Abstract
Oblique plane microscopy (OPM) is a single objective light-sheet microscopy that performs three dimensional (3D) imaging by axial scan of the generated light-sheet. Recently, multiple techniques for lateral scanning of the generated light-sheet in OPM have emerged. However, their suitability for geometrically distortion free 3D imaging, which essentially requires a constant tilt light-sheet scan, has not been evaluated. In this work, we use a geometrical optics approach and derive analytical relationship for the amount of tilt variance in planar mirror based scanned oblique plane illumination (SOPi) arrangement. We experimentally validate the derived relationship and use it to arrive at an optimized scanner geometry and to understand its associated limitations. We discuss the effects of scanning on optical aberrations and 3D field of view in optimized, tilt invariant, lateral scanning OPM systems. We also provide experimental strategies enabling precise scanner alignment for tilt invariance, as well as an open source platform for rapid design of new oblique light-sheet microscopes.
Highlights
Light-sheet microscopy is a powerful imaging technique based on optical sectioning
Oblique plane microscopy (OPM) is a unique setup which performs light-sheet microscopy with a single objective facing a given sample, providing maximum steric access to the sample [9]. It combines the concepts of aberration free remote focusing and selective plane illumination microscopy (SPIM) [10,11,12]
To combine the best features of the previous two arrangements, we introduced a plane mirror based scanned oblique plane illumination (SOPi) microscopy [17,18]
Summary
Light-sheet microscopy is a powerful imaging technique based on optical sectioning. The conventional light-sheet microscopy configuration consists of two objectives orthogonally arranged around a sample [1,2]. Oblique plane microscopy (OPM) is a unique setup which performs light-sheet microscopy with a single objective facing a given sample, providing maximum steric access to the sample [9]. To achieve this, it combines the concepts of aberration free remote focusing and selective plane illumination microscopy (SPIM) [10,11,12]. In addition to addressing tilt invariance in oblique light-sheet scanning, we evaluate optical aberrations and field of view in the optimized system during a lateral scan
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