Fluorescence microscopy is a tool which underpins the biosciences. We develop new fluorescence microscopy techniques, with an emphasis on improving resolution, synthesising data, and producing verifiable results. Our image analysis approaches, including Bayesian inference and deep learning, allow us to investigate the structure and dynamics of the cell cytoskeleton with improved resolution and accuracy. Our results give new insight into the complex processes regulating cell migration in 3D environments, yielding better model systems for biomedicine.