*Confocal Laser Scanning Microscopy for Spectroscopic Studies of Living Photosynthetic Cells DOI: http://dx.doi.org/10.5772/intechopen.84825*

Leica software (LAS AF Lite 2.6) was used to extract the fluorescence profiles across the bleaching area (orange rectangle) from the images, summing all the pixel values in the Y-direction for each X coordinate. And then MathCad 14 was used for curve fitting and further calculations.

(**Figure 12(b)**) shows a typical time dependence of fluorescence recovery after photobleaching. The open circles denote the integrated signals over the bleached area, marked by the light-blue vertical rectangle in the fluorescence intensity images (a), and the line represents a fitting curve. First three points correspond to prebleaching images. Then goes a 1-s gap corresponds to bleaching period. The next 20 points were recorded with step 1.3 s and another 10 points with step 5 s.

One pre-bleach and five post-bleach fluorescence profiles across the bleaching area (orange rectangle) at a series of time-points were aligned (**Figure 12(c)**). The post-bleach profiles (shown in thin lines) were corrected by subtracting the prebleach profile (shown in thick red line), and the Gaussian curves were then fitted to the corrected fluorescence profiles (**Figure 12(d)**). The fitted Gaussian curves were used to obtain corresponding depth (C) and the half-width (1/e2) (R) of the bleach. Note that the bleaching profile remains Gaussian, but becomes broader and shallower with time. The change in C and R with time and diffusion distance in one dimensional diffusion model can be described according to the equation given in [49]. Finally, a suitable function of C is plotted versus time in (**Figure 12(e)**) and the corresponding diffusion coefficient was obtained. Here, R0 and C0 are the values of R and C at time just after bleaching. For one-dimensional diffusion, the plot shown in (**Figure 12(e)**) should be linear with gradient equal to the diffusion coefficient. The calculations for the diffusion coefficient of phycobilisomes give a result of 6.8 × 10–11 cm2 /s, which is similar to those given in [49]. Together with the linearity of the plot in **Figure 12(e)**, this indicates that this procedure can be applied to spherical cells also and that our measurement does not greatly perturb the system.
