Research Papers: Imaging

Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk

[+] Author Affiliations
Nick Smisdom, Martin vandeVen, Jean-Michel Rigo, Marcel Ameloot

Hasselt University, Transnational University Limburg, Biomedical Research Institute, School of Life Sciences, Agoralaan Building C, Diepenbeek, 3590 Belgium

Kevin Braeckmans, Hendrik Deschout

Ghent University, Biophotonic Imaging Group, Laboratory of General Biochemistry & Physical Pharmacy, Faculty of Pharmacy, Harelbekestraat 72, Ghent, 9000 Belgium

Stefaan C. De Smedt

Ghent University, Ghent Research Group on Nanomedicines, Laboratory of General Biochemistry & Physical Pharmacy, Faculty of Pharmacy, Harelbekestraat 72, Ghent, 9000 Belgium

J. Biomed. Opt. 16(4), 046021 (April 20, 2011). doi:10.1117/1.3569620
History: Received December 15, 2010; Revised March 01, 2011; Accepted March 02, 2011; Published April 20, 2011; Online April 20, 2011
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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.

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© 2011 Society of Photo-Optical Instrumentation Engineers (SPIE)

Citation

Nick Smisdom ; Kevin Braeckmans ; Hendrik Deschout ; Martin vandeVen ; Jean-Michel Rigo, et al.
"Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk", J. Biomed. Opt. 16(4), 046021 (April 20, 2011). ; http://dx.doi.org/10.1117/1.3569620


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