长期高分辨率活体显微镜在肺与真空稳定成像窗口
1Department of Developmental and Molecular Biology,Albert Einstein College of Medicine, 2Department of Obstetrics/Gynecology and Woman’s Health,Albert Einstein College of Medicine, 3Department of Anatomy & Structural Biology,Albert Einstein College of Medicine, 4Gruss-Lipper Biophotonics Center Integrated Imaging Program,Albert Einstein College of Medicine, 5Medical Research Council Centre for Reproductive Health, Queen’s Medical Research Institute,University of Edinburgh
Summary
This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.
Abstract
转移到辅助站点，如肺，肝，骨，死亡率约90％1创伤性事件。这些网站，肺是最困难的使用活体光学成像评估由于身体，细腻的性质和维持适当的生理至关重要的作用范围内的封闭位置。而临床形式（正电子发射断层扫描（PET），磁共振成像（MRI）和计算机断层扫描（CT））能够提供该组织的非侵入性的图像，它们缺乏必要的可视化的最早播种事件分辨率，与单个像素由近千细胞。肺转移播种假设当前模型只是一个肿瘤细胞的到来之后发生的事件是确定性的生存和随后的生长。这意味着，与单细胞分辨率的2实时活体成像工具，以限定播种CEL的表型是必需ls和测试这些模型。虽然已使用各种体外制剂进行肺部的高分辨率光学成像，这些实验一般是单个时间点测定法和易受工件和可能的错误的结论，由于显着地改变的环境（温度，丛生，细胞因子， 等等。 ）从胸腔和循环系统3去除导致。最近的工作然而使用一真空稳定成像窗2,4,5中所示的完整的肺的那个时间推移活光学成像是可能的，典型的成像时间被限制在约6小时。在这里，我们描述了使用这种窗口历时12小时的肺进行长期活时间推移成像的协议。用这种方法得到的隔时图像的序列使在肺可视化和细胞 – 细胞相互作用的定量，膜动力学和血管灌注。我们进一步Ðescribe的图像处理技术，让肺微血管的空前明确的说法。
Introduction
高分辨率活光学成像已被证明是对理解许多生物过程，允许单细胞和亚细胞的参数被测量和量化的关键。在癌症研究，肿瘤和基质细胞的活体成像导致许多微环境相互作用6-11是仅在完整的动物本的发现。
关于体内单细胞分辨率的光学成像血管内和肿瘤细胞的传播在乳腺癌相关的微环境的发现甚至导致对预后和治疗反应的乳腺癌患者12-16新颖标记。适用于完整的内部重要器官的深处观赏的最佳成像技术是临床模式（MRI，PET，CT），它提供整个器官的美丽风景，并可以揭示病理它们产生临床症状之前也。他们无法，HH但是，揭示转移的驱动肿瘤进展的早期阶段和细胞机制由于缺乏单细胞分辨率。到时候肺转移，在这些模式可见，他们很好地建立和增殖。中的估计，90％的该到达肺要么不生存17或最初保持休眠18，它们比以前预期19，成像到达和生存的最早步骤更早到达播散的肿瘤细胞，并观察变得至关重要理解转移性接种和肿瘤生长的复发在偏远地点的过程。
表演肺这些意见已被证明不过非常困难;绝大部分影像学已经利用体外或植制剂20-23，只给个说法成单个时间点的肺。虽然这些准备工作确实提供了有用的信息细则第十五，它们不给微环境的各种部件之间发生的相互作用，因果关系，以及动态特性的完整的理解。缺乏适当的循环系统（与稳态伴随失调），并从人体的免疫系统的其余部分断开的使得它渴望来验证这些制剂在体内完整的组织产生的结论。
许多团体都进行了完整的肺2,4,5,24-33与Wearn和德国是第一个手术暴露胸膜层24和特里率先利用植入式成像窗口25的活体成像。
在肺高分辨率成像是由肺的恒定运动大大阻碍和几种技术已经开发了克服这种限制。瓦格纳和菲利浦27所研究的犬肺部的自然运动并设计了手术方案在一个相对固定的区域找到他们的植入窗口，而瓦格纳在他的窗口手术准备利用真空固定组织28。自那时以来，各种技术已被用于图像肺包括：支气管夹紧，顺序呼吸暂停和选通成像，过取样的采集，肺叶和真空34的胶合。每一种都有其优点和缺点，没有一种技术已成为优于其他34。例如，支气管夹紧和连续呼吸暂停改变气体在肺正常交流，并可能导致肺不张。门控成像和过采样收购不从这些缺点，但需要高速或专门的影像设备不普及。最后肺既胶合和真空技术避免两者上述缺点的，但是可能表现出剪切力引起的损伤，如果服务不是德恩。近年来，真空窗口已被小型化，并适于在使用共聚焦和多光子显微镜4,5,33和优异的高分辨率成像的小鼠用已经达到2。 表1概括了本丰富的历史，并强调那些描述新颖的论文进步在利用活体肺显像窗口。
本协议描述在现场，完整的肺使用较长时间推移多光子活体显微镜的图像转移具有最高分辨率亚细胞可能。图像使用配备有高数值孔径物镜和多个光电倍增管（PMT）探测器多光子显微镜获得长达12小时。转基因小鼠模型被用于荧光标记天然的巨噬细胞用荧光高分子葡聚糖和荧光蛋白转染的肿瘤细胞（标记脉管和肿瘤细胞respectivel沿Y）。虽然这种选择荧光标记的细胞使肿瘤细胞，内皮细胞 – 巨噬细胞的相互作用和动态可视化，该协议将荧光或无荧光，鼠标的任何应变工作。采集后，残余漂移运动（如果有的话）是使用斐济插件35,36和自定义宏时间平均血管通道，以消除由未标记的循环血细胞闪烁引起的消除。
虽然该协议的重点成像转移，该技术适用于观察到的与在肺高分辨率单细胞成像的许多其它生物过程。
Protocol
在本协议中所述的所有程序都按照为使用脊椎动物，包括医药机构动物护理和使用委员会的爱因斯坦医学院的事先批准的准则和条例被执行。 1.生成荧光标记的小鼠模型和肿瘤细胞通过将0.1g的BSA用100ml的PBS混合制备100ml的0.1％（重量/体积）牛血清白蛋白/磷酸盐缓冲盐水（BSA / PBS）缓冲液。 通过稳定转染制备荧光标记的肿瘤细胞。 注意：在这里，我们使用E…
Representative Results
为了证明可以用这种方法可以实现的结果的类型，我们注射标记有荧光蛋白三叶草成MacBlue小鼠44的尾静脉在手术前不同时间点E0771-LG的肿瘤细胞。手术后，155 kD的罗丹明标记的葡聚糖注射IV标记进行血管和时间推移成像。 当成像的小鼠后24小时注射，单细胞是血管内可见，与巨噬细胞和单核细胞相互作用。这种情况的一…
Discussion
体内光学成像用荧光标记的功能标记，如蛋白质和抗体结合高分辨率急剧增加了转移级联的理解。它使直接可视化和单细胞，并在肿瘤细胞中的亚细胞参数，宿主细胞及其微环境的定量。原发性肿瘤内这种成像已经导致，例如，到离散的微环境是支持或者生长侵袭或传播6,7的发现。在入侵的情况下， 在体内成像揭示巨噬细胞和肿瘤细胞的共的迁徙流的在血管内7,50优…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This research was supported by NIH-CA100324, Einstein National Cancer Institute’s cancer center support grant P30CA013330, R01CA172451 to JWP and the Integrated Imaging Program. This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at the Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work. The authors thank Mike Rottenkolber, Ricardo Ibagon and Anthony Leggiadro of the Einstein machine shop for their skilled and timely craftsmanship, the laboratory of Matthew Krummel for generously sharing their window design drawings, Kevin Elicieri and Jeremy Bredfeldt for their expertise in microscopy and their amplifier recommendations and Allison Harney and Bojana Gligorijevic for informative discussions.
Materials
| Nickel-Plated Brass Vacuum Regulator 1/8 NPT Female, w/ Gauge, 0 – 20" Hg Vacuum | McMaster Carr | 4172K12 | Vacuum Regulator |
| Brass Barbed Hose Fitting Adapter for 1/4" Hose ID X 1/8" NPTF Male Pipe | McMaster Carr | 5346K13 | Vacuum Regulator Hose Adapter |
| Pyrex Brand Filtering Flasks with Tubulation; Neck tooled for rubber stopper No. 4; Capacity: 50mL | Corning Life Sciences Glass | 5360-50 | Vacuum Flask |
| Round Glass Coverslips Thickness #1.5, 0.16-0.19mm 10mm dia. | Ted Pella, Inc. | 260368 | Cover slips |
| Exel International Disposable Safelet I.V. Catheters; 22gx1 in. | Exel International | 26746 | Tracheal Catheter |
| PERMA-HAND Black Braided Silk Sutures, ETHICON LIGAPAK Dispensing Reel Size 2-0 | VWR | 95056-992 | String |
| Liquid Super Glue, Clear, 0.14oz | Hendel Corp. | LOC1647358 | Cyano-acrylate Glue |
| Tetramethylrhodamine isothiocyanate–Dextran | Sigma-Aldrich | T1287-500MG | 155kD Dextran |
| Laboratory Clear Tygon PVC Tubing, 1/16" ID, 1/8" OD, 1/32" Wall Thickness, 25 ft. Length | McMaster Carr | 5155T12 | Thin Tubing & Tubing for Luer |
| Crack-Resistant Polyethylene Tubing, 1/8" ID, 1/4" OD, 1/16" Wall Thickness, White, 50 ft. Length | McMaster Carr | 5181K24 | Thick Tubing |
| Depillatory Lotion | Nair | – | |
| Micro Medical Tubing 95 Durometer LDPE | Scientific Commodities Inc. | BB31695-PE/1 | Tubing for tail vein catheter |
| 30 G x 1 in. BD PrecisionGlide Needle | BD | 305128 | Needles for tail vein catheter |
| Puritan Nonsterile Cotton-Tipped Swabs | Fisher Scientific | 867WCNOGLUE | |
| Clear Polycarbonate Barbed Tube Fitting, Reducing Straight for 3/32" x 1/16" Tube ID | McMaster Carr | 5117k51 | Connectors between tubes |
| One-Hole Rubber Stoppers | Fisher Scientific | 14-135F | Stopper for Vacuum Flask |
| SHARP Precision Barrier Tips, For P-100, 100µl | Denville Scientific Inc. | P1125 | Pipette Tip |
| Laboratory tape | Fisher Scientific | 159015R | |
| Puralube | Henry Schein Animal Health | 008897 | Opthalmic Ointment |
| Gemini Cautery Kit | Harvard Apparatus | 726067 | Cautery Pen |
| Graefe Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4" Length | Roboz Surgical | RS-5135 | Forceps |
| Extra Fine Micro Dissecting Scissors 4" Straight Sharp/Sharp 24mm | Roboz Surgical | RS-5912 | Sharp Scissors |
| Micro Dissecting Scissors 4" Straight Blunt/Blunt | Roboz Surgical | RS-5980 | Blunt Scissors |
| Wipes | Fisher Scientific | 06-666-A | Harness |
| PhysioSuite System | Kent Scientific | PhysioSuite | Vitals Monitor |
| 1 mL Syringe, Tuberculin Slip Tip | BD | 309659 | Syringe |
| Cyano acrylate | Staples | LOC1647358 | Cover Slip Adhesive |
| Petroleum Jelly | Fisher Scientific | 19-086291 | Water Barrier |
| Adapter Luer Cannulla 1.5-2.2mm | Harvard Apparatus | 734118 | Catheter Connector |
| MouseOx oximeter, software and sensors | STARR Life Sciences | MouseOx | Pulse Oximeter |
| Isoethesia (isoflurane) | Henry Schein Animal Health | 50033 | 250 mL |
| Oxygen | TechAir | OX TM | |
| 1 x PBS | Life Technologies | 10010-023 | |
| PVC Ball Valve, Push to Connect, 1/4 In | Grainger | 3CGJ7 | Vacuum Valve |
| Small Animal Ventilator | Harvard Apparatus | 683 | Alternative is available from Kent Scientific: MouseVent |
| OptiMEM Reduced Serum Medium | ThermoFisher Scientific | 31985062 | |
| Lipofectamine 2000 Transfection Reagent | ThermoFisher Scientific | 11668019 | |
| MacBlue Tg(Csf1r*-GAL4/VP16,UAS-ECFP)1Hume/J Mice | Jackson Laboratory | 026051 | |
| Multiphoton Microscope | Olympus | Fluoview FV1000 | Alternative to custom built scope |
| Environmental Enclosure | Precision Plastics | Chamber for FV1000 | Alternative to custom built enclosure |
| Phosphate Buffered Saline | ThermoFisher Scientific | 14190136 | |
| Laser Power Meter | Coherent | FieldMaxIITOP | |
| Laser Power Meter Head | Coherent | PM10 | |
| pcDNA3-Clover Fluorescent Protein Vector | Addgene | 40259 | |
| G418 Sulfate Selective Antibiotic | ThermoFisher Scientific | 10131027 | |
| MoFlo Fluorescent-Activate Cell Sorter | Beckman Coulter | XDP | |
| Trypsin EDTA 1X | Corning | 25-052-Cl | |
| 40 µm Mesh | Falcon | 352235 | |
| 96 Well Plate | Costar | 3599 | |
| 60 mm Culture Dish | Corning | 430196 | |
| 10 cm Culture Dish | Corning | 353003 | |
| Bovine Serum Albumin | Sigma-Aldrich | A4503 | |
| Dulbecco's Phosphate Buffered Saline 1X | Corning | 21-031-CV | |
| C57BL/6J Mouse | Jackson Laboratory | 000664 | |
| Kim Wipes | Fisher Scientific | 06-666-A |
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Citazione di questo articolo
Rodriguez-Tirado, C., Kitamura, T., Kato, Y., Pollard, J. W., Condeelis, J. S., Entenberg, D. Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window. J. Vis. Exp. (116), e54603, doi:10.3791/54603 (2016).