where and are the dimensions in the radial and axial orientation. Note that the same rb zb Gaussian approximation is equally used for two-photon photobleaching (Brown et al., 1999, Mazza et al., 2008, Mazza et al., 2007). The 3-D concentration distribution of fluorophores after photobleaching with a stationary light beam for a time can be readily obtained from Eq. t and : . The very same expression holds for the two-photon case: It was experimentally tested whether Eqs. and accurately describe the 3-D bleach profile of a focused laser beam. Figure 8 shows the radial and axial sections of the 3-D distribution of photobleached molecules in single photon excitation (Figure 8A and B) and two-photon excitation (Figure 8C and D). Figure 8E and F show the comparison between the 3-D effective bleached volumes (black lines) and the corresponding 3-D model based on the Gaussian approximation (red lines). It is clear that the 3-D Gaussian approximation of the effective bleaching intensity distribution works better in the two-photon excitation regime than in the single photon excitation one, since the confinement of the excitation volume due to the two-photon absorption process minimizes effects from the side lobes, thus reducing the extent of the 3-D bleached region. In single-photon FRAP, the Gaussian description should be used only in the 2-D case of lateral diffusion. Figure 7 shall be inserted here. 3.4 Effective bleaching intensity distribution As stated above, the Gaussian description is not suitable for the axial dimension in the case of single-photon photobleaching. It is worthwhile to take a closer look at the 2-D case as well,
where rb and zb are the dimensions in the radial and axial orientation. Note that the same Gaussian approximation is equally used for two-photon photobleaching (Brown et al., 1999, Mazza et al., 2008, Mazza et al., 2007). The 3-D concentration distribution of fluorophores after photobleaching with a stationary light beam for a time t can be readily obtained from Eq. and : . The very same expression holds for the two-photon case: It was experimentally tested whether Eqs. and accurately describe the 3-D bleach profile of a focused laser beam. Figure 8 shows the radial and axial sections of the 3-D distribution of photobleached molecules in single photon excitation (Figure 8A and B) and two-photon excitation (Figure 8C and D). Figure 8E and F show the comparison between the 3-D effective bleached volumes (black lines) and the corresponding 3-D model based on the Gaussian approximation (red lines). It is clear that the 3-D Gaussian approximation of the effective bleaching intensity distribution works better in the two-photon excitation regime than in the single photon excitation one, since the confinement of the excitation volume due to the two-photon absorption process minimizes effects from the side lobes, thus reducing the extent of the 3-D bleached region. In single-photon FRAP, the Gaussian description should be used only in the 2-D case of lateral diffusion. Figure 7 shall be inserted here. 3.4 Effective bleaching intensity distribution As stated above, the Gaussian description is not suitable for the axial dimension in the case of single-photon photobleaching. It is worthwhile to take a closer look at the 2-D case as well,
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