Axial Scanning of Dual Focus to Improve Light Sheet Microscopy

Axially swept light sheet microscopy (ASLM) is an emerging technique that enables isotropic, subcellular resolution imaging with high optical sectioning capability over a large field-of-view (FOV). Due to its versatility across a broad range of immersion media, it has been utilized to image specimens that may range from live cells to intact chemically cleared organs. However, because of its design, the performance of ASLM-based microscopes is impeded by a low detection signal and the maximum achievable frame-rate for full FOV imaging. Here we present a new optical concept that pushes the limits of ASLM further by scanning two staggered light sheets and simultaneously synchronizing the rolling shutter of a scientific camera. For a particular peak-illumination-intensity, this idea can make ASLMs image twice as fast without compromising the detection signal. Alternately, for a particular frame rate our method doubles the detection signal without requiring to double the peak-illumination-power, thereby offering a gentler illumination scheme compared to tradition single-focus ASLM. We demonstrate the performance of our instrument by imaging fluorescent beads and a PEGASOS cleared-tissue mouse brain.