Fig 1.
(a) Brain extracted after cardiac perfusion. (b) Brain placed in mouse brain slicer matrix. (c) Two 1 mm thick coronal sections removed from midpoint of brain. Red dashed lines in (b) denote approximate locations at which each section is removed. Each of the two sections were bisected, resulting in four samples that were then exposed to one of the four optical clearing agents. Red solid lines denote regions of interest probed and analyzed for attenuation measurements and confocal microscopy.
Fig 2.
Representative photos of brain samples taken during immersion in each OCA.
Time points of 0,3,6, and 24h are shown as well as 24h after re-immersion of samples in PBS-sodium azide.
Table 1.
Change in volume and thickness of brain samples during optical clearing.
Fig 3.
Normalized attenuation coefficients measured for each OCA over the 420–720 nm wavelength range.
Data points represent median values of measurements. Error bars representing spread are omitted for clarity.
Table 2.
Optical attenuation coefficient (λ = 640nm) during optical clearing.
Fig 4.
Attenuation reduction significance between time points.
Immersion in each optical clearing agent led to a significant reduction in attenuation coefficient. * and *** denote p<0.05 and p<0.001, respectively. PBS-SA = solution of PBS and sodium azide.
Fig 5.
Attenuation reduction significance between OCAs.
Of the four optical clearing agents, ScaleSQ had the highest and sRIMS the lowest optical clearing potential at each evaluated time point (3, 6, and 24h). *, **, and *** denote p<0.05, p<0.01, and p<0.001, respectively.
Fig 6.
(A) Representative confocal fluorescence microscope scans and (B) vascular density at specific immersion time points and at three imaging depths.
Note that the colorbar differs for each row of images, to facilitate comparison of the visibility of microvasculature among clearing agents. Only ScaleSQ enables visualization of microvessels at 800μm depth.