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

Nick Smisdom, Kevin Braeckmans, Hendrik Deschout, Martin vandeVen, Jean-Michel Rigo, Stefaan C. De Smedt, Marcel Ameloot
2011 Journal of Biomedical Optics  
Nick Smisdom 1 +32 (0) 11 269342 +32 (0) 11 269299 nick.smisdom@uhasselt.be Kevin Braeckmans 2 +32 (0) 9 2648098 +32 (0) 9 2648189 Kevin.Braeckmans@UGent.be Hendrik Deschout 2 +32 (0) 9 2648074 +32 (0) 9 2648189 Hendrik.Deschout@UGent.be Martin vandeVen 1 +32 (0) 11 269233 +32 (0) 11 269299 martin.vandeven@uhasselt.be Jean-Michel Rigo 1 +32 (0) 11 269236 +32 (0) 11 269299 jeanmichel.rigo@uhasselt.be Stefaan De Smedt 2 +32 (0) 9 2648076 +32 (0) 9 2648189 Stefaan.Desmedt@UGent.be Marcel Ameloot 1
more » ... be Marcel Ameloot 1 +32 (0) 11 269233 +32 (0) 11 269299 marcel.ameloot@uhasselt.be 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 to extract the diffusion coefficient accurately. The new generalized disk model presented here 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 was introduced to allow for proper weighting in this global analysis approach by nonlinear least squares. Experiments were performed on two independent CLSMs on homogeneous samples providing validation over a large range of diffusion coefficients.
doi:10.1117/1.3569620 pmid:21529089 fatcat:izznj2fnuzc5fjyelmwbcn2bm4