The aim of this project is to use computer-aided fluorescence microscopy to probe the structure and compartmentalization of cytoplasm in living cells. The research is aimed at three broad goals: 1, to understand the physical- chemical basis for the gel properties of cytoplasm; 2, to measure the solvent viscosity of the fluid that fills the spaces in the gel; and 3, to understand the functional implications of apparent subcellular variations of cytoplasmic structure that lead to compartmentalization of the cytoplasmic volume. In pursuit of the first goal, the technique of fluorescence recovery after photobleaching (FRAP) will be used to study the tracer diffusion of size-fractionated, inert, fluorescent particles both in the living cell and in solutions and gels reconstituted from biological components. In order to map solvent viscosity in living cells, a novel ratio imaging approach will be employed, which has been recently developed by Dr. Luby-Phelps. In addition, the ratio imaging techniques will be applied to the study of cytoplasmic compartmentalization during a variety of cellular functions. Although it has been studied for over 150 years, the submicrosopic architecture of cytoplasm still is not completely understood. From these studies, it is hoped that a "picture" of cytoplasm can be built up that can serve as a framework for understanding cellular biochemistry and metabolism.
