Fast TIRF-SIM imaging of dynamic, lowfluorescent biological samples

Fluorescence microscopy is the standard imaging technique to investigate the structures and dynamics of living cells. However, increasing the spatial resolution comes at the cost of temporal resolution and vice versa. In addition, the number of images that can be taken in sufficiently high quality is limited by fluorescence bleaching. Hence, super-resolved imaging at several Hertz of low fluorescent biological samples is still a big challenge and, especially in structured illumination microscopy (SIM), is often visible as imaging artifacts. In this paper, we present a TIRF-SIM system based on scan-mirrors and a Michelson interferometer, which generates images at 110 nm spatial resolution and up to 8 Hz temporal resolution. High resolution becomes possible by optimizing the illumination interference contrast, even for low fluorescent, moving samples. We provide a framework and guidelines on how the modulation contrast, which depends on laser coherence, polarization, beam displacement or sample movements, can be mapped over the entire field of view. In addition, we characterize the influence of the signal-to-noise ratio and the Wiener filtering on the quality of reconstructed SIM images, both in real and frequency space. Our results are supported by theoretical descriptions containing the parameters leading to image artifacts. This study aims to help microscopists to better understand and adjust optical parameters for structured illumination, thereby leading to more trustworthy measurements and analyses of biological dynamics.