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Figure
- FRAP analysis of photosynthetic membranes

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Fig. 1. Extracting data from a FRAP experiment on a cell of Synechococcus 7942. This experiment shows fluorescence from the phycobilisomes. An image (a) is recorded prior to bleaching, by scanning the confocal spot in the XY mode. The scan is then switched to the X-mode, the laser power is increased by a factor of 8, and the confocal spot is scanned across the cell for about 2 s to bleach a line. The laser power is then reduced again, the scan is switched back to the XY mode, and a second image (b) is recorded. A series of further images is recorded, at time intervals appropriate to the rate of diffusion. To extract data, image analysis software is used to obtain a fluorescence profile along the long axis of the cell (in the Y-direction), summing all the pixel values in the X-direction for each Y co-ordinate (c). Pre-bleach and post-bleach profiles are aligned (d). The post-bleach profile (shown in black) is corrected by subtracting the pre-bleach profile (shown in grey). A Gaussian curve is then fitted to the corrected fluorescence profile (e). The fitted Gaussian curve is used to obtain the depth (C) and the half-width (1/e2) (R) of the bleach (e). Finally, a suitable function of C is plotted versus time and regression analysis is used to obtain the diffusion coefficient (f). R0 and C0 are the values of R and C at time t=0. For one-dimensional diffusion, the plot shown in (f) should be linear with gradient equal to the diffusion coefficient (Mullineaux et al., 1997).

 

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