High-speed fluctuation microscopy with acousto-optic illumination

Traditional optical microscopes face an inherent trade-off between speed, spatial resolution, and implementation complexity. A promising strategy to balance them relies on techniques that exploit temporal fluctuations in fluorescence images. Readily implemented in widefield systems without the need for specialized dyes or hardware, these methods enable enhanced resolution imaging, down to the nanoscale. However, they typically require the acquisition of a large number of frames, which imposes a fundamental limitation on imaging speed. Here, we address this issue by combining the high-speed fluctuation microscopy multiple signal classification algorithm (MUSICAL) with acousto-optic-enabled patterned illumination. The microsecond-scale pattern switching enabled by acousto-optic systems and MUSICAL’s ability to operate without precise knowledge of the fluctuation source are synergistically combined to deliver high-resolution imaging at timescales as short as 32 ms (using only 6 frames acquired at 185 fps). As our results demonstrate, our approach delivers lateral resolution enhancement with low implementation effort, paving the way for extending the usage of high spatiotemporal resolution microscopies.