Addressable scanning multifocal structured illumination microscopy using acousto-optic deflectors.

Multifocal structured illumination microscopy (MSIM) is a popular super-resolution imaging technique known for its good probe compatibility, low laser power requirements, and improved imaging depth, making it widely applicable in biomedical research. However, the speed of MSIM imaging is typically constrained by the approaches employed to generate and scan the laser foci across the sample. In this study, we propose a flexible two-photon excitation MSIM method using a pair of acousto-optic deflectors. By adopting addressable scanning (AS) and synchronized capturing, MSIM super-resolution imaging can be performed in multiple discrete regions of interest (ROIs) within the field of view. Notably, this AS-MSIM scheme not only enhances the speed of MSIM imaging but also alleviates photobleaching and phototoxicity to biological samples. We demonstrate its potential by achieving super-resolution imaging of selected mitochondria within cells at a frame rate of 4 Hz. Furthermore, we deliberate the possibility of even faster imaging.