Multifocus microscopy with optical sectioning and high axial resolution

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 improved axial resolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while fluorescence from each illuminated plane in the sample is mapped onto a line on the camera with a multifocus optical element. A synchronized rolling shutter readout realizes optical sectioning. We describe the technique theoretically and verify its optical sectioning and resolution improvement 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 visualize the distribution of mitochondria at high axial resolution. Our method manages to capture sub-diffraction sized mitochondria-derived vesicles up to 30 µm deep into the tissue.