Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion

The relaxation of fluorescence from diffraction-limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo-conversion was measured in solutions of varied viscosity (η), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point-spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D_ap. The model incorporates two novel features that make it of broad utility. First, the model includes the no-flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D_ap. Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D_ap and η^-1 were obtained for the two proteins: for PAGFP, D_aq = 89 ± 2.4 μm² s^-1 (mean ± 95% confidence interval), and for EGFP D_aq = 91 ± 1.8 μm² s^-1. In CHO cells, the application of the model yielded D_ap = 20 ± 3 μm² s^-1 (PAGFP) and 19 ± 2 μm² s^-1 (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked 'bound fraction' explanation of the deviation of the terminal fluorescence recovery from its pre-bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488-nm excitation, and the requirement for ∼1/200th the cumulative light energy to produce data of comparable signal/noise.