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

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 Sca/eSF. This clearing method is a modified version of Sca/eS 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, Sca/eSO solution, phosphate buffer saline (-), and Sca/eS4 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 Sca/eS4D25(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.

Automated summary

This article presents a detailed protocol for visualizing neuronal structures in brain tissues at different levels, from mesoscopic to microscopic. By utilizing a modified version of Sca/eSF, the neuronal structures in mouse brain slices are successfully visualized with high preservation of fluorescence signals and structural integrity. The protocol includes a 3D-printed imaging chamber for reliable mounting of cleared brain tissues and a sequential incubation process for tissue clearing. The imaging was carried out using a confocal laser scanning microscope. This protocol is valuable for understanding neuronal structures from circuit to subcellular levels.