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Journal of Biomedical Optics

Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk
Author(s): Nick Smisdom; Martin J. vandeVen; Jean-Michel Rigo; Marcel M. Ameloot; Kevin Braeckmans; Hendrik Deschout; Stefaan C. De Smedt
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Paper Abstract

Fluorescence recovery after photobleaching (FRAP) carried out on a confocal laser-scanning microscope (CLSM) performs well for photobleached disks that are large compared to the resolution of the bleaching beam. For smaller disks approaching this resolution, current FRAP models providing a closed-form solution do not allow one to extract the diffusion coefficient accurately. The new generalized disk model we present addresses this shortcoming by bringing into account the bleaching resolution and the total confocal imaging resolution. A closed-form solution is obtained under the assumption of linear photobleaching. Furthermore, simultaneous analysis of FRAP data collected at various disk sizes allows for the intrinsic determination of the instrumental resolution parameters, thereby obviating the need for an extrinsic calibration. A new method to estimate the variance of FRAP data is introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments are performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients.

Paper Details

Date Published: 1 April 2011
PDF: 9 pages
J. Biomed. Opt. 16(4) 046021 doi: 10.1117/1.3569620
Published in: Journal of Biomedical Optics Volume 16, Issue 4
Show Author Affiliations
Nick Smisdom, Univ. Hasselt (Belgium)
Martin J. vandeVen, Univ. Hasselt (Belgium)
Jean-Michel Rigo, Univ. Hasselt (Belgium)
Marcel M. Ameloot, Univ. Hasselt (Belgium)
Kevin Braeckmans, Univ. Gent (Belgium)
Hendrik Deschout, Univ. Gent (Belgium)
Stefaan C. De Smedt, Univ. Gent (Belgium)

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