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
1European Laboratory for Non-linear Spectroscopy (LENS),University of Florence, 2Division of Physiology, Department of Experimental and Clinical Medicine,University of Florence, 3Department of Physics,University of Pisa, 4National Research Council – National Institute of Optics (CNR-INO), 5Department of Physics and Astronomy,University of Florence, 6Department of Biology,University of Florence
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 在烧杯中制备 4% w/v 琼脂…
Representative Results
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，?…
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 2,2'-thiodiethanol | Merck Life Science S.R.L. | 166782 | |
| Acetamide >= 99.0% (GC) | Merck Life Science S.R.L. | 160 | |
| Agarose High EEO | Merck Life Science S.R.L. | A9793 | |
| Boric Acid | Merck Life Science S.R.L. | B7901 | |
| Compressome VF-900-0Z Microtome | Precisionary | / | |
| Coverslips | LaserOptex | / | customized |
| Ethylenediaminetetraacetic acid disodium salt dihydrate | Merck Life Science S.R.L. | E5134 | |
| Glutaraldehyde | Merck Life Science S.R.L. | G7651 | |
| Glycine | Santa Cruz Biotechnology | SC_29096 | |
| Hydrogen Peroxide 30% | Merck Life Science S.R.L. | ||
| Incubator ISS-4075 | Lab companion | / | |
| Light-sheet fluorescence microscopy (LSFM) | / | / | custom-made |
| Loctite Attak | Henkel Italia srl | / | |
| Microscope slides | Laborchimica | / | customized |
| Phospate buffer saline tablet | Merck Life Science S.R.L. | P4417 | |
| Picodent Twinsil | Picodent | 13005002 | out of production |
| Potassium Hydrogen Phtalate | Merck Life Science S.R.L. | P1088 | |
| Sodium Azide | Merck Life Science S.R.L. | S2002 | |
| Sodium Dodecyl Sulfate | Merck Life Science S.R.L. | L3771 | |
| Sodium Sulfite | Merck Life Science S.R.L. | S0505 | |
| Spacers | Microlaser srl | customized | |
| Sputum Containers (dishes with screw lids) | Paul Boettger GmbH & Co. KG | 07.061.2000 | |
| Tris Base | PanReac AppliChem (ITW reagents) | A4577,0500 | |
| Triton X-100 | Merck Life Science S.R.L. | T8787 | |
| Tubes | Sarstedt | 62 547254 | |
| Tween 20 | Merck Life Science S.R.L. | P9416 | |
| Vibratome VT1000S | Leica Biosystem | / | |
| Water bath | Memmert | WNB 7-45 | |
| Antibodies and Dyes | |||
| Alexa Fluor 488 AffiniPure Alpaca Anti-Rabbit IgG (H+L) | Jackson Immuno Reasearch | 611-545-215 | Dilution used, 1:200 |
| Alexa Fluor 488 AffiniPure Bovine Anti-Goat IgG (H+L) | Jackson Immuno Reasearch | 805-545-180 | Dilution used, 1:200 |
| Alexa Fluor 647 AffiniPure Alpaca Anti-Rabbit IgG (H+L) | Jackson Immuno Reasearch | 611-605-215 | Dilution used, 1:200 |
| Anti-NeuN Antibody | Merck Life Science S.R.L. | ABN91 | Dilution used, 1:100 |
| Anti-Parvalbumin antibody (PV) | Abcam | ab32895 | Dilution used, 1:200 |
| Anti-Vimentin antibody [V9] – Cytoskeleton Marker (VIM) | Abcam | ab8069 | Dilution used, 1:200 |
| Calretinin Polyclonal antibody | ProteinTech | 12278_1_AP | Dilution used, 1:200 |
| DAPI | ThermoFisher | D3571 | Dilution used, 1:100 |
| Donkey Anti-Mouse IgG H&L (Alexa Fluor 568) | Abcam | ab175700 | Dilution used, 1:200 |
| Donkey Anti-Mouse IgG H&L (Alexa Fluor 647) | Abcam | ab150107 | Dilution used, 1:200 |
| Donkey Anti-Rabbit IgG H&L (Alexa Fluor 568) | Abcam | ab175470 | Dilution used, 1:200 |
| Donkey Anti-Rat IgG H&L (Alexa Fluor 568) preadsorbed | Abcam | ab175475 | Dilution used, 1:200 |
| Goat Anti-Chicken IgY H&L (Alexa Fluor 488) | Abcam | ab150169 | Dilution used, 1:500 |
| Goat Anti-Chicken IgY H&L (Alexa Fluor 568) | Abcam | ab175711 | Dilution used, 1:500 |
| Goat Anti-Chicken IgY H&L (Alexa Fluor 647) | Abcam | ab150171 | Dilution used, 1:500 |
| Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) | Abcam | ab150077 | Dilution used, 1:200 |
| Recombinant Alexa Fluor 488 Anti-GFAP antibody | Abcam | ab194324 | Dilution used, 1:200 |
| Somatostatin Antibody YC7 | Santa Cruz Biotechnology | sc-47706 | Dilution used, 1:200 |
| Vasoactive intestinal peptide (VIP) | ProteinTech | 16233-1-AP | Dilution used, 1:200 |
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Diesen Artikel zitieren
Di Meo, D., Ramazzotti, J., Scardigli, M., Cheli, F., Pesce, L., Brady, N., Mazzamuto, G., Costantini, I., Pavone, F. S. Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging. J. Vis. Exp. (203), e65960, doi:10.3791/65960 (2024).