我们感谢马萨诸塞州总医院放射科 AA Martinos 生物医学成像中心的 Bruce Fischl 提供本研究中分析的人脑标本。该项目获得了欧盟地平线2020研究与创新框架计划（Laserlab-Europe）第654148号赠款协议的资助，欧盟地平线2020研究与创新框架计划根据第785907号（人脑项目SGA2）和第945539号（人脑项目SGA3）获得资助，来自美国国立卫生研究院综合医院公司中心，奖励编号为U01 MH117023， 以及意大利教育部在欧洲生物成像意大利节点（ESFRI研究基础设施）框架内。最后，这项研究是在“Fondazione CR Firenze”的贡献下进行的。本文内容完全由作者负责，并不一定代表美国国立卫生研究院的官方观点。 图 1 是使用 BioRender.com 创建的。

福尔马林固定的人体组织样本由马萨诸塞州总医院 （MGH） 尸检服务（美国波士顿）的神经病理学部门提供。根据合作伙伴机构生物安全委员会（PIBC，协议 2003P001937）批准的组织收集方案，在死亡前获得健康参与者的书面同意。授权文件保存在美国马萨诸塞州波士顿的 MGH 尸检服务处，并可根据要求提供。
1. 琼脂糖包埋和样品切割
<ol><li>在烧杯中制备 4% w/v 琼脂糖溶液，在 1x 磷酸盐缓冲盐水（PBS，pH 7.4）中，并将组织块嵌入其中。等到达到室温（RT），然后在4°C下储存24小时。</li>
	<li>为了获得高精度切片，将样品粘在振动切片机的样品盘上，并用冷的 1x PBS （pH 7.4） 填充托盘。根据要切片的组织类型调整振动切片机参数，例如频率、振幅和速度（使用的值：频率 = 5 （50 Hz）、振幅 = 0.4、速度 = 3 （0.15 mm/s）。 注意：应根据样品量使用不同的振动器。对于最大体积为 70 x 40 x 15 mm 的样品，可以使用振动切片机（例如徕卡 VT1000 S）;对于较大的样品，可以使用压缩机（例如，VF-900-0Z 切片机18）或定制设备21。在这里，我们展示了厚度为 400 μm 或 500 μm 的切片。</li>
	<li>切割后，将所有切片放入由1x PBS（pH 7.4）和0.01%w / v叠氮化钠（NaN3）组成的保存溶液中，并储存在4°C。 注意：包埋前，等到琼脂糖溶液达到37°C。 较高的温度可能会损坏试样。建议不要将样品留在 PFA 中超过 48 小时，因为长时间固定可能会破坏抗原和增加组织自发荧光，从而干扰标记过程。对于人体组织的长期储存，含有 NaN3 的 PBS 用于防止微生物污染。由于 NaN3 的毒性，在化学罩下制备防腐剂溶液。</li>
</ol>2.组织固定
注意：以下方案中使用的所有溶液均以大体积制备，足以处理同一组织块的所有切片，并最大限度地减少技术差异并减少单个步骤的时间。
<ol><li>用 50% v/v 1x PBS pH 3、25% v/v 0.1 M 盐酸 （HCl）、25% v/v 0.1 M 邻苯二甲酸氢钾 （KHP） 和新鲜的 4% v/v 戊二醛制备关闭溶液。将所有样品放入带螺旋盖（70 x 23 mm）的培养皿中，并用关闭溶液在4°C轻轻摇动孵育1天，用铝箔保护它们免受光照。</li>
	<li>用 1x PBS（pH 7.4）和新鲜的 1% v/v 戊二醛制备 Switch-on 溶液。将所有样品放入新培养皿中，并用Switch-on溶液孵育，在4°C下轻轻摇动1天，用铝箔保护它们免受光线照射。 注意： 由于戊二醛的高毒性和光敏性，关闭和打开溶液都必须始终新鲜制备并保存在冰上的化学罩下。始终用每种溶液 20 mL 填充培养皿，并用封口膜密封。</li>
</ol>3. 灭活和清除
注意：样品中的反应性戊二醛必须通过与由 1x PBS pH 7.4、4% w/v 乙酰胺、4% w/v 甘氨酸、pH 9.0 组成的灭活溶液孵育来灭活。该溶液可在4°C下储存长达3个月。为了去除脂质并使组织透明，我们使用由 200 mM 十二烷基硫酸钠 （SDS）、20 mM 亚硫酸钠 （Na2SO3） 和 20 mM 硼酸 （H3BO3） 组成的透明溶液，pH 值为 9.0。溶液必须储存在室温下，因为SDS在4°C下沉淀。 以下所有步骤都将在充满溶液的试管中完成。
<ol><li>将样品从培养皿移至试管中，并在室温下用1x PBS pH 7.4轻轻摇动洗涤3×2小时。 注意：对于步骤 3.1，由于戊二醛毒性，有必要在化学罩下工作。乙酰胺、SDS和硼酸也是有毒试剂，必须在化学罩下处理。始终用每种溶液 50 mL 填充试管。</li>
	<li>将样品在37°C的灭活溶液中在水浴中孵育过夜（o / n）。</li>
	<li>在室温下在1x PBS pH 7.4中轻轻摇动3 x 2小时。</li>
	<li>将样品在55°C的透明溶液中在水浴中孵育3-7天。每 2 天更换一次溶液（表 1）。</li>
</ol>4. 免疫标记
注意：在标记步骤之前，有必要去除残留的 SDS，减少自发荧光，并用由 10 mM Tris 碱基、1 mM EDTA 和 0.05% v/v 吐温 20、pH 9 组成的抗原修复溶液揭开表位。该溶液的 pH 值为 9，经过优化，可实现高效的检索过程;Tris碱作为缓冲剂，以维持稳定的pH环境;EDTA是一种螯合剂，可增强抗原的可及性。非离子洗涤剂 Tween 20 有助于改善组织的通透性。该溶液可以储存在4°C。 需要注意的是，过氧化氢与抗原修复步骤相结合，通过分解和/或修饰负责非特异性背景信号的内源性荧光团（如脂褐素），在降低自发荧光信号方面具有协同作用。
<ol><li>用0.1%v / v Triton X-100（PBST）制备1x PBS，将其预热至37°C，并在培养箱中在37°C下轻轻摇动中洗涤样品3×3小时。保持最后一次洗涤 o/n。 注意：将PBST储备溶液储存在4°C。 从组织中去除SDS至关重要，因为它会在低温下形成不溶性沉淀物，这可能会干扰后续的采集过程。</li>
	<li>第二天，向每个样品中加入 10 mL 30% v/v 过氧化氢 （H2O2），并在室温下轻轻摇动 45 分钟。</li>
	<li>用 1x PBS（pH 7.4）洗涤 3 x 10 分钟，并在室温下轻轻摇动。</li>
	<li>在水浴中将抗原修复溶液预热至95°C;将样品转移到加热的溶液中，并在95°C下放置10分钟。</li>
	<li>让试样在轻轻摇晃中冷却至室温40分钟。</li>
	<li>用去离子水轻轻摇晃洗涤 3 x 5 分钟。</li>
	<li>在4°C下在PBS中平衡样品15分钟。</li>
	<li>用0.01%w / v NaN3 制备由PBST制成的新鲜抗体溶液（参见步骤1.3的注释）并加入一抗。将样品与溶液一起在带螺旋盖的培养皿中在37°C下孵育n天（1-7），在培养箱中轻轻摇晃并避光（表2）。 注：孵育时间取决于大小和厚度（表 1 和表 2）。</li>
	<li>n天后，将样品移入试管中，并在培养箱中用37°C预热的PBST洗涤3×2小时，轻轻摇动。</li>
	<li>在PBST中加入0.01%w / v NaN3 的二抗，并将样品在37°C的螺旋盖新培养皿中孵育n天（1-6），在培养箱中轻轻摇晃并避光（表1和表2）。</li>
	<li>n天后，将样品转移到试管中，并在37°C下用预热的PBST在温和的摇动培养箱中洗涤3 x 3小时。保持最后一次洗涤 o/n。 注意： 由于 Triton X-100 和 Tween 20 是粘性的，请缓慢移液以使吸头充满。始终用每种溶液 50 mL 填充试管。每个样品使用 10 mL 抗体溶液，并用封口膜密封培养皿以防止溶液蒸发。由于抗体溶液含有NaN3， 因此它们可以储存在4°C下，并在2周内重复用于标记新标本。</li>
</ol>5.折射率匹配
注意：为了达到高水平的透明度，有必要使组织折射率均匀化。在这里，我们使用在 1x PBS 中稀释的 2,2'-硫代二乙醇 （TDE）。TDE 溶液必须储存在室温下。
<ol><li>将样品转移到新培养皿中，加入30%v / v TDE，并在室温下轻轻摇动4小时。</li>
	<li>取出 30% v/v TDE 溶液，向样品中加入 68% v/v TDE，并在室温下轻轻摇动。 注意： 由于 TDE 是粘性的，请缓慢移液以使吸头充满。吸入TDE蒸气或雾气会刺激呼吸系统。因此，建议在通风良好的地方工作或使用通风橱以尽量减少暴露。TDE 溶液必须在室温下储存，每个样品使用 10 mL TDE 溶液。</li>
</ol>6. 样品安装
注：为了便于样品安装和 LSFM 图像采集，我们使用定制的密封样品架（称为“三明治”），它由三部分组成：显微镜载玻片、垫片和盖玻片。
<ol><li>将钢垫片（56×56×0.5mm3）放在显微镜载玻片（60×60×1mm3）上，并将样品放在显微镜载玻片上。</li>
	<li>小心地放入石英盖玻片（60 x 60 x 0.5 mm3），将所有东西夹在一起，并以 1：1 的比例准备双组分硅胶。使用石英盖玻片来匹配折射率并减少采集过程中的光学像差。</li>
	<li>使用1mL注射器，用双组分硅胶填充垫片和盖玻片之间的空间，并使其干燥10分钟。</li>
	<li>取下夹子，用一根小针慢慢用 68% v/v TDE 填充整个三明治。</li>
	<li>制作新鲜的胶水并密封三明治。 注意： 如果 TDE 在步骤 6.1 中意外到达垫片，则胶水将无法固化。确保在步骤 6.2 期间没有形成气泡。</li>
</ol>7. 剥离
注：SHORT的结构保留和增强的表位可及性允许通过去除抗体并用其他标记物重新染色样品来对切片进行多轮标记。
<ol><li>用刀片打开三明治，将标本放入装有30%v / v TDE的螺旋盖的新培养皿中，并放置3小时。</li>
	<li>将样品转移到试管中，在室温下在1x PBS（pH 7.4）中轻轻摇动洗涤3 x 2小时，并保留最后一次洗涤o/n。</li>
	<li>将样品置于80°C水浴中的透明溶液中4小时。</li>
	<li>在37°C下在1x PBST（pH 7.4）中轻轻摇晃洗涤3×2小时，并保留最后一次洗涤o/n。现在，从步骤4.8开始，样品已准备好再次标记。 注意：始终用每种溶液 50 mL 填充试管。每个样品使用 10 mL TDE 溶液。</li>
</ol>

使用 SHORT 和 LSFM 对人脑进行高分辨率 3D 重建

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作者声明，该研究是在没有任何可能被解释为潜在利益冲突的商业或财务关系的情况下进行的。

大面积人脑的高分辨率成像和 3D 重建需要机械组织切片，然后对单个切片进行光学透明化和免疫标记。这里介绍的协议描述了如何使用 SHORT 组织转化方法快速同时处理多个人脑厚切片，以便使用 LSFM 进行亚细胞分辨率的 3D 脑重建。
与其他方法不同，使用SHORT方法，清算和多重标签步骤不需要复杂的定制设备。可以完全处理大量标本，从而减少大型人脑组织块的处理时间，并...

分析大量人脑的 3D 分子组织和细胞结构需要标本的光学透明度，这是通过具有大量处理时间的方案实现的。开发了光学清除技术以最大限度地减少组织内折射率 （RI） 的异质性，从而减少光散射并增加高分辨率成像的光穿透深度 1,2,3,4,5。目前清除和深层组织标记方法的进展允许通过利用尖端显微镜技术对完整的啮齿动物器官和胚胎进行体积成像 6,7,8,9,10,11,12。
然而，与模式生物相比，死后人脑大面积的体积3D重建仍然是一项具有挑战性的任务。复杂的生物组成和多变的死后固定和储存条件会影响组织清除效率、抗体渗透深度和表位识别13、14、15、16、17、18、19。此外，与模式生物相比，仍然需要机械组织切片和随后的每个切片的清除和标记，以实现大面积人脑区域的高效清除和均匀标记，导致处理时间长，并且需要复杂的定制设备15,20,21,22。
SWITCH - H2O2 - antigen Retrieval -TDE （SHORT） 组织转化技术是专门为分析大量人脑而开发的18,23。该方法采用SWITCH方案11的组织结构保存和高浓度的过氧化氢来减少组织自发荧光，并结合表位恢复。SHORT 允许使用不同神经元亚型、神经胶质细胞、脉管系统和有髓纤维的标记物对人脑切片进行均匀染色18,24。其结果与低密度和高密度蛋白质的分析兼容。由此产生的高透明度水平和均匀的标记使得用荧光显微镜对厚切片进行体积重建，特别是对于快速采集光片的设备，可以使用18,24,25,26,27。
在这项工作中，我们描述了如何使用 SHORT 组织转化技术同时清除和多轮标记数十个福尔马林固定的人脑切片。四种不同的荧光标记可以一起使用，从而鉴定不同的细胞亚群。清除后，可以使用荧光显微镜进行高分辨率体积成像。在这里，我们使用了定制的倒置 LSFM18、24、25、26、27，它可以对样品进行快速光学切片并并行快速采集多个通道。通过这种常规的高通量方案，可以获得全面的细胞和结构表征，并具有人脑大面积的亚细胞分辨率，正如在整个布罗卡区域23 的映射中已经证明的那样。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,2&#39;-thiodiethanol</td>
			<td>Merck Life Science S.R.L.</td>
			<td>166782</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetamide &#62;= 99.0% (GC)</td>
			<td>Merck Life Science S.R.L.</td>
			<td>160</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose High EEO</td>
			<td>Merck Life Science S.R.L.</td>
			<td>A9793</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Merck Life Science S.R.L.</td>
			<td>B7901</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressome VF-900-0Z Microtome</td>
			<td>Precisionary</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>LaserOptex</td>
			<td>/</td>
			<td>customized</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
			<td>Merck Life Science S.R.L.</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Merck Life Science S.R.L.</td>
			<td>G7651</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC_29096</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide 30%</td>
			<td>Merck Life Science S.R.L.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator ISS-4075</td>
			<td>Lab companion&#160;</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Light-sheet fluorescence microscopy (LSFM)</td>
			<td>/</td>
			<td>/</td>
			<td>custom-made</td>
		</tr>
		<tr>
			<td>Loctite Attak</td>
			<td>Henkel Italia srl</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Laborchimica</td>
			<td>/</td>
			<td>customized</td>
		</tr>
		<tr>
			<td>Phospate buffer saline tablet</td>
			<td>Merck Life Science S.R.L.</td>
			<td>P4417</td>
			<td></td>
		</tr>
		<tr>
			<td>Picodent Twinsil</td>
			<td>Picodent</td>
			<td>13005002</td>
			<td>out of production</td>
		</tr>
		<tr>
			<td>Potassium Hydrogen Phtalate</td>
			<td>Merck Life Science S.R.L.</td>
			<td>P1088</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Merck Life Science S.R.L.</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Merck Life Science S.R.L.</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Sulfite</td>
			<td>Merck Life Science S.R.L.</td>
			<td>S0505</td>
			<td></td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>Microlaser srl</td>
			<td></td>
			<td>customized</td>
		</tr>
		<tr>
			<td>Sputum Containers (dishes with screw lids)</td>
			<td>Paul Boettger GmbH &#38; Co. KG</td>
			<td>07.061.2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>PanReac AppliChem (ITW reagents)</td>
			<td>A4577,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck Life Science S.R.L.</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubes</td>
			<td>Sarstedt</td>
			<td>62 547254</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Merck Life Science S.R.L.</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome VT1000S</td>
			<td>Leica Biosystem</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath&#160;</td>
			<td>Memmert</td>
			<td>WNB 7-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies and Dyes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Alpaca Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immuno Reasearch</td>
			<td>611-545-215</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Bovine Anti-Goat IgG (H+L)</td>
			<td>Jackson Immuno Reasearch</td>
			<td>805-545-180</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure Alpaca Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immuno Reasearch</td>
			<td>611-605-215</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Anti-NeuN Antibody</td>
			<td>Merck Life Science S.R.L.</td>
			<td>ABN91</td>
			<td>Dilution used, 1:100</td>
		</tr>
		<tr>
			<td>Anti-Parvalbumin antibody (PV)</td>
			<td>Abcam</td>
			<td>ab32895</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Anti-Vimentin antibody [V9] - Cytoskeleton Marker (VIM)</td>
			<td>Abcam</td>
			<td>ab8069</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Calretinin Polyclonal antibody</td>
			<td>ProteinTech</td>
			<td>12278_1_AP</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher</td>
			<td>D3571</td>
			<td>Dilution used, 1:100</td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG H&#38;L (Alexa Fluor 568)</td>
			<td>Abcam</td>
			<td>ab175700</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IgG H&#38;L (Alexa Fluor 647)</td>
			<td>Abcam</td>
			<td>ab150107</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L (Alexa Fluor 568)</td>
			<td>Abcam</td>
			<td>ab175470</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rat IgG H&#38;L (Alexa Fluor 568) preadsorbed</td>
			<td>Abcam</td>
			<td>ab175475</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Goat Anti-Chicken IgY H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150169</td>
			<td>Dilution used, 1:500</td>
		</tr>
		<tr>
			<td>Goat Anti-Chicken IgY H&#38;L (Alexa Fluor 568)</td>
			<td>Abcam</td>
			<td>ab175711</td>
			<td>Dilution used, 1:500</td>
		</tr>
		<tr>
			<td>Goat Anti-Chicken IgY H&#38;L (Alexa Fluor 647)</td>
			<td>Abcam</td>
			<td>ab150171</td>
			<td>Dilution used, 1:500</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Recombinant Alexa Fluor 488 Anti-GFAP antibody</td>
			<td>Abcam</td>
			<td>ab194324</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Somatostatin Antibody YC7</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-47706</td>
			<td>Dilution used, 1:200</td>
		</tr>
		<tr>
			<td>Vasoactive intestinal peptide (VIP)</td>
			<td>ProteinTech</td>
			<td>16233-1-AP</td>
			<td>Dilution used, 1:200</td>
		</tr>
	</tbody>
</table>

optical clearing and labeling for light-sheet fluorescence microscopy in large-scale human brain imaging

尽管在过去十年中出现了许多清除技术，但由于其尺寸和复杂性，处理死后人类大脑仍然是一项具有挑战性的任务，这使得微米分辨率的成像特别困难。本文提出了一种方案，通过使用 SHORT （SWITCH - H2O2 - Antigen Retrieval - 2,2'-硫代二乙醇 [TDE]） 组织转化方案同时处理数十个切片来执行人脑体积部分的重建，该协议能够使用光片荧光显微镜 （LSFM） 对样品进行透明、标记和顺序成像。SHORT提供快速的组织清除和具有多个神经元标记的厚切片的均质多标记，能够识别白质和灰质中的不同神经元亚群。清除后，通过LSFM以微米分辨率同时在多个通道中对切片进行成像，以进行快速的3D重建。通过在常规高通量方案中将SHORT与LSFM分析相结合，可以在短时间内以高分辨率获得大体积区域的3D细胞结构重建，从而实现人脑的全面结构表征。

Analyzing the 3D molecular organization and cytoarchitecture of large volumes of the human brain requires optical transparency of specimens, achieved through protocols with extensive processing time. Optical clearing techniques were developed to minimize heterogeneity in refractive index (RI) within the tissues, thereby reducing light scattering and increasing the light penetration depth for high-resolution imaging1,2,3,4,5. Current advances in clearing and deep tissue-labeling methods allow volumetric imaging of intact rodent organs and embryos by exploiting cutting-edge microscopy techniques6,7,8,9,10,11,12.
However, volumetric 3D reconstruction of large areas of the postmortem human brain still represents a challenging task compared with model organisms. The complex biological composition and the variable postmortem fixation and storage conditions can compromise the tissue clearing efficiency, the antibody penetration depth, and the epitope recognition13,14,15,16,17,18,19. Moreover, mechanical tissue sectioning and subsequent clearing and labeling of each slice is still required to achieve an efficient clearing and uniform labeling of large human brain areas, resulting in long processing times and the need for sophisticated custom equipment,&#160;compared with model organisms15,20,21,22.
The SWITCH - H2O2 - antigen Retrieval -TDE (SHORT) tissue transformation technique has been developed specifically to analyze large volumes of the human brain18,23. This method employs the tissue structural preservation of the SWITCH protocol11 and high concentrations of peroxide hydrogen to decrease tissue autofluorescence, in combination with epitope restoration. SHORT allows uniform staining of human brain slices with markers for different neuronal subtypes, glial cells, vasculature, and myelinated fibers18,24. Its results are compatible with the analysis of both low- and high-density proteins. The resulting high transparency levels and uniform labeling enable volumetric reconstruction of thick slices with fluorescence microscopy, in particular, for fast acquisition light-sheet apparatus can be used18,24,25,26,27.
In this work, we describe how the SHORT tissue transformation technique can be used for simultaneous clearing and multi-round labeling of tens of formalin-fixed human brain sections. Four different fluorescent markers can be used together, leading to the identification of different cellular sub-populations. After clearing, high-resolution volumetric imaging can be performed with fluorescence microscopy. Here, we used a custom-made inverted LSFM18,24,25,26,27, which enables fast optical sectioning of the sample and rapid acquisition of multiple channels in parallel. With this routinely high-throughput protocol, it is possible to obtain a comprehensive cellular and structural characterization with a sub-cellular resolution of large areas of the human brain as already demonstrated in the mapping of an entire Broca&#39;s area23.

作者聚焦：推进 3D 细胞结构分析 - 人脑的快速体积重建 

光片荧光显微镜在大规模人脑成像中的光学透明化和标记

这里描述的方案允许使用SHORT方法同时处理多个切片，厚度范围从100μm到500μm。这种方法大大缩短了整个过程的整体处理时间。在这项工作中，我们提供了整个管道（图1）的全面描述，用于同时处理多个死后人脑厚切片，并一次演示了24个切片的协议（图2A）。考虑到样品的大小和厚度（表1），清除和共标记的持续时间从11天到30天不等，?...

Watch this Scientific Journal Video about Advancing 3D Cytoarchitecture Analysis â€” Rapid Volumetric Reconstruction of the Human Brain at JoVE.com

本方案通过将 （SWITCH - H2O2 - Antigen Retrieval - 2,2'-硫代二乙醇 [TDE]） SHORT 组织转化技术与常规高通量方案中的光片荧光显微镜成像相结合，为数十个死后人脑切片的快速和同时光学清除、多轮标记和 3D 体积重建提供了分步程序。

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

人脑是一个复杂的器官，跨越不同的技能。要了解其功能，必须在整个大脑中构建详细的细胞传感器。我们的常规高通量方案允许以微米级分辨率分析人脑体积区域的 3D 细胞结构，从而实现其结构表征。人脑大面积的体积重建提出了一些实验挑战，这些挑战与大脑样本的巨大尺寸、复杂的生物成分、可变的死后固定和储存条件以及来自脂褐素型色素的自发荧光信号有关。所有这些都会影响光学清除效率和展示质量。与其他清除技术相比，短组织转化方法与光片显微镜成像相结合，可用于快速同时处理多个人脑切片。这些导致具有亚细胞分辨率的 3D 大脑重建。

optical-clearing-labeling-for-light-sheet-fluorescence-microscopy

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JoVE Journal

Neuroscience

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

1.4K 次观看.  University of Florence. 人脑是一个复杂的器官，跨越不同的技能。要了解其功能，必须在整个大脑中构建详细的细胞传感器。我们的常规高通量方案允许以微米级分辨率分析人脑体积区域的 3D 细胞结构，从而实现其结构表征。人脑大面积的体积重建提出了一些实验挑战，这些挑战与大脑样本的巨大尺寸、复杂的生物成分、可变的死后固定和储存条件以及来自脂褐素型色素的自发荧光信号有关...

视频: 作者聚焦：推进 3D 细胞结构分析 - 人脑的快速体积重建 

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.