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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 | Source: | 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. | Keywords: | fluorescence recovery after photobleaching; fluorescence photobleaching recovery; confocal laser-scanning microscopes; diffusion; artifacts; global analysis; weights | Document URI: | http://hdl.handle.net/1942/12018 | ISSN: | 1083-3668 | e-ISSN: | 1560-2281 | DOI: | 10.1117/1.3569620 | ISI #: | 000291031400027 | Category: | A1 | Type: | Journal Contribution | Validations: | ecoom 2012 |
Appears in Collections: | Research publications |
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