Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index

Abstract

Improvement of imaging quality has the potential to visualize previously unseen building blocks of the brain and is therefore one of the great challenges in neuroscience. Rapid development of new tissue clearing techniques in recent years have attempted to solve imaging compromises in thick brain samples, particularly for high resolution optical microscopy, where the clearing medium needs to match the high refractive index of the objective immersion medium. These problems are exacerbated in insect tissue, where numerous (initially air-filled) tracheal tubes branching throughout the brain increase the scattering of light. To date, surprisingly few studies have systematically quantified the benefits of such clearing methods using objective transparency and tissue shrinkage measurements. In this study we compare a traditional and widely used insect clearing medium, methyl salicylate combined with permanent mounting in Permount (“MS/P”) with several more recently applied clearing media that offer tunable refractive index (n): 2,2′-thiodiethanol (TDE), “SeeDB2” (in variants SeeDB2S and SeeDB2G matched to oil and glycerol immersion, n = 1.52 and 1.47, respectively) and Rapiclear (also with n = 1.52 and 1.47). We measured transparency and tissue shrinkage by comparing freshly dissected brains with cleared brains from dipteran flies, with or without addition of vacuum or ethanol pre-treatments (dehydration and rehydration) to evacuate air from the tracheal system. The results show that ethanol pre-treatment is very effective for improving transparency, regardless of the subsequent clearing medium, while vacuum treatment offers little measurable benefit. Ethanol pre-treated SeeDB2G and Rapiclear brains show much less shrinkage than using the traditional MS/P method. Furthermore, at lower refractive index, closer to that of glycerol immersion, these recently developed media offer outstanding transparency compared to TDE and MS/P. Rapiclear protocols were less laborious compared to SeeDB2, but both offer sufficient transparency and refractive index tunability to permit super-resolution imaging of local volumes in whole mount brains from large insects, and even light-sheet microscopy. Although long-term permanency of Rapiclear stored samples remains to be established, our samples still showed good preservation of fluorescence after storage for more than a year at room temperature.

GFAP splice variants fine-tune glioma cell invasion and tumour dynamics by modulating migration persistence

“…2a). Upon fixation of the ex vivo slices, we applied whole-mount immunofluorescent staining for laminin and used RapiClear tissue clearing 32 . Subsequently, we used confocal imaging to create a 3D-reconstruction of the invasion patterns of the cells in the brain slice (Fig.…”

Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

“…It should be noted, however, that depending on the residual, postclearing RI of the sample, lower index TOC solutions and objective lenses matched to glycerol might offer better results than the oil immersion. This was recently presented by Bekkouche et al [59] in the case of deep insect brain imaging, where Rapiclear1.49 and the glycerol-immersion objective was superior to Rapiclear1.52 and the oil-immersion system (with both protocols overcoming results obtained with TDE). Interestingly, they presented additional incubation with the ethanol, before actual TOC and RI-matching, resulting in a tremendous improvement of insect brain transparency.…”

“…In a recent work, Glaser et al [60] presented an extensive list of combinations between TOC methods and compatible materials for the development of cuvettes. Finally, Szczurek et al [42] point out that the viscosity of the clearing/imaging media should also be perceived as an important factor during SRM, with less viscous solutions obviously penetrating samples better and thus offering advantageous imaging conditions (but at a risk of inducing sample drift when compared to solutions of a higher viscosity [59]).…”

Modelling nonlinear dendritic processing of facilitation in a dragonfly target-tracking neuron

“…Farah et al, 2017) or one from another neuronal subtype (Shoemaker, 2015). This also highlights both the need for, and the challenges in obtaining high-resolution 3-dimensional imagery for complete reconstruction of dendritic trees using state-of-the-art tissue clearing and confocal imaging techniques (Bekkouche et al, 2020).…”