Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging

Abstract

One Sentence Summary: Glaser et al. describe a light-sheet microscopy architecture that enables passive multidirectional illumination with confocal line detection to enable both uniform fluorescence excitation and contrast-enhanced imaging of fluorescently labeled samples.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/270207 doi: bioRxiv preprint first posted online Feb. 23, 2018; Abstract Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method for rapid and optically efficient 3D microscopy. Initial LSFM designs utilized a static sheet of light, termed selective plane illumination microscopy (SPIM), which exhibited shadowing artifacts and deteriorated contrast due to light scattering. These issues have been addressed, in part, by multidirectional selective plane illumination microscopy (mSPIM), in which rotation of the light sheet is used to mitigate shadowing artifacts, and digital scanned light-sheet microscopy (DSLM), in which confocal line detection is used to reject scattered light. Here we present a simple passive multidirectional digital scanned light-sheet microscopy (mDSLM) architecture that combines the benefits of mSPIM and DSLM. By utilizing an elliptical Gaussian beam with increased angular diversity in the imaging plane, mDSLM provides shadow-free contrastenhanced imaging of fluorescently labeled samples.peer-reviewed)

Lightsheet Microscopy

“…A simple solution is to rotate the sample, which is sequentially illuminated from alternate sides, and fuse the image into a single dataset (Verveer et al, 2007); however, this slows image acquisition and can potentially overexpose the center of the sample. Another solution is to add a third objective lens so that the sample can be illuminated from two sides (mSPIM; Glaser et al, 2018;Hu, Bolus, & Brown, 2017;Huisken & Stainier 2007;Wu et al, 2011) by rapidly tilting or pivoting the beam (Huisken & Stainier, 2007). This is useful when the sample cannot be rotated, and it offers isotropic resolution.…”