SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples
Kevin Braeckmans 1, Katrien Remaut 1, Roosmarijn E Vandenbroucke 1, Bart Lucas 1, Stefaan C De Smedt 1* and Joseph Demeester 1
1 Ghent University
* To whom correspondence should be addressed. E-mail: stefaan.desmedt{at}ugent.be.
Submitted on October 21, 2006
Revised on November 9, 2006
Accepted on 28 November 2006
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Abstract |
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We present a truly quantitative FRAP model for use with the CLSM based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before (1). 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 (2). 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 in order 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 3-D samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free GFP diffusion in the cytoplasm and nucleus of living A549 cells.
Key Words:
FRAP, confocal scanning laser microscopy, diffusion, microphotolysis, photobleaching
