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

Abstract

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 pipeline on various cleared tissue samples and demonstrated its robustness over a wide range of clearing protocols.

Axial de-scanning using remote focusing in the detection arm of light-sheet microscopy

“…Unlike the adaptive optics or DM-based approaches that require correcting the defocus plane-by-plane, pupil-matched remote focusing (pmRF), pioneered by Botcherby et al 17,18 , instantaneously corrects defocus across 3D volumes for high-NA optics thereby conserving the microscope's temporal bandwidth [17][18][19][20][21][22][23][24][25][26][27] . In addition, because pmRF allows precise mapping of the wavefront coupled into the back-pupil of the objective, where the angular magnification is unity, such techniques have been routinely used to carry out aberration-free high-quality axial focus control [17][18][19][20][21][22][23][24][25][26][27] . In pmRF techniques, a fast axial scan is achieved by the translation of a small mirror in front of the remote objective using a focus actuator 19,20,24 or by a lateral scan of a galvo mirror in combination with a step or tilted mirror at the remote objective 28 .…”