聚焦激光扫描显微镜为基础的定量分析 阿斯珀吉卢斯熏蒸 孔尼迪亚分布在全山光学清除鼠肺
1Research Center for Molecular Mechanisms of Aging and Age-Related Diseases,Moscow Institute of Physics and Technology, 2Department of Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry,Russian Academy of Sciences, 3Institute of Biological Information Processing (IBI-7: Structural Biochemistry),Forschungszentrum Jülich
Summary
我们描述了对小鼠气道中 阿斯珀吉卢斯·福米加图斯 孔尼迪亚（3微米大小）分布进行定量分析的方法。该方法还可用于分析各种病理条件模型中气道中的微粒和纳米粒子聚集分布。
Abstract
阿斯珀吉卢斯·福米加图斯 ·科尼迪亚是空气中的病原体，可以穿透人类的气道。免疫能力不过敏的人表现出抵抗力和免疫耐受性，而在免疫功能低下的患者中，针叶可以殖民气道并导致严重的侵入性呼吸系统疾病。不同气道舱内的各种细胞参与防止真菌入侵的免疫反应：然而，病原体消除的时空方面尚未完全了解。光学清除的全安装器官，特别是实验鼠的肺部的三维（3D）成像，允许在感染后不同时间点在气道中检测荧光标记的病原体。在本研究中，我们描述了一个实验设置，对气道中的 A.富米加图斯 针叶分布进行定量分析。利用荧光聚焦激光扫描显微镜（CLSM），我们追踪了在向小鼠应用咽喉后6小时，支气管支部和肺泡室中荧光标记的针叶的位置。此处描述的方法以前用于检测准确的病原体位置和识别免疫反应不同阶段的病原体相互作用细胞。实验设置可用于估计不同病理条件下病原体消除的动能。
Introduction
每天，人们吸入空气中的病原体，包括机会性真菌阿斯珀吉卢斯烟熏（A.富米加图斯针叶）孢子，可以穿透呼吸道1。哺乳动物的呼吸道是不同代的气道系统，其特征是气道壁2、3、4的不同结构。气管支气管壁由多种细胞类型组成，其中细胞是提供粘膜间隙5的细胞。在藻类中，没有细胞，渗透性藻类空间病原体不能通过粘血清除6来消除。此外，每个气道生成是多个免疫细胞群的利基，这些群体的子集对于某些气道隔间来说是独一无二的。因此，藻类巨噬细胞位于气管室中，而气管和导管都与腹内树突状细胞7，8衬里。
A.富米加图斯针叶的大致大小为2-3.5μm9。由于人类甚至小鼠小气道的直径超过3.5μm，因此建议conidia可以穿透2、10、11的藻类空间。事实上，组织学检查显示，患有腹血病12的患者在骨泡中真菌生长。科尼迪亚也被发现在受感染小鼠的肺泡使用厚肺片13的活成像。同时，在14号小鼠支气管上皮的亮侧检测到锥形。
光学清除的全安装鼠肺的三维（3D）成像允许对气道15进行形态分析。特别是使用光学清除小鼠肺标本15对内脏胸神经分布进行定量分析。最近，Amich等人利用光学清除小鼠肺标本的光片荧光显微镜，对针 叶在免疫功能低下的小鼠体内应用后的真菌生长进行了调查。感染后不同时间点在气道中休息的conidia的精确位置对于识别细胞群非常重要，这些细胞群可以在炎症的某些阶段提供足够的抗真菌防御。然而，由于体积相对较小，气道中 A.fumigatus 针叶分布的时空分布特征较差。
在这里，我们提出了一个实验设置，用于定量分析受感染小鼠气道中的A.fumigatus针叶分布。我们使用荧光聚焦激光扫描显微镜（CLSM），对接受荧光标记为A.fumigatusconidia的鼻咽应用的小鼠的肺部进行光学清除，我们获得3D图像并执行图像处理。使用全安装肺叶的3D成像，我们之前已经显示了A.fumigatusconidia在小鼠的导流气道分布72小时后，针叶应用8。
Protocol
这里描述的所有实验室动物方法都已得到俄罗斯科学院舍米亚金和奥夫钦尼科夫生物有机化学研究所机构动物护理和使用委员会（协议编号226/2017）的批准。 1. A. 富米加图斯·科尼迪亚申请 要获得荧光标记 的A.富米加图斯 针叶，修复5×108 针叶，通过添加1mL的3%的甲醛针叶颗粒。在室温下在 50 mL 试管中孵育 2 小时。 用 20 mL 磷酸盐缓冲盐水 …
Representative Results
按照上述协议，获得了显示小鼠肺叶气道和A.富米加图斯针叶的3D图像（图1A）。斯特雷普塔维丁（用于气道可视化）标记支气管和支气管15。此外，大型船只，很容易从气道的形态区分，和普鲁拉可视化在气道通道（图1A-C）。气道表面和面罩的创建允许在气道通道中移除容器和普鲁拉投影;然而，气道表面的完整性?…
Discussion
全器官三维成像允许在不解剖标本的情况下获取数据，这对研究生物体中病原体解剖分布的空间方面具有重要意义。组织光学清除有几种技术和修改，有助于克服激光散射，并允许全器官成像15，16，18，19。其中一种定制组织清除方法包括甲醇组织脱水和脱皮，然后是与BABB的光学清除。这种方法是在1…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢斯文·克拉普曼教授（埃尔兰根大学医院和德国富阿·埃尔兰根-纽恩伯格大学医院）提供了 阿斯珀吉卢斯·富米加图斯 针叶菌株AFS150。作者感谢MIPT新闻办公室。五.B承认俄罗斯联邦科学和高等教育部（#075-00337-20-03项目，FSMG-2020-0003项目）。关于 A.富米加图斯 针叶成像和量化的工作得到了RSF第19-75-00082号的支持。气道成像工作由RFBR No 20-04-60311支持。
Materials
| Alexa Fluor 594 NHS Ester | ThermoFisher | A20004 | |
| Aspergillus fumigatus conidia | ATCC | 46645 | The strain AfS150, a ATCC 46645 derivative |
| Benzyl alcohol | Panreac | 141081.1611 | 98.0-100 % |
| Benzyl benzoate | Acros | AC10586-0010 | 99+% |
| C57Bl/6 mice | Pushchino Animal Breeding Centre (Russia) | Male. 12 – 30 week old. | |
| Catheter | Venisystems | G715-A01 | 18G |
| Cell imaging coverglass-bottom chamber | Eppendorf | 30742028 | 4 or 8 well chamber with coverglass bottom |
| Centrifuge | Eppendorf | 5804R | Any centrifuge provided 1000 g can be used |
| Confocal laser scanning microscope | ZEISS | ZEISS LSM780 | |
| Dimethyl sulfoxide | Sigma-Aldrich | 276855 | ≥99.9% |
| FIJI image processing package | FIJI | Free software | |
| Forcep | B. Braun Aesculap | BD557R | Toothed |
| Forcep | B. Braun Aesculap | BD321R | Fine-tipped |
| Forcep | Bochem | 1727 | Smooth |
| Glass bottle | DURAN | 242101304 | With groung-in lid |
| Graphic Editor Photoshop | Adobe Inc | Adobe Photoshop CS | |
| GraphPad Software | GraphPad | Prism 8 | |
| Imaris Microscopy Imaging Software | Oxford Instruments | Free trial is avalable https://imaris.oxinst.com/microscopy-imaging-software-free-trial | |
| Isoflurane | Karizoo | ||
| NaHCO3 | Panreac | 141638 | |
| Objective | ZEISS | 420640-9800-000 | Plan-Apochromat, 10 × (NA = 0.3) |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| PBS | Paneco | P060Π | |
| Pipette | ProLine | 722020 | 5 to 50 μL |
| Powdered milk | Roth | T145.2 | |
| Sample mixer | Dynal | MXIC1 | |
| Scissors | B. Braun | BC257R | Blunt |
| Shaker | Apexlab | GS-20 | 50-300 rpm |
| Skalpel | Bochem | 12646 | |
| Silk thread | B. Braun | 3 USP | |
| Streptavidin, Alexa Fluor 488 conjugate | ThermoFisher | S11223 | |
| Test tube | SPL Lifesciences | 50050 | 50 mL |
| Tris (hydroxymethyl aminomethane) | Helicon | H-1702-0.5 | Mr 121.14; CAS Number: 77-86-1 |
| Triton X-100 | Amresco | Am-O694-0.1 | |
| ZEN microscope software | ZEISS | ZEN2012 SP5 | https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html |
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Tags
Confocal Laser Scanning MicroscopyAspergillus FumigatusConidiaOptical ClearingMouse LungQuantitative AnalysisRespiratory TractMucociliary ClearanceAlveolar MacrophagesNeutrophilsOptical ClearingBenzyl AlcoholBenzyl BenzoateConfocal MicroscopyLaserSpectral RangeDetector GainPinholePixel ResolutionZ-stack
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Maslov, I. V., Bogorodskiy, A. O., Pavelchenko, M. V., Zykov, I. O., Troyanova, N. I., Borshchevskiy, V. I., Shevchenko, M. A. Confocal Laser Scanning Microscopy-Based Quantitative Analysis of Aspergillus fumigatus Conidia Distribution in Whole-Mount Optically Cleared Mouse Lung. J. Vis. Exp. (175), e62436, doi:10.3791/62436 (2021).