Kelley D. Sullivan

Improved Model of Fluorescence Recovery Expands the Application of Multiphoton Fluorescence Recovery after Photobleaching in Vivo

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.

Thermally activated diffusion in reversibly associating polymers

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.

Single- and Two-Photon Fluorescence Recovery after Photobleaching

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 introduction discusses the three basic FRAP methods: traditional FRAP, multiphoton FRAP (MPFRAP), and FRAP with spatial Fourier analysis (SFA-FRAP). Each discussion is accompanied by a description of the mathematical analysis appropriate for situations in which the recovery kinetics is dictated by free diffusion. In some experiments, the recovery kinetics is dictated by the boundary conditions of the system, and FRAP is then used to quantify the connectivity of various compartments. Because the appropriate mathematical analysis is independent of the bleaching method, the analysis of compartmental connectivity is discussed last, in a separate section.

Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching

Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro and in vivo biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).