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

摘要

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