Abstract
The analysis of Fluorescence Recovery After Photobleaching (FRAP) experiments involves mathematical modeling of the fluorescence recovery process. An important feature of FRAP experiments that tends to be ignored in the modeling is that there can be a significant loss of fluorescence due to bleaching during image capture. In this paper, we explicitly include the effects of bleaching during image capture in the model for the recovery process, instead of correcting for the effects of bleaching using reference measurements. Using experimental examples, we demonstrate the usefulness of such an approach in FRAP analysis.
Highlights
The Fluorescence Recovery After Photobleaching (FRAP) technique is a popular technique for investigating dynamics of protein diffusion and binding in living cells [1,2,3,4,5,6,7,8].FRAP experiments involve bleaching of fluorescently labeled proteins in a pre-chosen location inside the cell with a high intensity laser pulse
If an image is captured for an exposure time v, the fluorescence concentration in the cell will decrease from an initial value of C(t~0)~C0 in this time according to the kinetic expression [19]
The analysis of FRAP experiments is an ongoing area of research
Summary
The FRAP technique is a popular technique for investigating dynamics of protein diffusion and binding in living cells [1,2,3,4,5,6,7,8]. FRAP experiments involve bleaching of fluorescently labeled proteins in a pre-chosen location inside the cell with a high intensity laser pulse. The recovery curve can be fit to models to estimate transport and binding parameters. The accurate modeling of FRAP experiments and issues with parameter estimation are active areas of interest [2,9,10,11,12,13,14,15,16,17]. The approach to fit FRAP experiments to mathematical models involves a suitable normalization of the experimental data [18]. If F (t) is the fluorescence in a spot in the cytoplasm, and bleaching occurs at t~0, one way to normalize the signal is
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