Name: longitudinal intravital imaging through clear silicone windows
Uploaded: 2022-01-05T12:30:00
Description: Watch this Scientific Journal Video about Longitudinal Intravital Imaging Through Clear Silicone Windows at JoVE.com

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。

所有描述的程序均按照冷泉港实验室外科指南进行，并已获得冷泉港实验室机构动物护理和使用委员会的批准。
1.铸造硅胶窗 
<ol><li>通过将基弹性体和固化剂以10：1（v / v）的比例混合来制备有机硅聚合物（PDMS）。</li>
	<li>通过在无菌，光滑的表面上沉积少量PDMS来铸造窗口，并将体积面积比调整为所需的厚度。 注意：在24孔板盖的盖子上使用200mg聚合物溶液?...

<ol>
	<li>Dondossola, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+osteolytic+progression+and+therapy+response+of+cancer+lesions+in+the+bone.">Intravital microscopy of osteolytic progression and therapy response of cancer lesions in the bone.</a> Science Translational Medicine. 10 (452), (2018).</li><li>Haeger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cancer+invasion+forms+an+integrin-dependent+radioresistant+niche.">Collective cancer invasion forms an integrin-dependent radioresistant niche.</a> Journal of Experimental Medicine. 217 (1), 20181184 (2020).</li><li>Harper, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+early+dissemination+and+metastasis+in+Her2(+)+mammary+cancer.">Mechanism of early dissemination and metastasis in Her2(+) mammary cancer.</a> Nature. 540 (7634), 588-592 (2016).</li><li>Eickhoff, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+anti-viral+immunity+requires+multiple+distinct+T+cell-dendritic+cell+interactions.">Robust anti-viral immunity requires multiple distinct T cell-dendritic cell interactions.</a> Cell. 162 (6), 1322-1337 (2015).</li><li>Engelhardt, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginating+dendritic+cells+of+the+tumor+microenvironment+cross-present+tumor+antigens+and+stably+engage+tumor-specific+T+cells.">Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells.</a> Cancer Cell. 21 (3), 402-417 (2012).</li><li>Sammicheli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+monocytes+hinder+antiviral+B+cell+responses.">Inflammatory monocytes hinder antiviral B cell responses.</a> Science Immunology. 1 (4), (2016).</li><li>Entenberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+permanent+window+for+the+murine+lung+enables+high-resolution+imaging+of+cancer+metastasis.">A permanent window for the murine lung enables high-resolution imaging of cancer metastasis.</a> Nature Methods. 15 (1), 73-80 (2018).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mice+for+long-term+intravital+imaging+of+the+mammary+gland.">Preparation of mice for long-term intravital imaging of the mammary gland.</a> Cold Spring Harbor Protocols. 2011 (2), 5562 (2011).</li><li>Ritsma, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+implantation+of+an+abdominal+imaging+window+for+intravital+microscopy.">Surgical implantation of an abdominal imaging window for intravital microscopy.</a> Nature Protocols. 8 (3), 583-594 (2013).</li><li>Pittet, M. J., Garris, C. S., Arlauckas, S. P., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+the+wild+lives+of+immune+cells.">Recording the wild lives of immune cells.</a> Science Immunology. 3 (27), (2018).</li><li>Alieva, M., Ritsma, L., Giedt, R. J., Weissleder, R., van Rheenen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+windows+for+long-term+intravital+imaging:+General+overview+and+technical+insights.">Imaging windows for long-term intravital imaging: General overview and technical insights.</a> Intravital. 3 (2), 29917 (2014).</li><li>Heo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+soft,+transparent,+freely+accessible+cranial+window+for+chronic+imaging+and+electrophysiology.">A soft, transparent, freely accessible cranial window for chronic imaging and electrophysiology.</a> Scientific Reports. 6, 27818 (2016).</li><li>Anderson, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fracture Mechanics: Fundamental and Applications. , (2005).</li><li>Nakasone, E. S., Askautrud, H. A., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+drug+responses+in+the+tumor+microenvironment+in+mouse+models+of+breast+cancer.">Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer.</a> Journal of Visualized Experiments: JoVE. (73), e50088 (2013).</li><li>Sasmono, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+macrophage+colony-stimulating+factor+receptor-green+fluorescent+protein+transgene+is+expressed+throughout+the+mononuclear+phagocyte+system+of+the+mouse.">A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse.</a> Blood. 101 (3), 1155-1163 (2003).</li><li>Cole, R. W., Jinadasa, T., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+and+interpreting+point+spread+functions+to+determine+confocal+microscope+resolution+and+ensure+quality+control.">Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control.</a> Nature Protocols. 6 (12), 1929-1941 (2011).</li><li>Sobolik, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+novel+murine+mammary+imaging+windows+to+examine+wound+healing+effects+on+leukocyte+trafficking+in+mammary+tumors+with+intravital+imaging.">Development of novel murine mammary imaging windows to examine wound healing effects on leukocyte trafficking in mammary tumors with intravital imaging.</a> Intravital. 5 (1), 1125562 (2016).</li><li>Jacquemin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+high-resolution+imaging+through+a+flexible+intravital+imaging+window.">Longitudinal high-resolution imaging through a flexible intravital imaging window.</a> Science Advances. 7 (25), (2021).</li></ol>

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3M Medipore Soft Cloth Surgical Tape</td>
			<td>3M</td>
			<td>70200770819</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk suture 4-0 PERMA HAND BLACK 1 x 18&#34; RB-2</td>
			<td>Ethicon&#160;</td>
			<td>N267H</td>
			<td></td>
		</tr>
		<tr>
			<td>ACTB-ECFP mice</td>
			<td>Jackson Laboratory</td>
			<td>22974</td>
			<td></td>
		</tr>
		<tr>
			<td>AEC Substrate Kit, Peroxidase (HRP), (3-amino-9-ethylcarbazole)</td>
			<td>Vector Laboratories&#160;</td>
			<td>SK-4200</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>BD&#160;</td>
			<td>326895</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia system</td>
			<td>Molecular Imaging Products Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acqknowledge software and sensors&#160;</td>
			<td>BIOPAC</td>
			<td>ACK100W, ACK100M, TSD110</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine spray&#160;</td>
			<td>LORIS</td>
			<td>109-08</td>
			<td></td>
		</tr>
		<tr>
			<td>c-fms-EGFP (MacGreen) mice</td>
			<td>Jackson Laboratory</td>
			<td>18549</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (30-F11)</td>
			<td>Invitrogen</td>
			<td>14-0451-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD68 Antibody</td>
			<td>Abcam</td>
			<td>ab125212</td>
			<td></td>
		</tr>
		<tr>
			<td>Curity gauze sponges&#160;</td>
			<td>Covidien</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Goat IgG H&#38;L (HRP)&#160;</td>
			<td>Abcam</td>
			<td>ab6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IgG H&#38;L (HRP)&#160;</td>
			<td>Abcam</td>
			<td>ab97064</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Rat IgG H&#38;L (HRP)&#160;</td>
			<td>Abcam</td>
			<td>ab102182</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow SYLGARD 184 Silicone Encapsulant Clear</td>
			<td>Electron Microscopy Sciences</td>
			<td>24236-10</td>
			<td>Two-part, 10:1 mixing ratio</td>
		</tr>
		<tr>
			<td>Round Cover Glass, 8mm Diameter, #1.5 Thickness&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>72296-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ender-3 Pro 3D printer</td>
			<td>Shenzhen Creality 3D Technology Co., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Far Infrared Heated blanket</td>
			<td>Kent Scientific</td>
			<td>RT-0520</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc Receptor Blocker</td>
			<td>Innovex Biosciences</td>
			<td>NB309</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji imaging processing package</td>
			<td></td>
			<td></td>
			<td>https://imagej.net/software/fiji/</td>
		</tr>
		<tr>
			<td>FluoroSpheres carboxylate, 0.04&#181;m, yellow-green (505/515)</td>
			<td>Invitrogen</td>
			<td>F8795</td>
			<td></td>
		</tr>
		<tr>
			<td>Gating system:</td>
			<td>BIOPAC Systems Inc.</td>
			<td></td>
			<td rowspan="7">The components together allow monitoring mouse vitals during imaging and gating image acquisition on mouse respiration. All were acquired from BIOPAC systems.</td>
		</tr>
		<tr>
			<td>Acqknowledge software&#160;</td>
			<td></td>
			<td>ACK100W, ACK100M</td>
		</tr>
		<tr>
			<td>Diff. Amp. Module, C Series&#160;</td>
			<td></td>
			<td>DA100C</td>
		</tr>
		<tr>
			<td>Dual Gating Sys small animal</td>
			<td></td>
			<td>DTU200&#160;</td>
		</tr>
		<tr>
			<td>MP160 for Windows - Analysis system</td>
			<td></td>
			<td>MP160WSW&#160;</td>
		</tr>
		<tr>
			<td>MouseOx Plus 120V&#160;</td>
			<td></td>
			<td>MOX-120V;015000&#160;</td>
		</tr>
		<tr>
			<td>Pressure Pad&#160;</td>
			<td></td>
			<td>TSD110&#160;</td>
		</tr>
		<tr>
			<td>Gelfoam</td>
			<td>Pfizer</td>
			<td>9031508</td>
			<td>Absorbable gelatin sponge</td>
		</tr>
		<tr>
			<td>Hardened fine scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
			<td>Two pairs; stainless steel, sharp-sharp 
			tips, straight tip, 26 mm 
			cutting edge, 11 cm length</td>
		</tr>
		<tr>
			<td>Human/Mouse Myeloperoxidase/MPO Antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF3667</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td>Turn on approximately 30 min 
			before use; sterilize tools at &#62;200 
			&#176;C for 30 s</td>
		</tr>
		<tr>
			<td>Imaris&#160;</td>
			<td>Bitplane</td>
			<td>www.bitplane.com</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion medium Immersol W 2010</td>
			<td>Zeiss</td>
			<td>444969-0000-000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringes with BD Ultra-Fine needle 6mm x 31G 1 mL/cc</td>
			<td>BD</td>
			<td>324912</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane (Fluriso)</td>
			<td>VetOne</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight&#174; 594</td>
			<td>Vector Laboratories&#160;</td>
			<td>DL-1177-1</td>
			<td></td>
		</tr>
		<tr>
			<td>LysM-eGFP mice</td>
			<td>www.mmrrc.org</td>
			<td>012039-MU</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting forceps</td>
			<td>Roboz</td>
			<td>RS-5135</td>
			<td>Serrated, slight curve, 0.8 mm tip width; 4&#34; length</td>
		</tr>
		<tr>
			<td>Micro dissecting forceps</td>
			<td>Roboz</td>
			<td>RS-5153</td>
			<td>1 x 2 teeth, slight curve, 0.8 mm tip 
			width, 4&#34; length</td>
		</tr>
		<tr>
			<td>MTS MiniBionix II 808</td>
			<td>MTS Systems</td>
			<td></td>
			<td>Servohydraulic material testing machine</td>
		</tr>
		<tr>
			<td>Neutrophil Elastase 680 FAST probe</td>
			<td>PerkinElmer</td>
			<td>NEV11169</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>General Welding Supply Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>General Welding Supply Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polylactic acid filament</td>
			<td>Hatchbox</td>
			<td></td>
			<td>1.75 mm diameter</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube ophthalmic ointment</td>
			<td>Dechra&#160;</td>
			<td>NDC17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex 7 wound clips</td>
			<td>Roboz Surgical</td>
			<td>RS-9255</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel grid</td>
			<td>Fotofab</td>
			<td></td>
			<td>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.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Surface Treated SterileTissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>FB012929</td>
			<td>Lid used as curing surface for imaging windows</td>
		</tr>
		<tr>
			<td>TriM Scope Multiphoton Microscope&#160;</td>
			<td>LaVision BioTec</td>
			<td></td>
			<td>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</td>
		</tr>
		<tr>
			<td>Vetbond</td>
			<td>3M</td>
			<td>70200742529</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR micro cover glass</td>
			<td>VWR</td>
			<td>48404-453</td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal intravital imaging through clear silicone windows

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。

通过成像窗口的活体成像可用于在数小时至数周内以单细胞分辨率观察，跟踪和量化各种细胞和分子事件。成像窗口的理想特征包括：a）对小鼠健康和组织生理学的影响有限;b） 耐久性;c） 插入简单;d）清晰的标记，用于重复成像同一区域。结果是一个多功能的惰性硅胶窗口，易于生产和插入，并且可以配备网格，以在多个成像会话的范围内定位相同的视场。
该窗口由PDMS制?...

Watch this Scientific Journal Video about Longitudinal Intravital Imaging Through Clear Silicone Windows at JoVE.com

Longitudinal Intravital Imaging Through Clear Silicone Windows

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。

通过透明硅胶窗进行纵向活体成像

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

这种铸造和插入有机硅窗口的程序降低了纵向活体内成像的技术障碍。我们的实验方案可生产坚固、易于适应的窗户，动物对它们的耐受性良好。使用这种技术，我们对肝脏，胰腺和乳腺中的癌症和免疫细胞进行了成像。只需进行最小的调整，它就可以适应许多解剖位置和生物学问题。对于那些第一次尝试这种技术的人来说，练习从注射器中释放少量的胶水。涂上过多的胶水会使成像区域变得浑浊，并使手术区域难以使用。通过在干净光滑的表面上沉积少量新制备的PDMS有机硅聚合物聚二甲基硅氧烷，开始铸造硅胶窗口，并调整体积与面积比以获得所需的厚度。一旦PDMS沉积在所需的铸件表面上，将不锈钢网格轻轻地压入硅胶中，为相同组织区域的重复成像提供标记。要去除气泡并使有机硅聚合物脱气，请将涂层表面置于真空干燥器中45分钟。完成后，将聚合物在75至80摄氏度的烘箱中固化45分钟。将聚合物划在模具的边缘，并使用镊子从用于铸造的表面轻轻剥离固化的窗口。将鼠标放在左侧侧卧位。通过在剃须的手术部位连续三次施用10%体积的聚维酮碘溶液和70%体积的乙醇来制备用于手术的麻醉小鼠进行手术。必须注意保持无菌手术区域。接下来，使用无菌剪刀和镊子在肋弓下方三毫米处的右侧侧部做一个10毫米的切口。取出皮肤的一平方厘米部分，并使用第二对无菌剪刀和镊子去除腹膜部分略小于皮肤部分的部分。将少量手术粘合剂沉积在无菌孔板上，并用31号注射器将其取出，以涂敷在要成像区域边缘周围的肝脏表面上，然后将窗口定位在所需区域上，并将其牢固地固定在肝脏上约两分钟，直到粘合剂干燥。将窗的边缘折叠在腹膜下后，将少量的手术粘合剂沉积在窗的边缘上，这些粘合剂现在位于腹膜下方，用镊子向下推腹膜以将其固定在窗户上。同样，在向下推皮肤之前，通过沉积少量的手术粘合剂来密封腹膜，然后在窗户边缘周围涂上胶水以形成一个边缘，以防止皮肤在窗户上长回来。要将窗口插入胰腺，请将鼠标放在右侧外侧褥位，并在左侧侧翼做切口，然后切除腹膜的一部分，如前所述。使用浸泡有无菌盐水的无菌棉签，轻轻拉动脾脏以观察胰腺。用湿润的棉签重新定位胰腺，使更多的表面积通过切口可见。进入胰腺后，如前所述，将成像窗口插入并密封在所需区域。要进入乳腺，将小鼠置于仰卧位，并使用无菌剪刀和镊子在其中一个腹股沟上做一个 10 毫米的切口。取下乳腺上方0.5平方厘米的皮肤部分，然后通过在两个表面之间铺开无菌剪刀以破坏粘附，轻轻地将乳腺与皮肤分开。使用31号注射器在要成像的区域边缘周围的乳腺表面上施加少量的手术粘合剂。将窗口定位并牢牢地靠在乳腺上，直到粘合剂变干。将窗户的边缘折叠在皮肤下后，在现在皮肤下方的窗口边缘上沉积少量手术粘合剂，并用镊子向下推皮肤以将皮肤固定在窗口上。在窗口边缘周围涂上胶水以创建一个边缘，以防止皮肤在窗口上长回来。在开始成像之前，用浸有70%乙醇的棉签轻轻擦拭窗口，以清除任何残留的碎屑。使用水浸透镜时，将超声波凝胶涂抹在窗户上，避免气泡。要开始成像，请通过目镜定位并放置对焦网格。然后通过将网格的底部设置为零来确定近似的组织成像深度。该信息将有助于在后续成像会话中定位相应的Z平面。要在多个成像会话中识别同一位置，请使用网格上的方块作为参考点。在成像会话期间，请注意网格方向和网格内成像的每个视场的位置。网格可用于在映像会话成功时导航回特定的网格区域和成像区域。每次成像后，用浸有70%乙醇的棉签轻轻擦拭窗口，去除任何残留的镜头浸入介质和碎屑。硅胶窗口可以捕获随时间推移的动态免疫细胞浸润。具有代表性的分析说明了第二天和第四天重塑乳腺组织内的中性粒细胞浸润。硅胶窗口允许在窗口插入后几周内在多个时间点可视化肿瘤。用200毫克PDMS铸造的窗户通常更适合手术植入，因为它们表现出比150毫克铸造的窗户更大的韧性。值得注意的是，PDMS窗户不像标准玻璃那样刚性，其表现为玻璃窗的杨氏模量明显大于PDMS。不同器官的窗口插入耐受性良好，因为大多数小鼠随着时间的推移体重继续增加。尝试此过程时要记住的最重要的事情是使用胶水完全密封窗户。这可以防止感染，组织干燥和窗户移位。手术后，可以通过硅胶窗口进行原位注射，使研究人员能够研究各种主题，例如细胞增殖，细胞 - 细胞相互作用和治疗效果。

Research

JoVE Journal

Cancer Research

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

长期高分辨率活体显微镜在肺与真空稳定成像窗口

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

科研

癌症研究

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

转移性的型号同源小鼠肾细胞癌的临床前疗法的定量和纵向评估

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

核素荧光报告基因成像在啮齿动物肿瘤模型中跟踪肿瘤进展

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

脑肿瘤细胞行为的纵向生命成像,以响应侵入性手术活检

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

利用生命内成像和固定组织分析评估与癌细胞传播相关的转移门道介导血管渗透性的肿瘤微环境

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

比较在小鼠肾脏和鸡胆膜上建立的转移性透明细胞肾细胞癌模型

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

肝细胞癌患者的跨径向获取化疗

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

https://cloudfront.jove.com/CDNSource/teasers/61109.jpg

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

通过颅内注射和磁共振成像对大脑转移进行建模

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

使用快速和定量的细胞内胆碱膜模型发现转移调节器

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...