Tomina, Y.; Ishijima, A.; Toyoshima, Y.; Shishido, H.; Hirooka, R.; Mukumoto, K.; Wen, C.; Kanamori, M.; Kuze, K.; Murakami, Y.; Oe, S.; Tanaka, S.; Yonamine, Y.; Nishigami, Y.; Goda, K.; Ijiro, K.; Nakagaki, T.; Arakawa, K.; Ishihara, T.; Onami, S.; Iino, Y.; Mikami, H.

Volumetric fluorescence microscopy is a powerful method for studying complex biological systems because it enables comprehensive observation of structural and physiological dynamics. In particular, light-sheet microscopy (LSM) is a leading option for real-time volumetric fluorescence imaging as it combines high imaging speed, low phototoxicity, minimal photobleaching, high spatiotemporal resolution, and low computational burden. To capture fast biological events, various efforts have been made to improve the imaging speed of volumetric fluorescence microscopy, including LSM. However, existing approaches entail significant trade-offs that make routine volumetric imaging at and beyond video rates challenging under practical conditions. Here, we introduce image-scanning LSM, a method that substantially increases the volumetric imaging speed achievable with LSM while preserving key performance metrics, such as spatial resolution and photon efficiency, as well as accessibility. Our implementation, termed image-scanning oblique plane (ISOP) microscopy, enables volumetric fluorescence imaging at up to 1,000 volumes per second with submicrometer lateral spatial resolution. We demonstrate the broad utility of ISOP microscopy by recording and analyzing the dynamics of behaving and rapidly moving organisms.