Scan field rotation with a K-mirror for fast two-photon imaging along curved planes

In contrast to the curved geometry of biological structures, the imaging plane of conventional microscopes is flat. In principle, axial scanning can adjust the focus to follow curved structures. In resonant scanning microscopes, which are broadly used for video-rate functional imaging, however, such focus adjustment is typically possible at rates faster than the slow (~30 Hz) scan axis but not as fast as the fast (~8000 Hz) resonant axis. The fixed direction of the fast resonant axis therefore constrains how the imaging plane can be tilted or curved using axial scanning, and limits how well it can be aligned with biological structures of interest. Here, we present an optical modification that overcomes this constraint. Specifically, we combine scan field rotation using a K-mirror with fast axial scanning using an electrically tunable lens. We demonstrate the versatility of our approach for calcium imaging by aligning the imaging plane with the curved pyramidal cell layer in mouse hippocampus, increasing the number of simultaneously imaged neurons. In addition, we image dendritic arbors of hippocampal and cortical pyramidal neurons that would otherwise be only partially accessible for fast imaging, and we apply our method to the curved layers of cerebellar Purkinje cells. Furthermore, we demonstrate how scan field rotation can be used to optimally orient a dendritic branch for high-speed subcellular glutamate imaging. Our approach is based on a single module and can therefore be retrofitted to standard two-photon systems. We openly share design specifications, 3D-printed parts, and custom ScanImage-compatible code to facilitate rapid adoption.