Multifocus microscopy with optically sectioned axial superresolution

Abstract: Multifocus microscopy enables recording of entire volumes in a single camera exposure. In dense samples, multifocus microscopy is severely hampered by background haze. Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers axial superresolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while generated fluorescence is linearised onto the camera with a multifocus optical element. A synchronised rolling shutter readout realised optical sectioning. We describe the technique theoretically and verify its optical sectioning and superresolution capabilities. We demonstrate a prototype system with a multifocus beam splitter cascade and record monolayers of endothelial cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue and visualise the distribution of mitochondria at axial superresolution. Our method manages to capture sub-diffraction sized mitochondria-derived vesicles up to 30 um deep into the tissue.