Name: a tissue clearing method for neuronal imaging from mesoscopic to microscopic scales
Uploaded: 2022-05-10T16:00:00
Description: Watch this Scientific Journal Video about A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales at JoVE.com

The authors thank Yoko Ishida (Juntendo University) for AAV vector production and Kisara Hoshino (Juntendo University) for technical assistance. This study was supported by JSPS KAKENHI (JP20K07231 to K.Y.; JP21H03529 to T.F.; JP20K07743 to M.K.; JP21H02592 to H.H.) and Scientific Research on Innovative Area &#8220;Resonance Bio&#8221; (JP18H04743 to H.H.). This study was also supported by the Japan Agency for Medical Research and Development (AMED) (JP21dm0207112 to T.F. and H.H.), Moonshot R&#38;D from the Japan Science and Technology Agency (JST) (JPMJMS2024 to H.H.), Fusion Oriented Research for disruptive Science and Technology (FOREST) from JST (JPMJFR204D to H.H.), Grants-in-Aid from the Research Institute for Diseases of Old Age at the Juntendo University School of Medicine (X2016 to K.Y.; X2001 to H.H.), and the Private School Branding Project.

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...

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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...

Tissue clearing methods have improved depth-independent imaging of biological and clinical samples with light microscopy, allowing for extraction of structural information on intact tissues1,2. 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 methods1,2. Hydrophilic approaches surpass in preserving fluorescence signals and tissue integrity and are less toxic compared to the other two approaches3,4.
A hydrophilic clearing method, ScaleS, holds a distinctive position with its preservation of structural and molecular integrity as well as potent clearing capability (clearing-preservation spectrum)5. In a previous study, we developed a rapid and isometric clearing protocol, ScaleSF, for tissue slices (~1-mm thickness) by modifying the clearing procedure of ScaleS6. This clearing protocol requires sequential incubations of brain slices in three solutions for 10.5-14.5 h. The method is featured with a high clearing-preservation spectrum, which is compatible even with electron microscopy (EM) analysis (Supplementary Figure 1), allowing for multi-scale high-resolution three dimensional (3D) imaging with accurate signal reconstruction6. Thus, ScaleSF should be effective especially in the brain, where neuronal cells elaborate exuberant processes of tremendous length, and arrange specialized fine subcellular structures for transmitting and receiving information. Extracting structural information with scales from circuit to subcellular levels on neuronal cells is quite useful toward better understanding of brain functions.
Here, we provide a detailed protocol to visualize neuronal structures with scales from the mesoscopic/circuit to microscopic/subcellular level using ScaleSF. The protocol includes tissue preparation, tissue clarification, handling of cleared tissues, and confocal laser scanning microscopy (CLSM) imaging of cleared tissues. Our protocol focuses on interrogating neuronal structures from circuit to subcellular component scales. For a detailed procedure for preparation of the solutions and stereotaxic injection of adeno-associated virus (AAV) vectors into mouse brains, refer to Miyawaki et al. 20167 and Okamoto et al. 20218, respectively.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16x multi-immersion objective lens</td>
			<td>Leica Microsystems</td>
			<td>HC FLUOTAR 16x/0.60 IMM CORR VISIR</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Nacalai Tesque</td>
			<td>01028-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>TaKaRa Bio</td>
			<td>L03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Nacalai Tesque</td>
			<td>13407-45</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Nacalai Tesque</td>
			<td>06286-55</td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-cyclodextrin</td>
			<td>Wako Pure Chemical Industries</td>
			<td>037-10643</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens Essential</td>
			<td>Scientific Volume Imaging</td>
			<td>ver. 18.10.0p8/21.10.1p0 64b</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td>ver. 9.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X</td>
			<td>Leica Microsystems</td>
			<td>LAS X, ver. 3.5.5.19976</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl-&#946;-cyclodextrin</td>
			<td>Tokyo Chemical Industry</td>
			<td>M1356</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck Millipore</td>
			<td>1.04005.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10x; pH 7.4)</td>
			<td>Nacalai Tesque</td>
			<td>27575-31</td>
			<td>10x PBS(&#8211;)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Nacalai Tesque</td>
			<td>31233-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Kyoritsu Seiyaku</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>TCS SP8</td>
			<td>Leica Microsystems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque</td>
			<td>35501-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Nacalai Tesque</td>
			<td>35940-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating tissue slicer</td>
			<td>Dosaka EM</td>
			<td>PRO7N</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 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 (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;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.

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% &#177; 1.3%).&#160;The preservation of fluorescence and structural integrity of the tissues...

Watch this Scientific Journal Video about A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales at JoVE.com

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

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.

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

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