This dissertation focuses on novel fluorescence microscopy techniques and the biophysical analysis of cell biology enabled by such techniques. Modern cell biology research benefits greatly from the ability to accurately visualize the inner workings of cells. Fluorescence microscopy is particularly well suited to imaging live cells, as it is gentle enough to avoid damaging cells, provides sufficient spatial resolution to image small cellular features, and targets and visualizes specific cell structures and processes with high contrast. An additional feature that is often desirable in fluorescence microscopy is the ability to image rapidly enough to freeze the motion of dynamic cell processes, yet technical limitations make imaging with both high spatial and temporal resolution challenging. In this thesis I address methods for improving the speed, spatial resolution, and optical sectioning of fluorescence microscopy techniques. I then apply some of these innovations to study actin structures and dynamics in epithelial cells. Because of its role in driving cellular motion, targeted studies of the actin cytoskeleton using fluorescence microscopy can be used to examine cell migration dynamics. In both in vivo and in vitro experiments, I use high spatiotemporal resolution fluorescence microscopy techniques to provide insight into the role of the actin cytoskeleton in responding to external structural stimuli.