Multiphoton fluorescence recovery after photobleaching is a well-established microscopy technique used to measure the diffusion of macromolecules in biological systems. We have developed an improved model of the fluorescence recovery that includes the effects of convective flows within a system. We demonstrate the validity of this two-component diffusion-convection model through in vitro experimentation in systems with known diffusion coefficients and known flow speeds, and show that the diffusion-convection model broadens the applicability of the multiphoton fluorescence recovery after photobleaching technique by enabling accurate determination of the diffusion coefficient, even when significant flows are present. Additionally, we find that this model allows for simultaneous measurement of the flow speed in certain regimes. Finally, we demonstrate the effectiveness of the diffusion-convection model in vivo by measuring the diffusion coefficient and flow speed within tumor vessels of 4T1 murine mammary adenocarcinomas implanted in the dorsal skinfold chamber.

Mass transport of a small molecule dye through dynamic polymer networks containing reversibly associating side-groups was directly compared to steady-shear viscosity measurements suggesting that viscous relaxation is limited more by the frequency of dissociation events while mass transport is determined primarily by the degree of association.

Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to measure diffusion coefficients of macromolecules in biological systems. The three-dimensional resolution and superior depth penetration within scattering samples offered by MP-FRAP make it an important tool for investigating both in vitro and in vivo systems. However, biological systems frequently confine diffusion within solid barriers, and to date the effect of such barriers on the measurement of absolute diffusion coefficients via MP-FRAP has not been studied. We have used Monte Carlo simulations of diffusion and MP-FRAP to understand the effect of barriers of varying geometries and positions relative to the two-photon focal volume. Furthermore, we supply ranges of barrier positions within which MP-FRAP can confidently be employed to measure accurate diffusion coefficients. Finally, we produce two new MP-FRAP models that can produce accurate diffusion coefficients in the presence of a single plane boundary or parallel infinite plane boundaries positioned parallel to the optical axis, up to the resolution limit of the multiphoton laser scanning microscope.

Fluorescence recovery after photobleaching (FRAP) is a microscopy technique for measuring the kinetics of fluorescently labeled molecules, and can be applied both in vitro and in vivo for two-and three-dimensional systems. This chapter discusses the three basic FRAP methods: traditional FRAP, multi-photon FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the appropriate mathematical analysis appropriate for situations in which the recovery kinetics are dictated by free diffusion. In some experiments, the recovery kinetics are dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Since the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.

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