期刊
JOURNAL OF BIOMEDICAL OPTICS
卷 16, 期 4, 页码 -出版社
SPIE-SOC PHOTOPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.3569620
关键词
fluorescence recovery after photobleaching; fluorescence photobleaching recovery; confocal laser-scanning microscopes; diffusion; artifacts; global analysis; weights
资金
- Research Council of the UHasselt
- tUL
- Agency for Innovation by Science and Technology in Flanders (IWT)
- Functional Supramolecular Systems (BELSPO) [IAP P6/27]
- FWO-onderzoeksgemeenschap Scanning and Wide Field Microscopy of (Bio)-organic Systems
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]
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