ScaleS: an optical clearing palette for biological imaging

Abstract

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer's disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer's disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

On-chip clearing of arrays of 3-D cell cultures and micro-tissues

“…As described by Hama et al, 35 a solution of 9.1M urea and 22.5% (w/v) sorbitol in ultrapure H 2 O was prepared by dissolving urea (Anachemia, Richmond, BC) and sorbitol (Bio Basic) in warm water. The ScaleSQ(0) solution was chosen for our clearing experiments due to its rapid clearing and absence of detergents.…”

“…The ScaleSQ(0) solution was chosen for our clearing experiments due to its rapid clearing and absence of detergents. As suggested by Hama et al, 35 the solution was kept warm (>30 C) at all times by incubating at 37 C in an incubator before use, and by using portable hot packs as well as a lens-warmer setup to warm the solution during the on-chip clearing experiment. We did not observe urea precipitation during our experiments under these conditions.…”

“…32,37 lScaleSQ(0) also first shrinks and then expands, as was described in its original demonstration. 35 This shrinkage-expansion process for lClear T2 and lScaleSQ(0) is likely due to an initial osmotic shrinkage followed by swelling due to hyperhydration; formamide (which is similar to urea) may clear tissues by a similar hyperhydration mechanism. 47 Awareness of responses like this could be important to the users of this clearing method as the shrinkage-expansion process has the potential to induce imaging artefacts.…”

“…Scale requires long incubation times and swells the tissue, while a sorbitol-based variation (ScaleS) has been demonstrated to alleviate these issues through the use of a solution combining sorbitol and urea. 35 ScaleSQ, a variant of ScaleS designed for faster clearing in a single-step process, uses a high concentration of urea along with sorbitol to clear 1-2 mm thick brain slices. Ke et al have demonstrated SeeDB, a hydrophilic fructose-based clearing agent to match the RI of tissue lipids and the media, without quenching the fluorophores, shorter incubation time (days) than the urea-based methods (weeks to months), and size preservation in brain samples.…”

“…For example, CUBIC relies on chemical lipid removal followed by RI matching, 29 while FRUIT has combined the use of fructose and urea for reduced solution viscosity and easier clearing of brain samples. 34 Among all the aforementioned clearing techniques, SeeDB, 33 Clear T2 , 32 and ScaleS 35 are the only ones demonstrated to be compatible with visualizing both intrinsically expressed fluorescent proteins and lipophilic dyes, 36 two types of fluorophores with wide applicability in phenotypic drug screening research.…”