A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

Summary

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Abstract

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (–), and ScaleS4 solution, for a total of 10.5–14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

Introduction

Tissue clearing methods have improved depth-independent imaging of biological and clinical samples with light microscopy, allowing for extraction of structural information on intact tissues^1,2. Optical clearing techniques could also potentially speed up, and reduce the cost for histological analysis. Currently, three major clearing approaches are available: hydrophilic, hydrophobic, and hydrogel-based methods^1,2. Hydrophilic approaches surpass in preserving fluorescence signals and tissue integrity and are less toxic compared to the other two approache....

Protocol

All the experiments were approved by the Institutional Animal Care and Use Committees of Juntendo University (Approval No. 2021245, 2021246) and performed in accordance with Fundamental Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan (2006). Here, male C57BL/6J mice injected with AAV vector carrying enhanced green fluorescent protein (EGFP) gene and parvalbumin (PV)/myristoylation-EGFP-low-density lipoprotein receptor C-terminal bacterial artificial chromosome (BAC) transgenic mice (PV-FGL mice)⁹ were used. PV-FGL mice were maintained in C57BL/6J background. No sex-based differences were found with regard to ....

Results

Optical clearing of a mouse brain slice of 1-mm thickness was achieved using this protocol. Figure 1B represents transmission images of a mouse brain slice before and after the clearing treatment. The tissue clearing method rendered a 1-mm-thick mouse brain slice transparent. A slight expansion in final sizes of brain slices was found after the incubation in the clearing solution for 12 h (linear expansion: 102.5% ± 1.3%). The preservation of fluorescence and structural integrity of the tissues.......

Discussion

Critical steps within the protocol
There are a few critical steps in the protocol that should be conducted with utmost caution to obtain meaningful results. Uniform fixation of samples is imperative for 3D imaging within large-scale tissues. The objective lens, sample, and immersion fluid should have matching RI. RI-mismatch among them will lead to highly disturbed imaging of EGFP-expressing cells within the cleared brain slices (Figure 3). The correction collar adjustment of the o.......

Materials

Name	Company	Catalog Number	Comments
16x multi-immersion objective lens	Leica Microsystems	HC FLUOTAR 16x/0.60 IMM CORR VISIR
Agar	Nacalai Tesque	01028-85
Agarose	TaKaRa Bio	L03
Dimethyl sulfoxide	Nacalai Tesque	13407-45
D-Sorbitol	Nacalai Tesque	06286-55
γ-cyclodextrin	Wako Pure Chemical Industries	037-10643
Glycerol	Sigma-Aldrich	G9012
Huygens Essential	Scientific Volume Imaging	ver. 18.10.0p8/21.10.1p0 64b
Imaris	Bitplane	ver. 9.0.0
Leica Application Suite X	Leica Microsystems	LAS X, ver. 3.5.5.19976
Methyl-β-cyclodextrin	Tokyo Chemical Industry	M1356
Paraformaldehyde	Merck Millipore	1.04005.1000
Phosphate Buffered Saline (10x; pH 7.4)	Nacalai Tesque	27575-31	10x PBS(–)
Sodium azide	Nacalai Tesque	31233-55
Sodium pentobarbital	Kyoritsu Seiyaku	N/A
TCS SP8	Leica Microsystems	N/A
Triton X-100	Nacalai Tesque	35501-15
Urea	Nacalai Tesque	35940-65
Vibrating tissue slicer	Dosaka EM	PRO7N