Multifocus microscopy with optical sectioning and high axial resolution

Abstract

Multifocus microscopy enables recording of entire volumes in a single camera 11 exposure. In dense samples, multifocus microscopy is severely hampered by background haze. 12 Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers 13 improved axial resolution capabilities. In our method, a dithered oblique light-sheet scans the 14 sample volume during a single exposure, while fluorescence from each illuminated plane in the 15 sample is mapped onto a line on the camera with a multifocus optical element. A synchronized 16 rolling shutter readout realizes optical sectioning. We describe the technique theoretically and 17 verify its optical sectioning and resolution improvement capabilities. We demonstrate a 18 prototype system with a multifocus beam splitter cascade and record monolayers of endothelial 19 cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue 20 and visualize the distribution of mitochondria at high axial resolution. Our method manages to 21 capture sub-diffraction sized mitochondria-derived vesicles up to 30 µm deep into the tissue.