溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像
Summary
新颖的免疫染色兼容组织清除技术,如溶剂清除器官的终极 3D 成像,允许狂犬病病毒脑感染及其复杂的细胞环境的 3D 可视化。厚厚的抗体标记脑组织切片在光学上透明,以增加成像深度,并通过共聚焦激光扫描显微镜实现 3D 分析。
Abstract
通过免疫标记对组织和器官的感染过程进行可视化是现代感染生物学的关键方法。观察和研究器官组织内病原体分布、病变和丰度的能力为疾病发展和进展提供了关键数据。使用传统的显微镜方法,免疫标签主要限于从石蜡嵌入或冷冻样品获得的薄部分。然而,这些薄截面的有限2D图像平面可能导致关于受感染器官复杂结构和感染细胞背景的关键信息丢失。现代多色、免疫染色兼容的组织清除技术现在提供了一种相对快速和廉价的方式来研究受病毒感染的器官组织的大容量 3D 图像堆栈。通过将组织暴露于有机溶剂中,它变得光学透明。这与样品的折射率相匹配,最终导致光散射的显著减少。因此,结合长距离自由工作距离目标,传统的共聚焦激光扫描显微镜(CLSM)可以以高分辨率成像高达1毫米的大组织部分。在这里,我们描述了一个协议,在组织清除后应用深层组织成像来可视化受感染大脑中的狂犬病病毒分布,以便研究病毒发病机制、传播、肌动和神经入侵等主题。
Introduction
传统的组织学技术主要依靠器官组织的薄部分,这本质上只能提供对复杂 3D 环境的 2D 见解。虽然原则上是可行的,但串行薄截面的 3D 重建需要苛刻的技术管道,以便对采集的图像1进行切片和后续的硅对齐。此外,在微缩切片后无缝重建 z 体积至关重要,因为机械和计算伪影可能由于非重叠图像平面、染色变化和物理物理造成的图像配准不理想而保留破坏组织,例如,微托姆刀片。相比之下,对完整厚组织样本进行纯光学切片允许获取重叠的图像平面(过采样),从而有助于 3D 重建。这反过来又对分析复杂细胞群(例如,周围胶质和免疫细胞的神经元网络)中的感染过程非常有益。然而,厚组织部分的固有障碍包括光散射和有限的抗体渗透到组织。近年来,开发和优化了各种技术,以克服这些问题2,3,4,5,6,7,8 ,9,10,11,12,13.基本上,目标组织通过处理水性2、3、4、5、6、7进行光学透明化 ,8,9或有机溶剂基10,11,12,13溶液。引入 3DISCO (溶剂清除器官的 3D 成像)11、12及其后续 uDISCO(溶剂清除器官的最终 3D 成像)13提供了一个相对快速、简单且廉价的工具,出色的清算能力。清除协议的主要成分是有机溶剂特-丁醇(TBA)、苯基醇(BA)、苯甲酸酯(BB)和二苯醚(DPE)。iDISCO(溶剂清除器官的免疫标记支持3D成像)14的开发和添加,这是一种兼容的免疫染色方案,与现有方法形成另一个优势,使抗原的深层组织标记得以实现以及免疫染色样本的长期储存。因此,iDISCO14和 uDISCO13的组合允许使用传统的 CLSM 在大组织部分(高达 1 mm)对抗体标记蛋白进行高分辨率成像。
在所有三个维度中保存器官的复杂结构对脑组织尤其重要。神经元构成一个非常异质的细胞亚群,其基于其神经质投影的3D形态高度多样化(由马斯兰15审查)。此外,大脑由多个隔间和子隔间组成,每个隔间和子隔间由不同的细胞亚群及其比例组成,包括胶质细胞和神经元(由冯·巴托尔德等人审查)。作为一种神经性病毒,狂犬病病毒(RABV,由Fooks等人17审查)主要感染神经元,利用他们的运输机械沿着轴向方向从感染的主要部位到达中枢神经系统(CNS)。此处描述的协议(图1A)允许在从受感染的脑组织获得的大型、连贯的图像堆栈中,对RABV和RABV感染的细胞进行免疫染色辅助检测和可视化。这样就可以对感染环境进行无偏见的 3D 高分辨率评估。它适用于各种物种的脑组织,可在固定后或长期储存在甲醛(PFA)中的样品后立即进行,并允许对染色和清除的样品进行数月的储存和再成像。
Protocol
使用RABV感染,PFA固定存档脑材料。各自的动物实验研究由国家农业、食品安全和渔业办公室负责的动物护理、使用和道德委员会(LALFF M-V)进行评估,并获得许可。7221.3-2.1-002/11(小鼠)和7221.3-1-068/16(雪铁龙)。根据批准的指南进行动物实验中使用的一般护理和方法。 注意:本议定书使用各种有毒和/或有害物质,包括PFA、甲醇(MeOH)、过氧化氢(H2O2)、阿齐德钠(NaN3)…
Representative Results
iDISCO14和 uDISCO13结合高分辨率 CLSM,可深入了解脑组织 RABV 感染和周围细胞环境的时空分辨率和可塑性。 使用RABV磷蛋白(P)的免疫染色,在小鼠大脑的厚部分可以可视化受感染神经元细胞的复杂层(图3)。随后,可以重建采集图像堆栈的无缝 3D 投影(图 3A、B、右侧面板;动画图 1?…
Discussion
近年来组织清除技术的复苏和进一步发展2、3、4、5、6、7、89,10,11,12,13,<sup class="x…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢托马斯·梅滕莱特和维雷纳·特·坎普批判性地阅读了手稿。这项工作得到了梅克伦堡西波美拉尼亚联邦卓越倡议和欧洲社会基金(ESF)格兰特·科因费克特(ESF/14-BM-A55-0002/16)的支持,以及弗里德里希-勒弗勒研究所(Ri-0372)。
Materials
| Reagents | |||
| Benzyl alcohol | Alfa Aesar | 41218 | Clearing reagent |
| Benzyl benzoate | Sigma-Aldrich | BB6630-500ML | Clearing reagent |
| Dimethyl sulfoxide | Carl Roth | 4720.2 | Various buffers |
| Diphenyl ether | Sigma-Aldrich | 240834-100G | Clearing reagent |
| DL-α-Tocopherol | Alfa Aesar | A17039 | Antioxidant |
| Donkey serum | Bio-Rad | C06SBZ | Blocking reagent |
| Glycine | Carl Roth | 3908.2 | Background reduction |
| Goat serum | Merck | S26-100ML | Blocking reagent |
| Heparin sodium salt | Carl Roth | 7692.1 | Background reduction |
| Hydrogen peroxide solution (30 %) | Carl Roth | 8070.2 | Sample bleaching |
| Methanol | Carl Roth | 4627.4 | Sample pretreatment |
| Paraformaldehyde | Carl Roth | 0335.3 | Crystalline powder to make fixative solution |
| Sodium azide | Carl Roth | K305.1 | Prevention of microbial growth in stock solutions |
| tert-Butanol | Alfa Aesar | 33278 | Sample dehydration for tissue clearing |
| TO-PRO-3 | Thermo Fisher | T3605 | Nucleic acid stain |
| Triton X-100 | Carl Roth | 3051.2 | Detergent |
| Tween 20 | AppliChem | A4974,0500 | Detergent |
| Miscellaneous | |||
| 5 mL reaction tubes | Eppendorf | 0030119401 | Sample tubes |
| Coverslip, circular (diameter: 22 mm) | Marienfeld | 0111620 | Part of imaging chamber |
| Coverslip, circular (diameter: 30 mm) | Marienfeld | 0111700 | Part of imaging chamber |
| Hypodermic needle (27 G x ¾” [0.40 mm x 20 mm]) | B. Braun | 4657705 | Filling of the imaging chamber with clearing solution |
| RTV-1 silicone rubber | Wacker | Elastosil E43 | Adhesive for the assembly of the imaging chamber |
| Ultimaker CPE 2.85 mm transparent | Ultimaker | 8718836374869 | Copolyester filament for 3D printer to print parts of the imaging chamber |
| Technical equipment and software | |||
| 3D printer | Ultimaker | Ultimaker 2+ | Printing of imaging chamber |
| Automated water immersion system | Leica | 15640019 | Software-controlled water pump |
| Benchtop orbital shaker | Elmi | DOS-20M | Sample incubation at room temperature (~ 150 rpm) |
| Benchtop orbital shaker, heated | New Brunswick Scientific | G24 Environmental Shaker | Sample incubation at 37 °C (~ 150 rpm) |
| Confocal laser scanning microscope | Leica | DMI 6000 TCS SP5 | Inverted confocal microscope for sample imaging |
| Fiji | NIH (ImageJ) | open source software (v1.52h) | Image processing package based on ImageJ |
| Long working distance water immersion objective | Leica | 15506360 | HC PL APO 40x/1.10 W motCORR CS2 |
| Vibratome | Leica | VT1200S | Sample slicing |
| Workstation | Dell | Precision 7920 | CPU: Intel Xeon Gold 5118 GPU: Nvidia Quadro P5000 RAM: 128 GB 2666 MHz DDR4 SSD: 2 TB |
| Primary antibodies | |||
| Goat anti-RABV N | Friedrich-Loeffler-Institut | Monospecific polyclonal goat anti-RABV N serum, generated by goat immunization with baculovirus-expressed and His-tag-purified RABV nucleoprotein N Dilution: 1:400 |
|
| Rabbit anti-GFAP | Dako | Z0334 | Polyclonal antibody (RRID:AB_10013382) Dilution: 1:100 |
| Rabbit anti-MAP2 | Abcam | ab32454 | Polyclonal antibody (RRID:AB_776174) Dilution: 1:250 |
| Rabbit anti-RABV P 160-5 | Friedrich-Loeffler-Institut | Monospecific polyclonal rabbit anti-RABV P serum, generated by rabbit immunization with baculovirus-expressed and His-tag-purified RABV phosphoprotein P (see reference 23: Orbanz et al., 2010) Dilution: 1:1,000 |
|
| Secondary antibodies | |||
| Donkey anti-goat IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed Dilution: 1:500 |
| Donkey anti-mouse IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed Dilution: 1:500 |
| Donkey anti-rabbit IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed Dilution: 1:500 |
| Goat anti-mouse IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed Dilution: 1:500 |
| Goat anti-rabbit IgG | Thermo Fisher Scientific | depending on conjugated fluorophore | Highly cross-absorbed Dilution: 1:500 |
References
- Pichat, J., Iglesias, J. E., Yousry, T., Ourselin, S., Modat, M. A Survey of Methods for 3D Histology Reconstruction. Medical Image Analysis. 46, 73-105 (2018).
- Chung, K., et al. Structural and molecular interrogation of intact biological systems. Nature. 497 (7449), 332-337 (2013).
- Hama, H., et al. ScaleS: an optical clearing palette for biological imaging. Nature Neuroscience. 18 (10), 1518-1529 (2015).
- Ke, M. T., Fujimoto, S., Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature Neuroscience. 16 (8), 1154-1161 (2013).
- Kuwajima, T., et al. ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development. 140 (6), 1364-1368 (2013).
- Susaki, E. A., et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell. 157 (3), 726-739 (2014).
- Susaki, E. A., et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nature Protocols. 10 (11), 1709-1727 (2015).
- Yang, B., et al. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell. 158 (4), 945-958 (2014).
- Treweek, J. B., et al. Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nature Protocols. 10 (11), 1860-1896 (2015).
- Dodt, H. U., et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nature Methods. 4 (4), 331-336 (2007).
- Erturk, A., et al. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nature Medicine. 18 (1), 166-171 (2011).
- Erturk, A., et al. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nature Protocols. 7 (11), 1983-1995 (2012).
- Pan, C., et al. Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nature Methods. 13 (10), 859-867 (2016).
- Renier, N., et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell. 159 (4), 896-910 (2014).
- Masland, R. H. Neuronal cell types. Current Biology. 14 (13), 497-500 (2004).
- von Bartheld, C. S., Bahney, J., Herculano-Houzel, S. The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting. The Journal of Comparative Neurology. 524 (18), 3865-3895 (2016).
- Fooks, A. R., et al. Rabies. Nature Reviews Disease Primers. 3, 17091 (2017).
- WHO. WHO Expert Consultation on Rabies, Third Report. WHO Technical Report Series. , (2018).
- CDC. . Biosafety in Microbiological and Biomedical Laboratories, 5th Edition. US Department of Health and Human Services. , (2009).
- Arnold, M. M., et al. Effects of fixation and tissue processing on immunohistochemical demonstration of specific antigens. Biotechnic & Histochemistry. 71 (5), 224-230 (1996).
- Webster, J. D., Miller, M. A., Dusold, D., Ramos-Vara, J. Effects of prolonged formalin fixation on diagnostic immunohistochemistry in domestic animals. Journal of Histochemistry and Cytochemistry. 57 (8), 753-761 (2009).
- Werner, M., Chott, A., Fabiano, A., Battifora, H. Effect of formalin tissue fixation and processing on immunohistochemistry. The American Journal of Surgical Pathology. 24 (7), 1016-1019 (2000).
- Orbanz, J., Finke, S. Generation of recombinant European bat lyssavirus type 1 and inter-genotypic compatibility of lyssavirus genotype 1 and 5 antigenome promoters. Archives of Virology. 155 (10), 1631-1641 (2010).
Citer Cet Article
Zaeck, L., Potratz, M., Freuling, C. M., Müller, T., Finke, S. High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue. J. Vis. Exp. (146), e59402, doi:10.3791/59402 (2019).