| Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to probe the transport properties of macromolecules in biological systems. MP-FRAP utilizes two-photon fluorescence and photobleaching to produce a three-dimensionally resolved diffusion coefficient for an ensemble of molecules in the region of the two-photon focal volume. This thesis describes two fundamental improvements to the MP-FRAP technique, which are vital steps to enable MP-FRAP to be applied to the complex in vivo environment.;In Chapter 1, we lay the groundwork for our discussion of these advancements by introducing the MP-FRAP technique and the physics upon which it is based. We begin with a description of fluorescence and diffusion and discuss their importance in biomedical research. Next, we describe how two-photon fluorescence and photobleaching are applied to a diffusing system to measure the diffusion coefficient via fluorescence recovery after photobleaching (FRAP). Then, we take the reader through the evolution of FRAP, which leads to the application of two- photon fluorescence and photobleaching to produce MP-FRAP. Along the way, we highlight applications and advancements of the FRAP techniques, and introduce fluorescence correlation spectroscopy, a popular complement to FRAP.;In Chapter 2, we collect the experimental methods for the studies presented in Chapters 3 and 4. We begin with an in-depth discussion of our work to build and troubleshoot our MP-FRAP apparatus, followed by a detailed description of our data analysis protocol. Next, we delve into the specific methods for producing computer generated data and fits, as well as in vitro and in vivo experimental data, for our work in Chap. 3 on improving MP-FRAP to measure diffusion in the presence of convective flow. We end with a description of the Monte Carlo algorithm we developed for our work in Chap. 4 to model diffusion and multiphoton fluorescence recovery after photobleaching in the presence of reflective boundaries of various geometries.;In Chapter 3, we develop an improved analytical model of multiphoton fluorescence recovery after photobleaching that includes the effects of convective flows within a system. We use computer generated data and fits to explore the effect of convective flow on the shape and speed of fluorescence recovery, and to estimate the range of diffusion coefficients and flow speeds over which this new "diffusion-convection" model yields accurate diffusion coefficients (as compared to the diffusion-only model). We then demonstrate the validity of the diffusion-convection model through in vitro experimentation in systems with known diffusion coefficients and known flow speeds, and show that the diffusion-convection model enables accurate determination of the diffusion coefficient via MP-FRAP, even when significant flows are present. We conclude by demonstrating 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.;In Chapter 4, we present our work that allows MP-FRAP to be performed accurately near reflective boundaries of various geometries. Using Monte Carlo techniques, we first generate an initial distribution of bleached molecules, then simulate their diffusion away from the initial distribution, thereby producing fluorescence vs. time recovery curves in the region of the initial bleached distribution. These curves are then fit to the standard analytical MP-FRAP model to produce a diffusion coefficient. By introducing solid barriers into the model in the region of the initial bleached distribution, we learn how the presence of harriers of different geometries affects the measurement of diffusion via MP-FRAP. Finally, we supply ranges of barrier positions for each geometry within which MP-FR AP can confidently be employed to measure accurate diffusion coefficients. |
