility of Results Sensitivity and Specificity
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Braeckmans Kevin
Laboratory General Biochemistry and Physical Pharmacy, Ghent University, Ghent, Belgium.
Remaut Katrien
Vandenbroucke Roosmarijn E
Lucas Bart
De Smedt Stefaan C
Demeester Joseph
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Article Info
Journal
Biophysical journal
Abbr.
Biophys J
ISSN
0006-3495
DOI
10.1529/biophysj.106.099838
Published
2007-03-15
Epub
2007-00-05
Pages
2172-83
Language
English
Region
United States
NLM ID
0370626
PMCID
PMC1861797
Subset
IM
External Links
PubMed source DOI: 10.1529/biophysj.106.099838 PMC Full Text View journal details
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