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

作者感谢横子（顺天堂大学）的AAV载体生产以及Kisara Hoshino（顺天堂大学）的技术援助。本研究得到了JSPS KAKENHI（JP20K07231至K.Y.;JP21H03529 转 T.F.;JP20K07743 转 M.K.;JP21H02592 转 H.H.）和创新领域“共振生物”的科学研究（JP18H04743至H.H.）。本研究还得到了日本医学研究开发厅（AMED）（JP21dm0207112至T.F.和H.H.）、日本科学技术振兴机构（JST）的登月研发（JPMJMS2024至H.H.）、JST的颠覆性科学与技术聚变导向研究（FOREST）（JPMJFR204D至H.H.）、顺天堂大学医学院老年疾病研究所的助学金（X2016至K.Y.;X2001至H.H.），以及私立学校品牌项目。

所有实验均由顺天堂大学动物护理和使用委员会批准（批准号2021245，2021246），并根据日本科学理事会（2006年）的动物实验正确进行基本准则进行。这里，使用雄性C57BL / 6J小鼠注射携带增强绿色荧光蛋白（EGFP）基因的AAV载体和小白蛋白（PV）/肉豆蔻酰化-EGFP-低密度脂蛋白受体C端细菌人工染色体（BAC）转基因小鼠（PV-FGL小鼠）9 。PV-FGL小鼠维持在C57BL / 6J背景中。在这项研究中未?...

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协议中的关键步骤 协议中有一些关键步骤应该非常谨慎地进行，以获得有意义的结果。样品的均匀固定对于大规模组织内的3D成像至关重要。物镜、样品和浸入液应具有匹配的 RI。它们之间的RI不匹配将导致清除的脑切片内表达EGFP的细胞的高度干扰成像（图3）。物镜对浸入液的校正环调整可最大限度地减少深度引起的球面像差，从而最大限度地提高3D成像中的信号、对...

组织清除方法改进了光学显微镜对生物和临床样品的深度无关成像，允许提取完整组织的结构信息1，2。光学清除技术也可能潜在地加快速度，并降低组织学分析的成本。目前，有三种主要的清除方法可用：亲水，疏水和基于水凝胶的方法1，2。亲水方法在保持荧光信号和组织完整性方面超越了，并且与其他两种方法相比毒性更小3，4。
亲水性清除方法ScaleS以其保持结构和分子完整性以及强大的清除能力（清除 - 保存光谱）而具有独特的地位。在之前的一项研究中，我们通过修改ScaleS6的清除程序，为组织切片（〜1mm厚度）开发了一种快速等距清除方案ScaleSF。这种清除方案需要在三种溶液中连续孵育10.5-14.5小时的脑切片。该方法具有高清除保存光谱的特点，甚至与电子显微镜（EM）分析兼容（补充图1），允许多尺度高分辨率三维（3D）成像和准确的信号重建6。因此，ScaleSF应该有效，特别是在大脑中，神经元细胞精心设计了巨大的繁荣过程，并安排了用于传输和接收信息的专用精细亚细胞结构。从神经元细胞的回路到亚细胞水平提取具有尺度的结构信息对于更好地理解大脑功能非常有用。
在这里，我们提供了一个详细的方案，使用ScaleSF可视化神经元结构，其尺度从介观/电路到微观/亚细胞水平。该方案包括组织制备、组织澄清、清除组织的处理以及已清除组织的共聚焦激光扫描显微镜 （CLSM） 成像。我们的实验方案侧重于询问从电路到亚细胞组分量表的神经元结构。有关制备溶液和将腺相关病毒（AAV）载体注射到小鼠大脑中的详细程序，请分别参考Miyawaki等人20167 和Okamoto et al. 20218。

<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

这里提供了一个详细的方案，以可视化脑组织中从介观到微观水平的神经元结构。从神经回路到亚细胞神经元结构的神经元结构在用ScaleSF光学清除的小鼠脑切片中可视化。这种清除方法是ScaleS的改进版本，是一种用于组织切片的亲水性组织清除方法，可实现强大的清除能力以及高水平的荧光信号保存和结构完整性。可定制的三维（3D）打印成像室设计用于可靠地安装已清除的脑组织。注射携带增强绿色荧光蛋白基因的腺相关病毒载体的小鼠大脑用4%多聚甲醛固定，并用振动组织切片机切成1-mm厚的切片。通过遵循清除方案清除脑切片，其中包括在三种溶液中的顺序孵育，即ScaleS0溶液，磷酸盐缓冲盐水（-）和ScaleS4溶液，共10.5-14.5小时。将清除的脑切片安装在成像室上，并嵌入溶解在ScaleS4D25（0）溶液中的1.5%琼脂糖凝胶中。切片的3D图像采集是使用共聚焦激光扫描显微镜进行的，该显微镜配备了长工作距离的多浸入物镜。从介观神经元成像开始，我们成功地在光学清除的脑切片中可视化精细的亚细胞神经元结构，如树突状棘和轴突。该协议将有助于理解从电路到亚细胞组分尺度的神经元结构。

使用该协议实现1mm厚度的小鼠脑切片的光学清除。 图1B 表示清除治疗前后的小鼠脑切片的传输图像。组织清除方法使1毫米厚的小鼠脑切片透明。在清除溶液中孵育12小时后，发现脑切片的最终尺寸略有扩大（线性扩张：102.5%±1.3%）。在PV-FGL小鼠的质膜中，使用靶向EGFP表达评估组织的荧光和结构完整性的保存（图1C）。在这些小鼠中，体细胞突起膜靶向EGFP在PV阳?...

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

该协议提供了使用组织清除方法ScaleSF在脑切片中进行神经元成像的详细方法。该方案包括脑组织制备，组织澄清，清除切片的处理以及从介观到微观水平的神经元结构的共聚焦激光扫描显微镜成像。

一种从介观尺度到微观尺度的神经元成像组织清除方法

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

我们的协议将促进从电路到组件尺度的神经元结构的研究，这对于更好地理解大脑功能至关重要。我们的清除技术，ScaleSF发挥了强大的清除能力，以及同时可视化大型和小型结构所需的高水平组织保存。我们的方案在大脑中特别有效，其中神经元细胞表现出复杂的过程，巨大的联系，并安排专门的精细亚细胞结构。在开始实验之前，如本表所示，制备Sca / eS溶液并用箔覆盖样品。通过将ScaleS0和ScaleS4溶液各八毫升添加到六孔细胞培养板的两个独立孔中，并在培养箱中将板预热至37摄氏度来开始该过程。用刮刀将脑切片转移到预热的ScaleS0溶液中，并在37摄氏度下以90 RPM的振荡培养箱中将切片孵育两小时。接下来，将透化的脑切片转移到八毫升PBS减去六孔细胞培养板中，并通过保持在40至60 RPM的轨道振荡器中洗涤15分钟。重复此步骤两次。然后，将脑切片转移到8毫升预热的ScaleS4溶液中，并在37摄氏度下以90 RPM孵育8至12小时。对于腔室制备，3D打印腔室框架和显微镜载物台适配器。使用压敏粘合剂将腔室框架连接到盖玻片上。在ScaleS4D25（0）溶液中制备1.5%琼脂糖。混合溶液并用微波炉加热，直到琼脂糖完全溶解。然后让溶液冷却至37摄氏度。用刮刀将清除的脑切片安装到成像室的底部盖玻片上。使用干净的组织从清除的切片中取出多余的溶液。使用微量移液管将ScaleS4凝胶添加到脑切片中以填充成像室。用镊子在上面放另一个盖玻片，然后用一张纸巾和一张载玻片。将成像室转移到四摄氏度的冰箱中。将金属砝码放在载玻片上，静置 30 分钟。孵育后，从成像室中取出材料并擦去多余的凝胶。将成像室置于60毫米玻璃培养皿中，并用压敏粘合剂将成像室的边缘在多个点上连接到培养皿上。将ScaleS4溶液倒入培养皿中，在20至25摄氏度下以40至60 RPM温和摇动一小时。用新鲜溶液代替，并使用移液器吸头轻轻刮擦表面以去除凝胶表面上的气泡。将成像室安装在显微镜载物台上。准备一台共聚焦激光扫描显微镜，配备工作距离为2.5毫米，放大倍率为16倍，数值孔径为0.60的多浸入物镜。打开所有相关的成像设备，如工作站、显微镜、扫描仪、激光器、汞灯，然后启动成像软件。将多浸入式物镜的校正环设置为1.47。将物镜浸入溶液中，让它慢慢接近切片。清除物镜尖端的所有气泡。使用落射荧光在清除的组织中查找感兴趣的区域。要设置图像采集参数，首先，确定图像采集的位深度，因为图像的大小随着位的增加而增加。然后根据发射光谱设置检测波长。设置 XY 分辨率、扫描速度和针孔大小。还要调整激光功率、检测器放大器增益和偏移，直到获得合适的图像。根据 ROI 的大小确定平铺大小，确保捕获 ROI 的整个长度和宽度。在所有平面中的组织中导航，并设置堆栈的起点和终点。根据所需的 Z 分辨率设置 Z 步长。收集图像并使用图像分析软件进行处理。使用该协议，实现了一毫米厚度的小鼠脑切片的光学清除。EGFP在PV-FGL小鼠大脑皮层中的表达表明，组织的荧光和结构完整性得以保留。折射率失配引起的像差导致EGFP阳性神经元的图像亮度和分辨率的明显损失。当调整校正环以匹配此ScaleS4溶液时，用共聚焦激光扫描显微镜清楚地观察这些神经元。在一毫米厚的脑切片中完成小鼠初级躯体感觉皮层中EGFP标记神经元的3D重建。在更高的放大倍率下可以看到装饰有树突状棘的单个树突状乔木。在切片中还观察到轴突末端树艺化和轴突柱突。成像流体和物体透镜之间的反射指数匹配对于清除组织中的3D成像至关重要。我们的技术将有助于将神经元结构从电路整合到组件尺度，提供对神经元机制的见解，大脑中的处理信息。

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

生成高度丰富的神经文化皮层组织块冷冻保存

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

对于电生理和钙成像急性神经组织的长时间培养

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

重建神经元电路：快速神经延伸和功能性神经元连接的新方法

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

A Hydrophobic Tissue Clearing Method for Rat Brain Tissue

鼠脑组织疏水组织清除方法

Hydrophobic tissue clearing methods are easily adjustable, fast, and low-cost procedures that allows for the study of a molecule of interest in unaltered ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

在行为和刺激反应期间为整个大脑成像准备成人德罗索菲拉褪黑麦片加斯特

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

ROS Live Cell Imaging During Neuronal Development

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Immunostaining of Whole-Mount Retinas with the CLARITY Tissue Clearing Method

使用清晰组织清除方法对全山视网膜进行免疫染色

The tissue hydrogel delipidation method (CLARITY), originally developed by the Deisseroth laboratory, has been modified and widely used for immunostaining ...