Wesencraft, K. | Search bioRxiv

bioRxiv2022

Light-sheet mesoscopy with the Mesolens provides fast sub-cellular resolution imaging throughout large tissue volumes.

Rapid imaging of large biological tissue specimens such as ultrathick sections of mouse brain cannot easily be performed with a standard microscope. Optical mesoscopy offers a solution, but thus far imaging has been too slow to be useful for routine use. We have developed two different illuminators for light-sheet mesoscopy with the Mesolens and we demonstrate their use in high-speed optical mesoscale imaging of large tissue specimens. The first light-sheet approach uses Gaussian optics and is straightforward to implement. It provides excellent lateral resolution and high-speed imaging, but the axial resolution is poor. The second light-sheet is a more complex Airy light-sheet that provides sub-cellular resolution in three dimensions that is comparable in quality to point-scanning confocal mesoscopy, but the light-sheet method of illuminating the specimen reduces the imaging time by a factor of 14. This creates new possibilities for high-content, higher-throughput optical bioimaging at the mesoscale.

biophysics↗

bioRxiv2020

An evaluation of multi-excitation-wavelength standing-wave fluorescence microscopy (TartanSW) to improve sampling density in studies of the cell membrane and cytoskeleton

Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50% to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.

cell biology↗