Multifocus microscopy with optically sectioned axial superresolution

TL;DR

This paper introduces a scalable multifocus microscopy technique combining optical sectioning and axial superresolution, enabling rapid 3D imaging of dense samples with sub-diffraction resolution deep into tissue.

Contribution

The authors develop a novel multifocus microscopy method with optical sectioning and axial superresolution, verified theoretically and demonstrated on biological samples.

Findings

Achieved 35 volumes per second imaging rate.

Visualized mitochondria at axial superresolution.

Captured sub-diffraction mitochondria vesicles up to 30 μm deep.

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.