通过透明硅胶窗进行纵向活体成像
Summary
本文介绍了一种使用光学透明硅胶窗口进行长期活体内成像的方法，该窗口可以直接粘附在感兴趣的组织/器官和皮肤上。这些窗户比目前在该领域使用的其他窗户更便宜，用途更广，手术插入对动物造成有限的炎症和痛苦。
Abstract
活体显微镜 （IVM） 能够以单细胞分辨率可视化细胞运动、分裂和死亡。通过手术插入的成像窗口进行IVM特别强大，因为它允许在数天至数周内纵向观察同一组织。典型的成像窗口包括一个玻璃盖玻片，该盖玻片位于缝合到小鼠皮肤的生物相容性金属框架中。这些窗户会干扰小鼠的自由运动，引起强烈的炎症反应，并且由于玻璃破碎或缝合线撕裂而失效，其中任何一个都可能需要安乐死。为了解决这些问题，从聚二甲基硅氧烷（PDMS）薄膜中开发了用于长期腹部器官和乳腺成像的窗口，PDMS是一种以前用于颅骨成像窗口的光学透明硅胶聚合物。这些窗口可以直接粘在组织上，减少插入所需的时间。PDMS是灵活的，随着时间的推移，有助于其在小鼠中的耐久性 – 已经测试了长达35天。纵向成像是在不同会话期间对同一组织区域进行成像。在窗户内嵌入了一个不锈钢网格，以定位同一区域，从而可以在相同的位置可视化动态过程（如乳腺内卷积），相隔数天。该硅胶窗口还允许监测单个播散性癌细胞随时间发展成微转移瘤。本研究中使用的硅胶窗户比金属框架玻璃窗户更容易插入，并且会引起成像组织的有限炎症。此外，嵌入式网格允许在重复的成像过程中直接跟踪相同的组织区域。
Introduction
活体显微镜（IVM）是麻醉动物组织的成像，可以深入了解完整组织中细胞分辨率下生理和病理事件的动力学。该技术的应用差异很大，但IVM在癌症生物学领域发挥了重要作用，有助于阐明癌细胞如何侵入组织和转移，与周围的微环境相互作用，并对药物1，2，3作出反应。此外，IVM 通过提供与 离体 分析方法（例如流式细胞术）相辅相成的见解，成为促进对控制免疫反应的复杂机制理解的关键。例如，活体成像实验揭示了免疫功能的细节，因为它们与细胞迁移和细胞 – 细胞接触有关，并提供了一个平台来量化响应损伤或感染的时空动力学4，5，6，7。其中许多过程也可以通过组织学染色进行研究，但只有IVM允许跟踪动态变化。事实上，虽然组织学切片提供了给定时间组织的快照，但活体成像可以随着时间的推移跟踪同一组织内的细胞间和亚细胞事件。特别是，荧光标记的进展和分子报告基因的发展使分子事件与细胞行为相关，例如增殖，死亡，运动性以及与其他细胞或细胞外基质的相互作用。大多数IVM技术都基于荧光显微镜，由于光散射，这使得更深层组织的成像具有挑战性。因此，感兴趣的组织通常需要通过通常侵入性和终末期手术进行手术暴露。因此，根据器官部位的不同，组织可以连续成像一段时间，从几个到40小时8不等。或者，手术插入永久性成像窗口允许在几天到第7，9周的时间内按顺序对同一组织进行成像。
新型影像学窗口的开发已被强调为进一步改进活体成像方法的技术需求10.典型的活体成像窗口是一个金属环，其中包含用缝合线11固定在皮肤上的玻璃盖玻片。干扰自由运动，渗出物的积聚以及对玻璃盖玻片的损坏是使用此类窗户的常见问题。此外，原型窗口需要专门的生产，外科手术可能需要广泛的培训。为了解决这些问题，聚二甲基硅氧烷（PDMS）是一种有机硅聚合物，其先前已用于颅窗中用于大脑12的长期成像，被改编用于腹部器官和乳腺成像。在这里，介绍了生成基于PDMS的硅胶窗口的详细方法，包括如何将窗口投射到不锈钢网格周围，以便为相同组织区域的重复成像提供特征。此外，还描述了一种将窗户插入腹部器官或乳腺的简单，无缝合的外科手术。这种新方法克服了目前使用的成像窗口的一些最常见问题，并增加了纵向活体腔内成像的可及性。
Protocol
Representative Results
Discussion
活体内成像窗口是直接可视化细胞分辨率的生理和病理过程的重要工具，因为它们随着时间的推移而展开。所描述的用于在小鼠中投射和插入柔性硅胶成像窗口的新方法克服了当前使用的成像窗口的一些最常见问题（渗出，断裂和对正常移动性的干扰），为小鼠提供了额外的安全性，并增加了该技术的可访问性。
使用最广泛的成像窗口包括一个金属框架，里面装有玻璃盖玻?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢Rob Eifert在设计和优化激光切割不锈钢网格方面的帮助。这项工作得到了CSHL癌症中心（P30-CA045508）和美国国立卫生研究院（NIH）的医学博士基金（1R01CA2374135和5P01CA013106-49）的支持。CSHL和Northwell Health;汤普森家族基金会;游过美国;以及西蒙斯基金会向CSHL提供的赠款。M.S.得到了美国国家普通医学科学研究所医学科学家培训计划培训奖（T32-GM008444）和NIH国家癌症研究所的支持，奖项编号为1F30CA253993-01。L.M.由James S. McDonnell基金会博士后奖学金支持。J.M.A.是癌症研究所/欧文顿博士后奖学金（CRI奖#3435）的获得者。D.A.T.得到了Lustgarten基金会胰腺癌研究专用实验室和汤普森家庭基金会的支持。卡通是用 Biorender.com 创作的。
Materials
| 3M Medipore Soft Cloth Surgical Tape | 3M | 70200770819 | |
| Silk suture 4-0 PERMA HAND BLACK 1 x 18" RB-2 | Ethicon | N267H | |
| ACTB-ECFP mice | Jackson Laboratory | 22974 | |
| AEC Substrate Kit, Peroxidase (HRP), (3-amino-9-ethylcarbazole) | Vector Laboratories | SK-4200 | |
| Alcohol swabs | BD | 326895 | |
| Anesthesia system | Molecular Imaging Products Co. | ||
| Acqknowledge software and sensors | BIOPAC | ACK100W, ACK100M, TSD110 | |
| Betadine spray | LORIS | 109-08 | |
| c-fms-EGFP (MacGreen) mice | Jackson Laboratory | 18549 | |
| C57BL/6J mice | Jackson Laboratory | 664 | |
| CD45 Monoclonal Antibody (30-F11) | Invitrogen | 14-0451-82 | |
| CD68 Antibody | Abcam | ab125212 | |
| Curity gauze sponges | Covidien | ||
| Donkey Anti-Goat IgG H&L (HRP) | Abcam | ab6885 | |
| Donkey Anti-Rabbit IgG H&L (HRP) | Abcam | ab97064 | |
| Donkey Anti-Rat IgG H&L (HRP) | Abcam | ab102182 | |
| Dow SYLGARD 184 Silicone Encapsulant Clear | Electron Microscopy Sciences | 24236-10 | Two-part, 10:1 mixing ratio |
| Round Cover Glass, 8mm Diameter, #1.5 Thickness | Electron Microscopy Sciences | 72296-08 | |
| Ender-3 Pro 3D printer | Shenzhen Creality 3D Technology Co., LTD | ||
| Far Infrared Heated blanket | Kent Scientific | RT-0520 | |
| Fc Receptor Blocker | Innovex Biosciences | NB309 | |
| Fiji imaging processing package | https://imagej.net/software/fiji/ | ||
| FluoroSpheres carboxylate, 0.04µm, yellow-green (505/515) | Invitrogen | F8795 | |
| Gating system: | BIOPAC Systems Inc. | The components together allow monitoring mouse vitals during imaging and gating image acquisition on mouse respiration. All were acquired from BIOPAC systems. | |
| Acqknowledge software | ACK100W, ACK100M | ||
| Diff. Amp. Module, C Series | DA100C | ||
| Dual Gating Sys small animal | DTU200 | ||
| MP160 for Windows – Analysis system | MP160WSW | ||
| MouseOx Plus 120V | MOX-120V;015000 | ||
| Pressure Pad | TSD110 | ||
| Gelfoam | Pfizer | 9031508 | Absorbable gelatin sponge |
| Hardened fine scissors | Fine Science Tools | 14090-11 | Two pairs; stainless steel, sharp-sharp tips, straight tip, 26 mm cutting edge, 11 cm length |
| Human/Mouse Myeloperoxidase/MPO Antibody | R&D Systems | AF3667 | |
| Hot bead sterilizer | Fine Science Tools | 18000-45 | Turn on approximately 30 min before use; sterilize tools at >200 °C for 30 s |
| Imaris | Bitplane | www.bitplane.com | |
| Immersion medium Immersol W 2010 | Zeiss | 444969-0000-000 | |
| Insulin Syringes with BD Ultra-Fine needle 6mm x 31G 1 mL/cc | BD | 324912 | |
| Isoflurane (Fluriso) | VetOne | 502017 | |
| Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight® 594 | Vector Laboratories | DL-1177-1 | |
| LysM-eGFP mice | www.mmrrc.org | 012039-MU | |
| Micro dissecting forceps | Roboz | RS-5135 | Serrated, slight curve, 0.8 mm tip width; 4" length |
| Micro dissecting forceps | Roboz | RS-5153 | 1 x 2 teeth, slight curve, 0.8 mm tip width, 4" length |
| MTS MiniBionix II 808 | MTS Systems | Servohydraulic material testing machine | |
| Neutrophil Elastase 680 FAST probe | PerkinElmer | NEV11169 | |
| Nitrogen | General Welding Supply Corp. | ||
| Oxygen | General Welding Supply Corp. | ||
| Polylactic acid filament | Hatchbox | 1.75 mm diameter | |
| ProLong Diamond Antifade Mountant | Invitrogen | P36970 | |
| Puralube ophthalmic ointment | Dechra | NDC17033-211-38 | |
| Reflex 7 wound clips | Roboz Surgical | RS-9255 | |
| Stainless steel grid | Fotofab | One grid is 0.200 inches in diameter, with a total of 52 individual grid squares that are 0.016 x 0.016 inches. There is 0.003 inches of space between each square. | |
| Surface Treated SterileTissue Culture Plates | Fisher Scientific | FB012929 | Lid used as curing surface for imaging windows |
| TriM Scope Multiphoton Microscope | LaVision BioTec | Imaging was done on an upright 2-photon microscope (Trimscope, LaVision BioTec) equipped with two Ti:Sapphire lasers (Mai Tai and InSight, Spectra-Physics) and an optical parametric oscillator. The following Longpass Dichroic Beamsplitters (Chroma) were used to direct the signal towards four photomultipler tubes: T560LP T665LPXXR T495lxpr |
|
| Vetbond | 3M | 70200742529 | |
| VWR micro cover glass | VWR | 48404-453 |
References
- Dondossola, E., et al. Intravital microscopy of osteolytic progression and therapy response of cancer lesions in the bone. Science Translational Medicine. 10 (452), (2018).
- Haeger, A., et al. Collective cancer invasion forms an integrin-dependent radioresistant niche. Journal of Experimental Medicine. 217 (1), 20181184 (2020).
- Harper, K. L., et al. Mechanism of early dissemination and metastasis in Her2(+) mammary cancer. Nature. 540 (7634), 588-592 (2016).
- Eickhoff, S., et al. Robust anti-viral immunity requires multiple distinct T cell-dendritic cell interactions. Cell. 162 (6), 1322-1337 (2015).
- Engelhardt, J. J., et al. Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells. Cancer Cell. 21 (3), 402-417 (2012).
- Sammicheli, S., et al. Inflammatory monocytes hinder antiviral B cell responses. Science Immunology. 1 (4), (2016).
- Entenberg, D., et al. A permanent window for the murine lung enables high-resolution imaging of cancer metastasis. Nature Methods. 15 (1), 73-80 (2018).
- Ewald, A. J., Werb, Z., Egeblad, M. Preparation of mice for long-term intravital imaging of the mammary gland. Cold Spring Harbor Protocols. 2011 (2), 5562 (2011).
- Ritsma, L., et al. Surgical implantation of an abdominal imaging window for intravital microscopy. Nature Protocols. 8 (3), 583-594 (2013).
- Pittet, M. J., Garris, C. S., Arlauckas, S. P., Weissleder, R. Recording the wild lives of immune cells. Science Immunology. 3 (27), (2018).
- Alieva, M., Ritsma, L., Giedt, R. J., Weissleder, R., van Rheenen, J. Imaging windows for long-term intravital imaging: General overview and technical insights. Intravital. 3 (2), 29917 (2014).
- Heo, C., et al. A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology. Scientific Reports. 6, 27818 (2016).
- Anderson, T. L. . Fracture Mechanics: Fundamental and Applications. , (2005).
- Nakasone, E. S., Askautrud, H. A., Egeblad, M. Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer. Journal of Visualized Experiments: JoVE. (73), e50088 (2013).
- Sasmono, R. T., et al. A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse. Blood. 101 (3), 1155-1163 (2003).
- Cole, R. W., Jinadasa, T., Brown, C. M. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nature Protocols. 6 (12), 1929-1941 (2011).
- Sobolik, T., et al. Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging. Intravital. 5 (1), 1125562 (2016).
- Jacquemin, G., et al. Longitudinal high-resolution imaging through a flexible intravital imaging window. Science Advances. 7 (25), (2021).
