A Rapid Optical Clearing Protocol Using 2,2′-Thiodiethanol for Microscopic Observation of Fixed Mouse Brain

Abstract

Elucidation of neural circuit functions requires visualization of the fine structure of neurons in the inner regions of thick brain specimens. However, the tissue penetration depth of laser scanning microscopy is limited by light scattering and/or absorption by the tissue. Recently, several optical clearing reagents have been proposed for visualization in fixed specimens. However, they require complicated protocols or long treatment times. Here we report the effects of 2,2′-thiodiethanol (TDE) solutions as an optical clearing reagent for fixed mouse brains expressing a yellow fluorescent protein. Immersion of fixed brains in TDE solutions rapidly (within 30 min in the case of 400-µm-thick fixed brain slices) increased their transparency and enhanced the penetration depth in both confocal and two-photon microscopy. In addition, we succeeded in visualizing dendritic spines along single dendrites at deep positions in fixed thick brain slices. These results suggest that our proposed protocol using TDE solution is a rapid and useful method for optical clearing of fixed specimens expressing fluorescent proteins.

Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy

“…Previously, 2,2'-thiodiethanol (TDE) solutions were shown to quickly render fixed mouse brain tissues optically transparent. 7,8 TDE-treated fixed mouse brain slices exhibited a higher penetration depth in the visible and the near-infrared regions, and a stronger fluorescence signal was emitted from the deep layers. In this study, we used 51% TDE solutions to clear the 0.95-mm-thick fixed brain slices of YFP expressing mouse (Thy1-YFP-H mouse 28 ) and to adjust the refractive index to 1.42.…”

“…[3][4][5][6] On the other hand, several optical clearing reagents for fixed tissues have been proposed to increase the tissue penetration depth and enable much deeper imaging. [7][8][9] However, the spatial resolution and the fluorescence signal in laser scanning microscopy are frequently degraded by wavefront aberrations caused by refractive-index mismatches in the optical path. These aberrations and their effects have been numerically calculated and experimentally investigated by several authors.…”

Clearing of fixed tissue: a review from a microscopist’s perspective

“…12,13 A drawback of sugar solutions is their high viscosity; to solve this problem, alternative approaches based on organic compounds like 2,2′-thiodiethanol (TDE, n ¼ 1.42) 19,23,28 have been proposed. The family of water-based clearing includes also commercial solutions whose detailed composition is not known (FocusClear, n ¼ 1.45).…”

“…5,6,[8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] Here, we try to classify clearing approaches based on the microscopy technique where they can be applied, rather than on the clearing mechanism. 7 We believe that this new taxonomy can be a helpful starting point for microscopists willing to work with transparent tissue.…”

High-refractive index of acrylate embedding resin clarifies mouse brain tissue

“…Aqueous-based clearing of biological tissues tends to cause tissue expansion and requires an extended period of time, and some denaturing reagents, such as urea or sodium dodecyl sulfate, could cause the tissue to become antigen-insensitive. [6][7][8][9][10][11][12][13] Hydrogel embedding combined with electrophoresis clearing is a complex method that requires gel embedding and electrophoresis equipment, and the reproducibility of experimental results is poor. [14][15][16] Solvent-based reagents virtually quench endogenous fluorescent proteins.…”