BME PhD Thesis Defense Seminar: Javier Manzella-Lapeira
Multiphoton Fluorescence After Photobleaching with Shear Flow
Supervised by Professor Edward Brown
Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique that produces three-dimensionally resolved diffusion coefficients. This technique has been widely used to study diffusive properties of molecules in various biological environments. In this dissertation, we introduce an improvement to the MPFRAP technique that expands its applicability to environments where there is shear flow. This new model permits the study of diffusive transport of molecules in higher values of shear stress, that are physiologically relevant and inaccessible using the previous models, as well as providing a tool for the study of novel engineered materials with biomedical applications.
We have derived an improved mathematical model that incorporates shear stress in the flow of the solution under study and allows the calculation of diffusion coefficients in environments where there is a significant amount of flow speed and shear stress. Multiphoton fluorescence recovery after photobleaching is modeled with Monte Carlo simulations in order to test the new model and compare the results to the previous two models in a high-throughput manner. These results demonstrate the robustness of the shear-stress model when values of shear stress have caused the previous two models to fail to produce accurate diffusion coefficients. We then developed an in vitro experimental method to test the predictions made by the Monte Carlo simulations. We were able to produce a range of combinations of velocities and shear rates in experiments with physiologically relevant fluorescent dyes and confirm the predictions of the Monte Carlo simulations in which the shear stress model accurately fits diffusion coefficients beyond the previous models.