Name: whole-brain single-cell imaging and analysis of intact neonatal mouse brains using mri, tissue clearing, and light-sheet microscopy
Uploaded: 2022-08-01T14:00:00
Description: Watch this Scientific Journal Video about Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy at JoVE.com

这项工作得到了NIH（R01MH121433，R01MH118349和R01MH120125到JLS和R01NS110791到GW）和希望基金会的支持。我们感谢显微镜服务实验室的Pablo Ariel协助样品成像。病理学和检验医学系的显微镜服务实验室得到了癌症中心核心支持拨款P30 CA016086对北卡罗来纳大学（UNC）Lineberger综合癌症中心的部分支持。神经科学显微镜核心由拨款P30 NS045892支持。本出版物中报告的研究部分得到了北卡罗来纳州生物技术中心机构支持补助金2016-IDG-1016的支持。

所有小鼠均按照北卡罗来纳大学教堂山分校的机构动物护理和使用委员会（IACUC）使用并得到其批准。
1.小鼠解剖和灌注
注意：使用注射器对P4和P14小鼠进行以下解剖。灌注液的体积将根据动物的年龄而变化。
<ol><li>灌注 注意：多聚甲醛（PFA）是一种危险化学品。在化学通风橱中执行所有灌注步骤。<ol><li>手术前，通过腹腔注射（100mg / kg?...

<ol>
	<li>Dodt, H. U., 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=Ultramicroscopy:+Three-dimensional+visualization+of+neuronal+networks+in+the+whole+mouse+brain.">Ultramicroscopy: Three-dimensional visualization of neuronal networks in the whole mouse brain.</a> Nature Methods. 4 (4), 331-336 (2007).</li><li>H&#228;gerling, 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=A+novel+multistep+mechanism+for+initial+lymphangiogenesis+in+mouse+embryos+based+on+ultramicroscopy.">A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy.</a> The EMBO Journal. 32 (5), 629-644 (2013).</li><li>Tomer, R., Khairy, K., Keller, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shedding+light+on+the+system:+studying+embryonic+development+with+light+sheet+microscopy.">Shedding light on the system: studying embryonic development with light sheet microscopy.</a> Current Opinion in Genetics &amp; Development. 21 (5), 558-565 (2011).</li><li>Li, 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=Micro-optical+sectioning+tomography+to+obtain+a+high-resolution+atlas+of+the+mouse+brain.">Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain.</a> Science. 330 (6009), 1404-1408 (2010).</li><li>Stoner, 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=Patches+of+disorganization+in+the+neocortex+of+children+with+autism.">Patches of disorganization in the neocortex of children with autism.</a> The New England Journal of Medicine. 370 (13), 1209-1219 (2014).</li><li>Renier, N., 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=IDISCO:+A+simple,+rapid+method+to+immunolabel+large+tissue+samples+for+volume+imaging.">IDISCO: A simple, rapid method to immunolabel large tissue samples for volume imaging.</a> Cell. 159 (4), 896-910 (2014).</li><li>Susaki, E. 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=Whole-brain+imaging+with+single-cell+resolution+using+chemical+cocktails+and+computational+analysis.">Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.</a> Cell. 157 (3), 726-739 (2014).</li><li>Richardson, D. 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=Tissue+clearing.">Tissue clearing.</a> Nature Reviews. Methods Primers. 1 (1), 84 (2021).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+tissue+clearing.">Clarifying tissue clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Renier, N., 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=Mapping+of+brain+activity+by+automated+volume+analysis+of+immediate+early+genes.">Mapping of brain activity by automated volume analysis of immediate early genes.</a> Cell. 165 (7), 1789-1802 (2016).</li><li>Santi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+sheet+fluorescence+microscopy:+a+review.">Light sheet fluorescence microscopy: a review.</a> The Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society. 59 (2), 129-138 (2011).</li><li>Girkin, J. M., Carvalho, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+light-sheet+microscopy+revolution.">The light-sheet microscopy revolution.</a> Journal of Optics. 20 (5), 053002 (2018).</li><li>Budday, 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=Mechanical+properties+of+gray+and+white+matter+brain+tissue+by+indentation.">Mechanical properties of gray and white matter brain tissue by indentation.</a> Journal of the Mechanical Behavior of Biomedical Materials. 46, 318-330 (2015).</li><li>Johnson, G. 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=High-throughput+morphologic+phenotyping+of+the+mouse+brain+with+magnetic+resonance+histology.">High-throughput morphologic phenotyping of the mouse brain with magnetic resonance histology.</a> NeuroImage. 37 (1), 82-89 (2007).</li><li>Herculano-Houzel, S., Mota, B., Lent, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+scaling+rules+for+rodent+brains.">Cellular scaling rules for rodent brains.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (32), 12138-12143 (2006).</li><li>Matsumoto, K., 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=Advanced+CUBIC+tissue+clearing+for+whole-organ+cell+profiling.">Advanced CUBIC tissue clearing for whole-organ cell profiling.</a> Nature Protocols. 14 (12), 3506-3537 (2019).</li><li>F&#252;rth, D., 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=An+interactive+framework+for+whole-brain+maps+at+cellular+resolution.">An interactive framework for whole-brain maps at cellular resolution.</a> Nature Neuroscience. 21 (1), 139-149 (2018).</li><li>Chandrashekhar, V., 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=CloudReg:+automatic+terabyte-scale+cross-modal+brain+volume+registration.">CloudReg: automatic terabyte-scale cross-modal brain volume registration.</a> Nature Methods. 18 (8), 845-846 (2021).</li><li>Krupa, O., 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=NuMorph:+Tools+for+cortical+cellular+phenotyping+in+tissue-cleared+whole-brain+images.">NuMorph: Tools for cortical cellular phenotyping in tissue-cleared whole-brain images.</a> Cell Reports. 37 (2), 109802 (2021).</li><li>Petiet, A., Delatour, B., Dhenain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+neurodegenerative+disease+-+Alzheimer's+anatomical+and+amyloid+plaque+imaging.">Models of neurodegenerative disease - Alzheimer's anatomical and amyloid plaque imaging.</a> Methods in Molecular Biology. 771, 293-308 (2011).</li><li>Jenkinson, M., Beckmann, C. F., Behrens, T. E. J., Woolrich, M. W., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FSL.">FSL.</a> NeuroImage. 62, 782-790 (2012).</li><li>Jenkinson, M., Bannister, P., Brady, M., Smith, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+optimization+for+the+robust+and+accurate+linear+registration+and+motion+correction+of+brain+images.">Improved optimization for the robust and accurate linear registration and motion correction of brain images.</a> NeuroImage. 17 (2), 825-841 (2002).</li><li>Avants, B. B., Epstein, C. L., Grossman, M., Gee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetric+diffeomorphic+image+registration+with+cross-correlation:+evaluating+automated+labeling+of+elderly+and+neurodegenerative+brain.">Symmetric diffeomorphic image registration with cross-correlation: evaluating automated labeling of elderly and neurodegenerative brain.</a> Medical Image Analysis. 12 (1), 26-41 (2008).</li><li>. iDISCO method Available from: <a target="_blank" href="https://idisco.info/">https://idisco.info/</a> (2022)</li><li>Ariel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UltraMicroscope+II+-+A+User+Guide.">UltraMicroscope II - A User Guide.</a> University of North Carolina at Chapel Hill. , (2019).</li><li>. Anaconda Distribution - Anaconda documentation Available from: <a target="_blank" href="https://docs.anaconda.com/anaconda/">https://docs.anaconda.com/anaconda/</a> (2022)</li><li>Peng, 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=A+BaSiC+tool+for+background+and+shading+correction+of+optical+microscopy+images.">A BaSiC tool for background and shading correction of optical microscopy images.</a> Nature Communications. 8, 14836 (2017).</li><li>Klein, S., Staring, M., Murphy, K., Viergever, M. A., Pluim, J. P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elastix:+a+toolbox+for+intensity-based+medical+image+registration.">elastix: a toolbox for intensity-based medical image registration.</a> IEEE Transactions on Medical Imaging. 29, 196-205 (2010).</li><li>Lowe, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sift-the+scale+invariant+feature+transform.">Sift-the scale invariant feature transform.</a> International Journal. 2 (91-110), 2 (2004).</li><li>Young, D. M., 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+image+analysis+and+anatomical+atlas+3D+generation+using+MagellanMapper.">Whole-brain image analysis and anatomical atlas 3D generation using MagellanMapper.</a> Current Protocols in Neuroscience. 94 (1), 104 (2020).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Vel&#237;&#353;ek, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Under+the+(Light)+sheet+after+the+iDISCO.">Under the (Light) sheet after the iDISCO.</a> Epilepsy Currents / American Epilepsy Society. 16 (6), 405-407 (2016).</li><li>Young, D. M., 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=Constructing+and+optimizing+3D+atlases+from+2D+data+with+application+to+the+developing+mouse+brain.">Constructing and optimizing 3D atlases from 2D data with application to the developing mouse brain.</a> eLife. 10, (2021).</li><li>Yun, D. 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=Ultrafast+immunostaining+of+organ-scale+tissues+for+scalable+proteomic+phenotyping.">Ultrafast immunostaining of organ-scale tissues for scalable proteomic phenotyping.</a> bioRxiv. , (2019).</li><li>Silvestri, L., 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=Universal+autofocus+for+quantitative+volumetric+microscopy+of+whole+mouse+brains.">Universal autofocus for quantitative volumetric microscopy of whole mouse brains.</a> Nature Methods. 18 (8), 953-958 (2021).</li><li>Frasconi, P., 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=Large-scale+automated+identification+of+mouse+brain+cells+in+confocal+light+sheet+microscopy+images.">Large-scale automated identification of mouse brain cells in confocal light sheet microscopy images.</a> Bioinformatics. 30 (17), 587 (2014).</li><li>Kruskal, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+shortest+spanning+subtree+of+a+graph+and+the+traveling+salesman+problem.">On the shortest spanning subtree of a graph and the traveling salesman problem.</a> Proceedings of the American Mathematical Society. American Mathematical Society. 7 (1), 48-50 (1956).</li><li>Wang, Q., 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=The+allen+mouse+brain+common+coordinate+framework:+A+3D+reference+atlas.">The allen mouse brain common coordinate framework: A 3D reference atlas.</a> Cell. 181, 936-953 (2020).</li><li>Borland, D., 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=Segmentor:+a+tool+for+manual+refinement+of+3D+microscopy+annotations.">Segmentor: a tool for manual refinement of 3D microscopy annotations.</a> BMC Bioinformatics. 22 (1), 260 (2021).</li><li>Kumar, 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=Dual-view+plane+illumination+microscopy+for+rapid+and+spatially+isotropic+imaging.">Dual-view plane illumination microscopy for rapid and spatially isotropic imaging.</a> Nature Protocols. 9 (11), 2555-2573 (2014).</li></ol>

组织清除方法是测量大脑3D细胞组织的有用技术。文献中描述了许多组织清除方法，每种方法都有其优点和局限性6，7，8，9。用于分析组织清除图像中细胞类型的计算工具的选项相对有限。其他可用的工具已经实施到稀疏细胞群，其中分割难度较低10，35或?...

单细胞分辨率的全脑成像是神经科学中的一个重要挑战。细胞分辨率脑图像允许对脑回路进行详细分析和系统级映射，以及该回路如何被神经精神疾病的遗传或环境风险因素、发育中的胚胎中的细胞行为以及成人大脑中的神经回路破坏1，2，3。有多种组织学方法可以对重建的3D大脑进行高分辨率图像;然而，这些技术需要昂贵的专用设备，可能与免疫标记不兼容，并且某些方法的二维（2D）性质可能导致切片过程中的组织损伤和剪切4，5。
最近的进展提供了一种不需要组织切片的整个大脑成像的替代方法;它们涉及使用组织清除使大脑透明。在大多数组织清除方法中，通过去除脂质（因为它们是光散射的主要来源）以及在成像过程中将物体的折射率（RI）与样品浸没溶液的折射率（RI）相匹配来实现透明度。然后光可以穿过材料之间的边界而不会被散射6，7，8，9。
组织清除方法，如iDISCO+，通常与使用单光子激发显微镜的快速3D成像相结合，如LSFM6，7，10。在用荧光团标记的透明组织内，光片荧光显微镜通过用薄平面的光11激发对切片进行成像。LSFM的主要优点是一次照亮单个光学切片，该切片内分子的所有荧光都被激发，从而最大限度地减少了光漂白。此外，对整个光学切片进行成像可以基于相机检测激发切片，从而相对于点扫描12提高速度。LSFM无损地生产适合3D重建的配准良好的光学切片。
虽然iDISCO+方法允许在~3周内进行廉价的组织清除，但方案中的脱水步骤可能导致组织收缩和样品形态的潜在改变，从而影响体积测量6，10。在组织清除程序之前添加二次成像方法，例如MRI，可以测量整个样品的组织清除引起的收缩程度。在脱水步骤中，灰质和白质之间的机械性能差异可能导致脑物质变形不均匀，导致野生型和突变型样品之间不同的组织清除诱导的体积变形，并可能混淆对这些样品中体积差异的解释10，13.MRI是通过首先用造影剂（例如钆）灌注动物来进行的，然后在成像14之前将提取的目标组织在浸没溶液（例如fomblin）中孵育。MRI与组织清除和对同一样品进行LSFM兼容。
LSFM通常用于创建大规模显微镜图像，用于目标脑组织的定性可视化，而不是对脑结构进行定量评估（图1）。如果没有定量评估，就很难证明遗传或环境损害造成的结构差异。随着组织清除和成像技术的改进，以及存储和计算能力成本的降低，量化感兴趣组织内的细胞类型定位变得越来越容易，使更多的研究人员能够将这些数据纳入他们的研究中。
随着小鼠大脑中超过 1 亿个细胞15 和可以生成 TB 数据的全脑成像会话，对高级图像分析工具的需求不断增加，这些工具可以准确量化图像中的特征，例如细胞。对于组织清除图像存在许多分割方法，这些方法对核染色强度应用阈值并过滤具有预定义形状，大小或密度10，16，17，18的物体。然而，对结果的不准确解释可能是由于细胞大小、图像对比度和标记强度等参数的变化引起的。本文描述了我们建立的量化小鼠大脑细胞核的方案。首先，我们详细介绍了P4小鼠大脑组织收集的步骤，然后是根据公开可用的iDISCO+方法10优化的组织清除和免疫标记方案。其次，我们描述了使用MRI和光片显微镜进行的图像采集，包括用于捕获图像的参数。最后，我们描述并演示了NuMorph19，这是我们小组开发的一组图像分析工具，可以在组织清除后进行细胞类型特异性定量，使用核标记进行免疫标记以及注释区域的光片成像。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bruker 9.4T/30 cm MRI Scanner</td>
			<td>Bruker Biospec</td>
			<td></td>
			<td>Horizontal Bore Animal MRI System</td>
		</tr>
		<tr>
			<td>Dibenzyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>108014-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher-Scientific</td>
			<td>ICN19605590</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>S30-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EVO 860 4TB external SSD</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fomblin Y</td>
			<td>Speciality Fluids Company</td>
			<td>YL-VAC-25-6</td>
			<td>perfluoropolyether lubricant</td>
		</tr>
		<tr>
			<td>gadolinium contrast agent (ProHance)</td>
			<td>Bracco Diagnostics</td>
			<td>A9576</td>
			<td></td>
		</tr>
		<tr>
			<td>gadolinium contrast agent(ProHance)</td>
			<td>Bracco Diagnostics</td>
			<td>0270-1111-03</td>
			<td></td>
		</tr>
		<tr>
			<td>GeForce GTX 1080 Ti 11GB GPU</td>
			<td>EVGA</td>
			<td>08G-P4-6286-KR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-10KU</td>
			<td>Dissolved in H2O to 10 mg/mL</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution, 30%</td>
			<td>Sigma-Aldrich</td>
			<td>H1009-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ImSpector Pro</td>
			<td>LaVision BioTec</td>
			<td></td>
			<td>Microscope image acquisition software</td>
		</tr>
		<tr>
			<td>ITK Snap</td>
			<td></td>
			<td></td>
			<td>segmentation software</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher-Scientific</td>
			<td>A412SK-4</td>
			<td></td>
		</tr>
		<tr>
			<td>MVPLAPO 2x/0.5 NA Objective</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, powder, 95% (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td>Dissolved in 1x PBS to 4%</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline 10x (PBS)</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td>Diluted to 1x in H2O</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002-100G</td>
			<td>Dissolved in H2O to 10%</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tergitol type NP-40</td>
			<td>Sigma-Aldrich</td>
			<td>NP40S-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher-Scientific</td>
			<td>BP337 500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicroscope II Light Sheet Microscope</td>
			<td>LaVision BioTec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xeon Processor E5-2690 v4</td>
			<td>Intel</td>
			<td>E5-2690</td>
			<td></td>
		</tr>
		<tr>
			<td>Zyla sCMOS Camera</td>
			<td>Andor</td>
			<td></td>
			<td>Complementary metal oxide semiconductor camera</td>
		</tr>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td>Working concentration</td>
		</tr>
		<tr>
			<td>Alexa Fluor Goat 790 Anti-Rabbit</td>
			<td>Thermofisher Scientific</td>
			<td>A11369</td>
			<td>(1:50)</td>
		</tr>
		<tr>
			<td>Alexa Fluor Goat 568 Anti-Rat</td>
			<td>Thermofisher Scientific</td>
			<td>A11077</td>
			<td>(1:200)</td>
		</tr>
		<tr>
			<td>Rat anti-Ctip2</td>
			<td>Abcam</td>
			<td>ab18465</td>
			<td>(1:400)</td>
		</tr>
		<tr>
			<td>Rabbit anti-Brn2</td>
			<td>Cell Signaling Technology</td>
			<td>12137</td>
			<td>(1:100)</td>
		</tr>
		<tr>
			<td>To-Pro 3 (TP3)</td>
			<td>Thermofisher Scientific</td>
			<td>T3605</td>
			<td>(1:400)</td>
		</tr>
	</tbody>
</table>

whole-brain single-cell imaging and analysis of intact neonatal mouse brains using mri, tissue clearing, and light-sheet microscopy

组织清除后进行光片显微镜（LSFM）能够对完整的大脑结构进行细胞分辨率成像，从而可以定量分析由遗传或环境扰动引起的结构变化。全脑成像可以更准确地定量细胞，并研究物理切片组织的常用显微镜可能遗漏的区域特异性差异。与共聚焦显微镜相比，使用光片显微镜对清除的大脑进行成像大大提高了采集速度。尽管这些图像产生非常大量的大脑结构数据，但大多数在清除组织图像中进行特征量化的计算工具仅限于计数稀疏的细胞群，而不是所有细胞核。
在这里，我们展示了NuMorph（基于核的形态测量），一组分析工具，用于量化出生后第4天（P4）小鼠大脑注释区域内的所有细胞核和核标记物，在光片显微镜上清除和成像。我们描述了磁共振成像（MRI），以在组织清除脱水步骤引起的收缩之前测量脑体积，使用iDISCO+方法清除组织，包括免疫标记，然后使用市售平台进行光片显微镜，以细胞分辨率对小鼠大脑进行成像。然后，我们使用NuMorph演示了这个图像分析管道，该管道用于校正强度差异，拼接图像图块，对齐多个通道，计数细胞核，并通过注册到公开可用的图谱来注释大脑区域。
我们使用公开可用的协议和软件设计了这种方法，允许任何具有必要显微镜和计算资源的研究人员执行这些技术。这些组织清除、成像和计算工具允许测量和量化皮层中细胞类型的三维 （3D） 组织，应广泛适用于任何野生型/敲除小鼠研究设计。

由于iDISCO+协议引入了明显的组织收缩，肉眼很容易注意到（图2B），我们在组织清除之前向该管道中添加了一个MRI步骤，以量化组织清除引起的收缩。工作流程从从MR图像中去除非脑组织开始（图2C）。接下来，应用刚性变换（3个平移和3个旋转角度）将MR图像与光片图像对齐（图2D）。通过这样做，我们观察到组织清除程序引起的...

Watch this Scientific Journal Video about Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy at JoVE.com

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

该协议描述了使用iDISCO +对完整小鼠大脑进行磁共振成像，清除和免疫标记的方法，然后详细说明了使用光片显微镜成像和使用NuMorph的下游分析。

使用MRI，组织清除和光片显微镜对完整的新生小鼠大脑进行全脑单细胞成像和分析

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

小鼠大脑中约有1亿个细胞，全脑细胞分辨率图像的大小接近TB级，因此需要先进的图像分析工具来准确量化细胞。我们的计算管道可以预处理图像并量化小鼠皮层内的细胞核，同时在细胞检测精度、成像时间和计算资源之间保持合理的折衷。演示该程序的将是我实验室的研究生Felix Kyere和Ian Curtin。首先，将样品安装在正确的样品尺寸支架中，由于UltraMicroscope II显微镜的额定工作距离，样品的z尺寸深度不超过5.2毫米。然后将支架插入样品支架，使支架的螺钉与支架的支架成 45 度角。接下来，将支架放置到使样品垂直于光路的位置。之后，将显微镜上的变焦主体设置为4倍放大倍率或更高，每像素产生0.75微米。在 Inspector Pro 软件中，选择数值孔径值约为 0.08 的单个光片。为确保沿图像宽度保持轴向分辨率，请选择水平动态聚焦并根据激光波长应用建议的步数。然后根据配准通道调整每个通道的精细焦点，并根据通道属性调整每个通道的激光功率。接下来，将光片宽度调整为约 50%，以确保片材功效在样本大小的 y 维中最佳分布。然后，根据样本大小设置图块数，建议在图块之间重叠 15%，并在给定图块位置为每个堆栈按顺序捕获每个通道的图像。首先，下载并安装适用于Linux和NuMorph图像处理工具的Conda环境管理器。在命令行上，运行 matlab 并NM_setup。m从NuMorph下载并安装分析所需的图像分析软件包。然后通过编辑文件NM_samples.m来指定样本名称、输入和输出目录、通道信息和光片成像参数。对于强度调整，在NMp_template中，在处理一组新图像时，将“强度”调整设置为“真”，use_processed_images设置为“假”。接下来，将save_images和save_samples设置为 true。接下来，将拼贴着色设置为基本以使用基本算法应用阴影校正，或手动以使用UltraMicroscope II在特定光片宽度下的测量值应用平铺阴影校正。对于图像通道对齐，在NMp_template中，将channel_alignment设置为 true，将通道alignment_method设置为平移或松紧。接下来，将sift_refinement设置为 true，重叠值为 0.15，以匹配成像期间的切片重叠。要在 MATLAB 中运行预处理步骤，请指定样本名称并将配置设置为NM_config处理样本。然后，通过使用强度、对齐或拼接指定载物台NM_process指定配置针迹来运行任何预处理步骤，并检查输出目录中是否有每个阶段的输出文件。从 3D 地图集图像和关联的注释图像开始，该图像将每个体素分配给特定结构。对齐 Atlas 图像和注释文件，以确保它们在正确的方向上正确匹配。对齐后，通过执行命令处理 NuMorph 中的文件以指定手稿中所述的输入。在NMa_template中，将resample_images设置为 true，resample_resolution以匹配地图集。然后使用 resample_channels 指定要重新采样的通道号。然后，将register_images设置为 true，指定与 Atlas 目录中的文件匹配的atlas_file，并将registration_parameters设置为默认值。然后将save_registered_images设置为 true。对于细胞核检测、细胞计数和分类，请将count_nuclei和classify_cells都设置为 true。然后将count_method设置为 3dunet 和 min_intensity，为检测到的对象定义最小强度阈值。接下来，将classify_method设置为基于质心位置的非监督荧光强度的阈值或模拟监督线性支持向量机分类器的 SVM。要在 MATLAB 中执行分析步骤，请指定样本名称并设置配置以NM_config分析样本。接下来，通过指定配置阶段来运行任何分析步骤NM_analyze同时使用重采样、寄存器、计数或分类指定阶段，并检查每个阶段的输出文件的输出目录。在NMe_template中，将 update 设置为 true，compare_structures_by设置为任一索引。然后设置模板文件和结构表，其中指定所有可能的结构索引和结构，在指定细胞计数和细胞类型分类时进行评估。使用iDISCO+协议和神经元层特异性细胞核标记物进行组织清除，从而在等皮层中明确定义上层和下层神经元的细胞群。使用NuMorph的细胞计数取决于成功的预处理步骤，包括强度调整，通道对齐和拼接。但是，预处理步骤中的错误可能导致拼接不当，导致对齐和拼接不正确，从而导致图像具有对焦和失焦图案。为了计算来自特定大脑区域的细胞核，拼接的图像将使用Atlas进行注释，从而允许注释叠加在大脑区域。在NuMorph中使用经过训练的3D U-Net模型检测细胞核的质心，其中约1200万个总细胞核在等皮层中具有TO-PRO-3阳性，其中约260万个脑二阳性和160万个CTIP2阳性细胞核。分别在基底神经节和海马别特皮层中检测到约3.7和290万个TO-PRO-3阳性总核。然而，在这两个大脑区域中检测到的脑二阳性细胞可以忽略不计，在基底神经节和海马异体皮层中，每不到一百万个CTIP2阳性细胞中只有约1.5个被检测到。在采集过程中执行视觉质量检查，以实现良好的分割。并正确设置 Conda 环境，以确保在下游分析中不会遇到错误。除了细胞计数之外，该管道还允许与其他分割工具集成，这些工具可以测量细胞大小和形状，然后可以跨基因型组进行比较。通过我们的管道，我们可以确定大脑解剖结构在细胞分辨率下如何变化，从而识别对疾病风险很重要的细胞类型和大脑区域。

Research

JoVE Journal

Neuroscience

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Stereotaxic Surgery for Genetic Manipulation in Striatal Cells of Neonatal Mouse Brains

新生小鼠脑巴细胞基因调控的立体定向手术

Many genes are expressed in embryonic brains, and some of them are continuously expressed in the brain after birth. For such persistently expressed genes, ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

用光片荧光显微镜观察运动轴突导航和小鼠胚轴突树枝状的定量化

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

使用经颅功能超声成像的小鼠全脑3D激活和功能连接映射

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...

Imaging and Quantification of Intact Neuronal Dendrites via CLARITY Tissue Clearing

通过 CLARITY 组织清除对完整神经元树突进行成像和定量

Brain activity, the electrochemical signals passed between neurons, is determined by the connectivity patterns of neuronal networks, and from the ...

Deep-Tissue Three-Photon Fluorescence Microscopy in Intact Mouse and Zebrafish Brain

完整小鼠和斑马鱼大脑的深组织三光子荧光显微镜

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo ...

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

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 ...