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

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.