Research Article: Optical clearing potential of immersion-based agents applied to thick mouse brain sections

Date Published: May 10, 2019

Publisher: Public Library of Science

Author(s): Mathew Loren, Christian Crouzet, Adrian Bahani, Vitaly Vasilevko, Bernard Choi, Kristen C. Maitland.

http://doi.org/10.1371/journal.pone.0216064

Abstract

We have previously demonstrated that the use of a commercially-available immersion-based optical clearing agent (OCA) enables, within 3–6 hours, three-dimensional visualization of subsurface exogenous fluorescent and absorbing markers of vascular architecture and neurodegenerative disease in thick (0.5–1.0mm) mouse brain sections. Nonetheless, the relative performance of immersion-based OCAs has remained unknown. Here, we show that immersion of brain sections in specific OCAs (FocusClear, RIMS, sRIMS, or ScaleSQ) affects both their transparency and volume; the optical clearing effect occurs over the entire visible spectrum and is reversible; and that ScaleSQ had the highest optical clearing potential and increase in imaging depth of the four evaluated OCAs, albeit with the largest change in sample volume and a concomitant decrease in apparent microvascular density of the sample. These results suggest a rational, quantitative framework for screening and characterization of the impact of optical clearing, to streamline experimental design and enable a cost-benefit assessment.

Partial Text

Imaging thicker tissue sections is limited due to light scattering [1]. Recent progress in optical clearing methods modulates optical scattering to enable volumetric imaging without the need for sectioning. Entire organs can be cleared and imaged with minimal damage to structures of interest and without requiring extensive post-processing of data. Several optical clearing approaches, including variants of CLARITY [2,3], variants of Scale [4], FocusClear [5,6], RIMS/sRIMS [7], variants of 3DISCO [8], and SeeDB [9], are reported to reduce scattering within biological tissues and enable visualization of subsurface tissue structures, with a major emphasis on the brain.

We studied the following four OCAs: FocusClear (CelExplorer, Taiwan), refractive index matching solution (RIMS), sorbitol RIMS (sRIMS), and ScaleSQ. For RIMS, sRIMS, and ScaleSQ, we followed preparation protocols described in the respective papers with reagents purchased from Sigma Aldrich, Carlsbad, CA:

Our results reveal that immersion of brain sections in specific OCAs affects both their transparency and volume; the optical clearing effect occurs over the entire visible spectrum and is reversible; and that ScaleSQ had the highest optical clearing potential and increase in imaging depth of the four evaluated OCAs, albeit with the largest change in sample volume and a concomitant decrease in apparent microvascular density of the sample.

In summary, our results indicate that immersion of mouse brain sections in FocusClear, RIMS, sRIMS, or ScaleSQ lead to a decrease in optical attenuation coefficient over the visible spectrum, demonstrate the spatiotemporal dynamics associated with sample immersion, and show that confocal microscopy imaging depth trends with optical clearing potential. We present a rational, quantitative framework for screening and characterization of the impact of optical clearing. Selection of an OCA tailored to the requirements of a given set of experiments should help streamline experimental design and enable a cost-benefit assessment.

 

Source:

http://doi.org/10.1371/journal.pone.0216064

 

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