Development of Low Coherence Light Sheet Illumination Microscope for Fluorescence-free Bioimaging

摘要

Light Sheet Illumination Microscopy (LSIM) is an imaging modality featuring the novel arrangement with the illumination axis perpendicular to the detection axis. In this technology a well defined light sheet is generated and aligned precisely to the focal plane of the microscope objective and thus only the thin in-focus layer of the sample is illuminated and imaged, thereby avoiding out-of-focus light. Besides the inherent optical sectioning function, other advantages include fast imaging speed, high longitudinal resolution and decreased light-induced damage. Though promising, this microscopy is currently restricted to imaging fluorescently labeled tissue; in inspection of intact tissue using scattered light, the acquired images suffer from intense speckles because of the severe coherence in the illumination. This work aims to build a microscope capable of achieving intrinsic images of the fluorescence-free sample with reduced or eliminated speckles, by developing a low coherence light sheet illumination. To diminish the spatial coherence, the sample is illuminated with tens of independent sub-beams (without inter-coherence) illuminating the FOV (Field Of View) of the microscope with diverse incident angles. The temporal coherence is dramatically reduced by employing a supercontinuum laser with a broad spectrum as the light source. The new microscopy significantly extends the functionality of Light Sheet Illumination Microscopy and will enable many new bioimaging applications.