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...

<ol>
	<li>Susaki, E. A., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+and+whole-organ+clearing+and+imaging+techniques+with+single-cell+resolution:+Toward+organism-level+systems+biology+in+mammals.">Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: Toward organism-level systems biology in mammals.</a> Cell Chemical Biology. 23 (1), 137-157 (2016).</li><li>Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T., Ueda, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+principles+in+tissue+clearing+and+staining+protocols+for+whole-body+cell+profiling.">Chemical principles in tissue clearing and staining protocols for whole-body cell profiling.</a> Annual Reviews of Cell and Developmental Biology. 32, 713-741 (2016).</li><li>Ueda, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+profiling+of+cells+and+circuits+in+mammals+by+tissue+clearing+and+light-sheet+microscopy.">Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy.</a> Neuron. 106 (3), 369-387 (2020).</li><li>Ueda, H. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+clearing+and+its+applications+in+neuroscience.">Tissue clearing and its applications in neuroscience.</a> Nature Reviews. Neuroscience. 21 (2), 61-79 (2020).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScaleS:+an+optical+clearing+palette+for+biological+imaging.">ScaleS: an optical clearing palette for biological imaging.</a> Nature Neuroscience. 18 (10), 1518-1529 (2015).</li><li>Furuta, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-scale+light+microscopy/electron+microscopy+neuronal+imaging+from+brain+to+synapse+with+a+tissue+clearing+method,+ScaleSF.">Multi-scale light microscopy/electron microscopy neuronal imaging from brain to synapse with a tissue clearing method, ScaleSF.</a> iScience. 25 (1), 103601 (2022).</li><li>Miyawaki, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+imaging+of+cleared+brain+by+confocal+laser-scanning+microscopy.">Deep imaging of cleared brain by confocal laser-scanning microscopy.</a> Protocol Exchange. , (2016).</li><li>Okamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exclusive+labeling+of+direct+and+indirect+pathway+neurons+in+the+mouse+neostriatum+by+an+adeno-associated+virus+vector+with+Cre/lox+system.">Exclusive labeling of direct and indirect pathway neurons in the mouse neostriatum by an adeno-associated virus vector with Cre/lox system.</a> STAR Protocols. 2 (1), 100230 (2021).</li><li>Kameda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parvalbumin-producing+cortical+interneurons+receive+inhibitory+inputs+on+proximal+portions+and+cortical+excitatory+inputs+on+distal+dendrites.">Parvalbumin-producing cortical interneurons receive inhibitory inputs on proximal portions and cortical excitatory inputs on distal dendrites.</a> The European Journal of Neuroscience. 35 (6), 838-854 (2012).</li><li>Sohn, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+vector+platform+for+high-level+gene+transduction+of+central+neurons:+Adeno-associated+virus+vector+equipped+with+the+Tet-off+system.">A single vector platform for high-level gene transduction of central neurons: Adeno-associated virus vector equipped with the Tet-off system.</a> PLoS One. 12 (1), 0169611 (2017).</li><li>Hama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale:+a+chemical+approach+for+fluorescence+imaging+and+reconstruction+of+transparent+mouse+brain.">Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain.</a> Nature Neuroscience. 14 (11), 1481-1488 (2011).</li><li>Stepanyants, A., Martinez, L. M., Ferecsko, A. S., Kisvarday, Z. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fractions+of+short-+and+long-range+connections+in+the+visual+cortex.">The fractions of short- and long-range connections in the visual cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (9), 3555-3560 (2009).</li><li>Kuramoto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+thalamocortical+projections+from+the+motor+thalamic+nuclei+of+the+rat:+a+single+neuron-tracing+study+using+viral+vectors.">Two types of thalamocortical projections from the motor thalamic nuclei of the rat: a single neuron-tracing study using viral vectors.</a> Cerebral Cortex. 19 (9), 2065-2077 (2009).</li><li>Matsuda, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nigrostriatal+dopaminergic+neurons+form+widely+spread+and+highly+dense+axonal+arborizations+in+the+neostriatum.">Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 29 (2), 444-453 (2009).</li><li>Lin, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type-specific+and+projection-specific+brain-wide+reconstruction+of+single+neurons.">Cell-type-specific and projection-specific brain-wide reconstruction of single neurons.</a> Nature Methods. 15 (12), 1033-1036 (2018).</li><li>Winnubst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+1,000+projection+neurons+reveals+new+cell+types+and+organization+of+long-range+connectivity+in+the+mouse+brain.">Reconstruction of 1,000 projection neurons reveals new cell types and organization of long-range connectivity in the mouse brain.</a> Cell. 179 (1), 268-281 (2019).</li><li>Neckel, P. H., Mattheus, U., Hirt, B., Just, L., Mack, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+tissue+clearing+(PACT):+Technical+evaluation+and+new+perspectives+in+immunofluorescence,+histology,+and+ultrastructure.">Large-scale tissue clearing (PACT): Technical evaluation and new perspectives in immunofluorescence, histology, and ultrastructure.</a> Scientific Reports. 6, 34331 (2016).</li></ol>

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 ...

Our protocol would facilitate investigation of neuronal structures from circuit to component scales which is essential for better understanding of brain functions. Our clearing technique, ScaleSF exerts potent clearing capability, as well as a high level of tissue preservation that is required for simultaneous visualization of both large and small scale structures. Our protocol is especially effective in the brain where neuronal cells exhibit elaborate processes, tremendous links, and arrange specialized fine subcellular structures.Before beginning the experiment, prepare Sca/eS solutions as shown in this table and cover the samples with foil. Start the procedure by adding eight milliliters each of ScaleS0 and ScaleS4 solutions to two separate wells of a six well cell culture plate and pre-warm the plate to 37 degrees Celsius in an incubator. Transfer the brain slices to the pre-warmed ScaleS0 solution with a spatula and incubate the slices for two hours at 37 degrees Celsius in a shaking incubator at 90 RPM.Next, transfer the permeabilized brain slices in eight milliliters of PBS minus in a six well cell culture plate and wash for 15 minutes by keeping in an orbital shaker at 40 to 60 RPM. Repeat this step twice. Then, transfer the brain slices in eight milliliters of pre-warmed ScaleS4 solution and incubate for eight to 12 hours at 90 RPM at 37 degrees Celsius.For chamber preparation, 3D print the chamber frame and microscope stage adapters. Attach the chamber frame to a coverslip using a pressure sensitive adhesive. Prepare 1.5%agarose in ScaleS4D25(0)solution.Mix the solution and microwave it until the agarose is fully dissolved. Then allow the solution to cool to 37 degrees Celsius. Mount the cleared brain slice onto the bottom coverslip of the imaging chamber with a spatula.Remove the excess solution from the cleared slice using a clean tissue. Add the ScaleS4 gel to the brain slice using a micropipette to fill the imaging chamber. Place another coverslip on top with forceps followed by a piece of tissue paper and a glass slide.Transfer the imaging chamber to a refrigerator at four degrees Celsius. Place metal weights on the glass slide and leave them for 30 minutes. After the incubation, remove the material from the imaging chamber and wipe off excess gel.Place the imaging chamber in a 60 millimeter glass Petri dish and attach the rim of the imaging chamber at multiple points to the dish with a pressure sensitive adhesive. Pour the ScaleS4 solution into the dish and shake gently for one hour at 40 to 60 RPM at 20 to 25 degrees Celsius. Substitute with fresh solution and remove air bubbles on the gel surface by gently scraping the surface using a pipette tip.Mount the imaging chamber on a microscope stage. Prepare a confocal laser scanning microscope equipped with a multi-immersion objective lens of 2.5 millimeter working distance, 16x magnification and 0.60 numerical aperture. Turn on all the relevant imaging equipment, such as the workstation, microscope, scanner, lasers, mercury lamp, and launch an imaging software.Set the correction collar of the multi-immersion objective lens to 1.47. Immerse the objective lens in the solution and let it approach the slice slowly. Remove all air bubbles trapped on the tip of the objective lens.Find regions of interest in cleared tissues using epifluorescence. To set image acquisition parameters, first, determine the bit depth for image acquisition as the size of the image increases with the bit. Then set the detection wavelength according to the emission spectrum.Set the XY resolution, scan speed, and pinhole size. Also adjust the laser power, detector amplifier gain, and offset until a suitable image is obtained. Determine the tiling size based on the size of the ROI, ensuring that the entire length and width of the ROI are captured.Navigate through the tissue in all planes and set the start and endpoints of the stack. Set the Z step size, according to the desired Z resolution. Collect the images and process them using image analysis software.Using this protocol, optical clearing of a mouse brain slice of one millimeter thickness was achieved. EGFP expression in the cerebral cortex of PV-FGL mice showed that the fluorescence and structural integrity of the tissues was preserved. Refractive index mismatch induced aberrations caused a noticeable loss of image brightness and resolution of EGFP positive neurons.These neurons were clearly visualized with the confocal laser scanning microscope when the correction collar was adjusted to match this ScaleS4 solution. 3D reconstruction of EGFP labeled neurons in the mouse primary somatosensory cortex was done in a one millimeter thick brain slice. Individual dendritic arbors decorated with dendritic spines were seen at higher magnification.Axon terminal arborization and axonal boutons were also observed in the slices. Reflective index matching between imaging fluid and object lens is critical for 3D imaging in cleared tissues. Our technique would facilitate integrating neuron structure from circuit to component scales, providing insights into neuron mechanisms, information of processing in the brain.

a-tissue-clearing-method-for-neuronal-imaging-from-mesoscopic-to

Research

JoVE Journal

Neuroscience

2.7K Views.  Juntendo University Graduate School of Medicine. Il nostro protocollo faciliterebbe lo studio delle strutture neuronali dal circuito alle scale dei componenti, che è essenziale per una migliore comprensione delle funzioni cerebrali. La nostra tecnica di compensazione, ScaleSF, esercita una potente capacità di compensazione, nonché un alto livello di conservazione dei tessuti che è necessario per la visualizzazione simultanea di strutture su larga e piccola scala. Il nostro protocollo è particolarmente efficace nel cervello dove le cell...