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Please use this identifier to cite or link to this item: http://hdl.handle.net/1942/12018

Title: Fluorescence recovery after photobleaching on the confocal laser-scanning microscope: generalized model without restriction on the size of the photobleached disk
Authors: SMISDOM, Nick
Braeckmans, Kevin
Deschout, Hendrik
VAN DE VEN, Martin
RIGO, Jean-Michel
De Smedt, Stefaan C.
AMELOOT, Marcel
Issue Date: 2011
Publisher: SPIE-SOC PHOTOPTICAL INSTRUMENTATION ENGINEERS
Citation: JOURNAL OF BIOMEDICAL OPTICS, 16(4)
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. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3569620]
Notes: [Smisdom, Nick; vandeVen, Martin; Rigo, Jean-Michel; Ameloot, Marcel] Transnatl Univ Limburg, Hasselt Univ, Biomed Res Inst, Sch Life Sci, B-3590 Diepenbeek, Belgium. [Braeckmans, Kevin; Deschout, Hendrik] Univ Ghent, Fac Pharm, Biophoton Imaging Grp, Gen Biochem & Phys Pharm Labs, B-9000 Ghent, Belgium. [De Smedt, Stefaan C.] Univ Ghent, Fac Pharm, Ghent Res Grp Nanomed, Gen Biochem & Phys Pharm Labs, B-9000 Ghent, Belgium.
URI: http://hdl.handle.net/1942/12018
DOI: 10.1117/1.3569620
ISI #: 000291031400027
ISSN: 1083-3668
Category: A1
Type: Journal Contribution
Validation: ecoom, 2012
Appears in Collections: Bio-Physics
Physiology
Immunology - Biochemistry

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