153 / 2024-05-15 15:28:05

Fast and flexible two-photon multifocal structured illumination microscopy using acousto-optic deflectors

super-resolution imaging,multifocal structured illumination microscopy,addressable scanning

Super-resolution fluorescence microscopy bypasses the diffraction limitations in traditional optical imaging and has become an indispensable tool for high-resolution subcellular imaging. Among the various super-resolution techniques, structured illumination microscopy (SIM) offers only moderate resolution enhancement, but its advantages of good probe compatibility and low laser power requirements have made it widely applicable in biomedical research. However, SIM’s imaging depth is constrained by significant out-of-focus disturbance inherent in wide-field imaging. Multifocal structured illumination microscopy (MSIM), which utilizes laser focus arrays to excite specimens, substantially improves imaging depth. Nonetheless, the speed of MSIM imaging is often limited by the methods of generating and scanning the laser foci across the sample. To overcome these limitations, we have developed a fast and flexible two-photon MSIM system. This system is engineered through the synchronization of a pair of acousto-optic deflectors and a high speed sCMOS camera. By employing addressable scanning, we can rapidly and flexibly produce multifocal arrays and scan them across the specimen, allowing MSIM to conduct super-resolution imaging in multiple discrete and irregularly shaped regions of interest (ROIs) within biological samples. Our innovative approach to generate multifocal spot arrays exclusively within ROIs not only enhances the speed of MSIM super-resolution imaging but also mitigates photobleaching and phototoxicity of biological samples. We have successfully demonstrated super-resolution imaging of selected mitochondria within cells, achieving a resolution of 140 nm at a frame rate of 4 Hz. These findings indicate the potential of our method to contribute to the study of dynamic functions, kinetics, and metabolic processes in biological research.