Signal Improved ultra-Fast Light-sheet Microscope (SIFT) for large tissue imaging.

Light-sheet fluorescence microscopy (LSFM) in conjunction with tissue clearing techniques enables morphological investigation of large tissues faster and with excellent optical sectioning. Recently, cleared tissue axially swept light-sheet microscope (ctASLM) demonstrated three-dimensional isotropic resolution in millimeter-scaled tissues. But ASLM based microscopes suffer from low detection signal and slow imaging speed. Here we report a simple and efficient imaging platform that employs precise control of two fixed distant light-sheet foci to carry out ASLM. This allowed us to carry out full field of view (FOV) imaging at 40 frames per second (fps) which is a four-fold improvement compared to the current state-of-the-art. In addition, in a particular frame rate, our method doubles the signal compared to the current ASLM technique. To augment the overall imaging performance, we also developed a deep learning based tissue information classifier that enables faster determination of tissue boundary. We demonstrated the performance of our imaging platform on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

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.