Name: intravital widefield fluorescence microscopy of pulmonary microcirculation in experimental acute lung injury using a vacuum-stabilized imaging system
Uploaded: 2022-04-06T14:40:00
Description: Watch this Scientific Journal Video about Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System at JoVE.

作者要感谢Pina Colarusso博士，他在编辑和修订本手稿方面提供了丰富的专业知识。

这里描述的所有程序都是在达尔豪斯大学实验动物委员会（UCLA）的事先批准下进行的。
1. 准备工作
<ol><li>肺部成像系统：为了准备窗口，在外圈顶部施用一层薄薄的真空润滑脂，同时避免真空通道的污染。将干净的8毫米玻璃盖玻片放在窗户上，然后轻轻按下以形成密封。</li>
	<li>宽视场荧光显微镜：使用配备20x/0.40长工作距离物镜和帧速率为25 FPS的黑白电荷耦?...

<ol>
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Potential consequences in chronic obstructive pulmonary disease.</a> Frontiers in Immunology. 8, 1766 (2017).</li><li>Condon, M. R., Kim, J. E., Deitch, E. A., Machiedo, G. 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R., Kuebler, W. M. <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+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> Journal of Applied Physiology. 104 (2), 338-346 (2008).</li><li>Butt, Y., Kurdowska, A., Allen, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+lung+injury:+A+clinical+and+molecular+review.">Acute lung injury: A clinical and molecular review.</a> Archives of Pathology and Laboratory Medicine. 140 (4), 345-350 (2016).</li><li>Park, I., 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=Neutrophils+disturb+pulmonary+microcirculation+in+sepsis-induced+acute+lung+injury.">Neutrophils disturb pulmonary microcirculation in sepsis-induced acute lung injury.</a> European Respiratory Journal. 53 (3), 1800786 (2019).</li><li>Kim, J. 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A., H&#252;ttmann, G., K&#246;nig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopic+optical+coherence+tomography+imaging+to+assess+mucus-mobilizing+interventions+for+muco-obstructive+lung+disease+in+mice.">Intravital microscopic optical coherence tomography imaging to assess mucus-mobilizing interventions for muco-obstructive lung disease in mice.</a> American Journal of Physiology - Lung Cellular and Molecular Physiology. 318 (3), 518-524 (2020).</li><li>Lamm, W. J. E., Bernard, S. L., Wiltz, W., Wagner, J., Glenny, R. 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P., 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=Effect+of+a+protective-ventilation+strategy+on+mortality+in+the+acute+respiratory+distress+syndrome.">Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome.</a> New England Journal of Medicine. 338 (6), 347-354 (2009).</li><li>Looney, M. R., Bhattacharya, J. <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+the+lung.">Live imaging of the lung.</a> Annual Review of Physiology. 76, 431-445 (2013).</li></ol>

这里介绍的协议需要练习并注意几个关键步骤。首先，在开始插管和手术之前准备成像窗口很重要。使用最少量的真空润滑脂涂覆成像窗口的外环，涂上盖玻片，并用一滴蒸馏水测试吸力。提前准备可以防止暴露的肺在设置过程中变干。虽然可以用温盐水冲洗，但这样做可能会损害脆弱的肺组织。
插管后，在将鼠标转移到IVM平台时，插管可能偶尔会移位。为了防止这种情况?...

活体显微镜（IVM）是一种有用的成像工具，用于可视化和研究 体内各种生物物理过程。肺 在体内 成像是非常具有挑战性的，因为它的封闭位置，其组织的脆弱性以及呼吸和心跳引起的运动伪影1，2。已经开发了各种活体显微镜（IVM）装置，用于肺微循环中白细胞 - 内皮相互作用的实时成像，以克服这些挑战。这些方法基于手术暴露和稳定肺部以进行成像。
动物通常通过外科手术为肺病媒体移植做准备。首先，对动物进行插管和通气，这允许手术切除胸窗并随后进行干预以稳定肺部以进行成像。一种技术涉及将实质粘附在玻璃盖玻片3上，该过程可能会对成像组织造成重大的物理创伤。更先进的是利用真空系统在玻璃窗4下稳定肺部。这种设置有助于通过分布在大面积区域的可逆真空将肺表面松散地粘附到盖玻片上，并扩大肺，同时仍然限制x，y和z尺寸4的运动。真空通过围绕装置成像区域的通道均匀施加，并将组织拉入面向成像级盖玻片4的浅锥形区域。通过这个观察窗，可以使用各种光学成像方式研究肺微循环。
肺病媒体能对多种微循环参数进行定量成像。这些包括诸如白细胞轨迹速度和长度5，红细胞流速6和氧合7，肿瘤转移8，免疫细胞亚群9，10，11的区别，微粒的可视化12，肺泡动力学13，14，血管通透性15和毛细血管功能16的测量.这里的重点是白细胞募集和毛细血管功能。肺微循环中白细胞募集的开始涉及白细胞和内皮细胞之间的瞬时滚动相互作用和牢固的粘附相互作用，两者在炎症条件下均增加16，17。通常，滚动通过操作员定义的参考线的白细胞数量来量化，而粘附则通过内皮16上不动的白细胞数量来量化。毛细血管功能也可能在炎症状态下受到影响，通常导致灌注减少。这可以归因于几个因素，包括红细胞变形性的降低18和内皮细胞诱导的NO合酶的杂交表达导致病理性分流19。通常，测量每个区域灌注毛细血管的聚集长度，并将其报告为功能性毛细血管密度（FCD）。
实时研究肺部白细胞募集需要用荧光染料或荧光标记抗体标记生物靶标20。或者，各种转基因小鼠菌株如溶菌酶M-green荧光蛋白（LysM-GFP）小鼠可用于对特异性免疫细胞亚群（如嗜中性粒细胞21，22）进行成像。然后可以使用宽视场荧光显微镜，共聚焦显微镜或多光子显微镜观察荧光标记的白细胞。这些技术通过利用特定的激发波长和检测发射的荧光来实现对比度，同时阻断激发波长的检测，从而突出显示标记的物体。
关于小鼠肺白细胞滚动、粘附和功能毛细血管密度量化的现有研究主要依赖于手动视频分析。这是通过开源软件（如斐济6，23）、专有软件（如CapImage12）或定制图像处理系统24实现的。相反，各种专有软件平台（例如，NIS Element，Imaris，Volocity，MetaMorph）可以自动测量各种其他生理参数，包括前面提到的许多5，6，7，8，9，10，11，12，13，15。
关于使用肺病媒的急性肺损伤 （ALI） 和急性呼吸窘迫综合征 （ARDS） 的病理学，已经做出了重要的观察结果。ARDS的特征在于肺部的一系列病理生理过程，包括由内皮和上皮屏障25功能障碍引起的肺水肿和肺泡损伤。使用小鼠模型，已经发现脓毒症诱导的ALI与肺环境中免疫细胞运输的显着有害变化有关26。发现招募到脓毒症诱导的ALI小鼠毛细血管的嗜中性粒细胞阻碍微循环，从而增加ALI26的缺氧。此外，IVM已被用于深入了解ARDS27发作后的潜在修复机制。肺病媒综合防治也是了解各种阻塞性肺疾病病理生理学变化的宝贵工具。例如，囊性纤维化（CF）和慢性阻塞性肺疾病（COPD）等疾病中粘液运输的可视化促进了对粘液清除的新型和现有治疗方法的研究28。这些条件下的白细胞运输也进行了分析17.
该协议扩展了Lamm等人最初描述的方法，以 使用常规荧光显微镜研究白细胞 - 内皮相互作用。所描述的程序采用 体内 肺成像系统，其中包括16.5 cm x 12.7cm金属底座，显微操作器和真空成像窗口（图1）。该系统安装在20 cm x 23.5 cm的3D打印平台（补充文件1）中，为通风机管和加热垫提供安全连接。该方法为小鼠肺微循环 在体内提供了可重复和可量化的成像。详细解释了手术准备的重要方面以及真空稳定肺成像系统的正确利用。最后，利用ALI实验模型对改变的白细胞滚动、白细胞粘连和与炎症相关的毛细血管灌注进行有代表性的成像和分析。使用该方案应有助于进一步重要研究急性疾病状态下肺部微循环的病理生理学变化。

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			<td>1 mL BD Luer Slip Tip Syringe sterile, single use</td>
			<td>Becton, Dickinson and Company</td>
			<td>309659</td>
			<td>1 mL syringe</td>
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			<td>ADSON Dressing Forceps, Tip width 0.6 mm, teeth length 11.5 mm, 12 cm</td>
			<td>RWD Life Science Co.</td>
			<td>F12002-12</td>
			<td>Blunt forceps</td>
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			<td>Albumin-Fluorescein Isothiocyanate</td>
			<td>Sigma-Aldrich</td>
			<td>A9771-1G</td>
			<td>FITC-albumin</td>
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			<td>Alcohol Swab Isopropyl Alcohol 70% v/v</td>
			<td>Canadian Custom Packaging Company</td>
			<td>80002455</td>
			<td>Alcohol wipe</td>
		</tr>
		<tr>
			<td>AVDC110 Advanced Digital Video Converter</td>
			<td>Canopus</td>
			<td>00631069602029</td>
			<td>Digital video converter</td>
		</tr>
		<tr>
			<td>B/W - CCD - Camera</td>
			<td>Horn Imaging</td>
			<td>BC-71</td>
			<td>Camera</td>
		</tr>
		<tr>
			<td>Bovie Deluxe High Temperature Cautery Kit</td>
			<td>Fine Science Tools</td>
			<td>18010-00</td>
			<td>Cauterizer</td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Charles River Laboratories International</td>
			<td>C57BL/6NCrl</td>
			<td>C57BL/6 Mice</td>
		</tr>
		<tr>
			<td>Cotton Tipped Applicators</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td>Cotton applicator</td>
		</tr>
		<tr>
			<td>CS-8R 8mm Round Glass Coverslip</td>
			<td>Warner Instruments</td>
			<td>64-0701</td>
			<td>Glass coverslip</td>
		</tr>
		<tr>
			<td>Digital Pressure Gauge</td>
			<td>ITM Instruments Inc.</td>
			<td>DG2551L0NAM02L0IM&#38;V</td>
			<td>Digital Pressure Gauge</td>
		</tr>
		<tr>
			<td>Dr Mom Slimline Stainless LED Otoscope</td>
			<td>Dr. Mom Otoscopes</td>
			<td>1001</td>
			<td>Otoscope</td>
		</tr>
		<tr>
			<td>Ethyl Alchohol 95% Vol</td>
			<td>Commercial Alcohols</td>
			<td>P016EA95</td>
			<td>95% ethanol</td>
		</tr>
		<tr>
			<td>Fine Scissors - Martensitic Stainless Steel</td>
			<td>Fine Science Tools</td>
			<td>14094-11</td>
			<td>Scissors</td>
		</tr>
		<tr>
			<td>Fisherbrand Colored Labeling Tape</td>
			<td>Fisher Scientific</td>
			<td>1590110</td>
			<td>Labeling tape</td>
		</tr>
		<tr>
			<td>Gast DOA-P704-AA High-Capacity Vacuum Pump</td>
			<td>Cole-Parmer Canada Company</td>
			<td>ZA-07061-40</td>
			<td>Vacuum pump</td>
		</tr>
		<tr>
			<td>Hartman Hemostats</td>
			<td>Fine Science Tools</td>
			<td>13003-10</td>
			<td>Hemostatic forceps</td>
		</tr>
		<tr>
			<td>High Vacuum Grease</td>
			<td>Dow Corning</td>
			<td>DC976VF</td>
			<td>Vacuum grease</td>
		</tr>
		<tr>
			<td>Isoflurane USP</td>
			<td>Fresenius Kabi</td>
			<td>CP0406V2</td>
			<td>Isoflurane</td>
		</tr>
		<tr>
			<td>LIDOcaine HCl Injection 1% 50 mg/5 mL</td>
			<td>Teligent Canada</td>
			<td>0121AD01</td>
			<td>Lidocaine HCl 1%</td>
		</tr>
		<tr>
			<td>Lung SurgiBoard</td>
			<td>Luxidea, Inc.</td>
			<td>IMCH-0001</td>
			<td>Designed for intravital microscopy of the lung</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Teva Canada</td>
			<td>00485802</td>
			<td>Mineral oil</td>
		</tr>
		<tr>
			<td>Mouse Endotracheal Intubation Kit</td>
			<td>Kent Scientific Corporation</td>
			<td>ETI-MSE</td>
			<td>Intubation stand, anesthesia mask, 20 G endotracheal cannula, fibre optic cable</td>
		</tr>
		<tr>
			<td>MST49 Fluorescence Microscope</td>
			<td>Leica Microsystems</td>
			<td>10 450 022</td>
			<td>Fluorescence Microscope</td>
		</tr>
		<tr>
			<td>N Plan L 20x/0.40 Long Working Distance Microscope Objective</td>
			<td>Leica Microsystems</td>
			<td>566035</td>
			<td>20x objective</td>
		</tr>
		<tr>
			<td>Non-Woven Sponges 2&#34; x 2&#34;</td>
			<td>AMD-Ritmed</td>
			<td>A2101-CH</td>
			<td>Gauze</td>
		</tr>
		<tr>
			<td>Optixcare Eye Lube Plus</td>
			<td>Aventix</td>
			<td>5914322</td>
			<td>Tear gel</td>
		</tr>
		<tr>
			<td>Original Prusa i3 MK3S+ 3D Printer</td>
			<td>Prusa Research</td>
			<td>PRI-MK3S-KIT-ORG-PEI</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>Oxygen, Compressed</td>
			<td>Linde Canada Inc.</td>
			<td></td>
			<td>Oxygen</td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 30 G x 1/2 (0.3 mm x 13 mm)</td>
			<td>Becton, Dickinson and Company</td>
			<td>305106</td>
			<td>30 G needle</td>
		</tr>
		<tr>
			<td>Pyrex 5340-2L 5340 Filtering Flasks, 2000 mL</td>
			<td>Cole-Parmer Canada Company</td>
			<td>5340-2L</td>
			<td>Vacuum flask</td>
		</tr>
		<tr>
			<td>Rhodamine 6 G</td>
			<td>Sigma-Aldrich</td>
			<td>252433</td>
			<td>Rhodamine 6G</td>
		</tr>
		<tr>
			<td>Secure Soft Cloth Medical Tape - 3&#34;</td>
			<td>Primed</td>
			<td>PM5-630709</td>
			<td>Cloth tape</td>
		</tr>
		<tr>
			<td>Silastic Medical Grade Tubing .040 in. ID x .085 in. OD</td>
			<td>Dow Corning</td>
			<td>602-205</td>
			<td>1.0 mm I.D. polyethylene tubing</td>
		</tr>
		<tr>
			<td>Somnosuite Low-Flow Anesthesia System</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-01, SS-04-module</td>
			<td>Small rodent ventilator, Low-flow anesthesia system, Heating pad, Rectal temperature probe, Pulse oximeter</td>
		</tr>
		<tr>
			<td>Tissue Forceps, 12.5cm long, Curved, 1 x 2 Teeth</td>
			<td>World Precision Instruments</td>
			<td>501216</td>
			<td>Toothed forceps</td>
		</tr>
		<tr>
			<td>Transpore Medical Tape, 1527-1, 1 in x 10 yd (2.5 cm x 9.1 m)</td>
			<td>3M</td>
			<td>7000002795</td>
			<td>Medical tape</td>
		</tr>
		<tr>
			<td>Tubing,Clear,3/8 in Inside Dia.</td>
			<td>Grainger Canada</td>
			<td>USSZUSA-HT3314</td>
			<td>1.0 cm I.D. polyethylene tubing</td>
		</tr>
		<tr>
			<td>Whatman 6720-5002 50 mm In-Line Filters, PTFE, 0.2 &#181;m</td>
			<td>Cole-Parmer Canada Company</td>
			<td>6720-5002</td>
			<td>Inline 0.2&#181;m filter</td>
		</tr>
	</tbody>
</table>

intravital widefield fluorescence microscopy of pulmonary microcirculation in experimental acute lung injury using a vacuum-stabilized imaging system

白细胞-内皮相互作用的活体成像为活体动物免疫介导的疾病提供了有价值的见解。急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）和其他体内 呼吸系统疾病 的研究由于肺部的可及性和固有的运动伪影有限而很困难。尽管如此，已经制定了各种方法来克服这些挑战。该协议描述了一种用于玻璃体内荧光显微镜的方法，用于在ALI的实验模型中研究肺微循环中的实时白细胞 - 内皮相互作用。使用 体内 肺成像系统和3D打印的活体内显微镜平台来保护麻醉小鼠并稳定肺部，同时最大限度地减少混杂的肺损伤。制备后，使用宽视场荧光显微镜研究白细胞粘附，白细胞滚动和毛细管功能。虽然这里介绍的方案侧重于炎症性肺病急性模型中的成像，但它也可能适用于研究肺部的其他病理和生理过程。

为了说明通过该方案可以获得的结果，在成像前6小时使用鼻内细菌脂多糖（LPS）滴注模型诱导急性肺损伤（ALI）。简而言之，用异氟醚麻醉小鼠（n = 3），并将来自铜 绿假单胞菌 在无菌盐水（10mg / mL）中的LPS小液滴以5mg / kg的剂量移液到左肾上腺素中。将其与幼稚小鼠（n = 3;无鼻内给药）进行比较。
在影像学检查后，成功的手术准备可以通过几个因素来识别。肺应相?...

Watch this Scientific Journal Video about Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System at JoVE.

Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System

活体荧光显微镜可用于实时研究白细胞 - 内皮相互作用和毛细血管灌注。该协议描述了使用真空稳定的肺成像系统在肺微循环中对这些参数进行成像和量化的方法。

使用真空稳定成像系统的实验性急性肺损伤中肺微循环的活体内宽视场荧光显微镜

Intravital imaging of leukocyte-endothelial interactions offers valuable insights into immune-mediated disease in live animals. The study of acute lung ...

该协议详细描述了手术制备和使用真空稳定系统对体内肺白细胞粘附和毛细血管灌注进行成像。这种技术的主要优点是，它允许对完整的镜像肺进行稳定的高分辨率活体内成像，同时尽量减少物理创伤。首先，通过在外圈顶部施用一层薄薄的真空润滑脂来准备成像窗口，同时避免污染真空通道。将干净的8毫米玻璃盖玻片放在窗户上，然后轻轻按下以形成密封。将成像窗口连接到配有数字压力表的真空泵，该真空泵能够提供50至60毫米的水银恒定抽吸。将一段长度的光纤电缆穿过20号气管插管，并将尖端浸入盐酸利多卡因中。麻醉后，插入改良的耳镜，使上门牙适合窥器的间隙。调整范围和舌头位置，直到会厌和声带清晰可见。将光纤电缆穿过窥器中的间隙，用小的圆周运动将电缆穿过声带之间，然后用光纤电缆作为引导，轻轻地将插管滑入气管中，使鼠标插管。用1.5%异氟醚开始通气后，通过测试踏板反射来确认麻醉深度。用医用胶带将套管固定在鼻子上。使用标签胶带将右前爪固定在九点钟位置的加热垫上，并将左后爪向尾部伸出，固定在六点钟位置。使用布带，将左前爪轻轻拉伸到12点钟位置，并将胶带的另一端固定到活体内显微镜平台的顶部，然后应用直肠温度探头和爪子脉搏血氧仪在整个实验过程中监测生命体征。为了准备手术，用70%酒精擦拭对胸部和腹部进行消毒，并涂上一层轻薄的矿物油来蘸湿小鼠左侧的头发。在肋骨笼底部附近做一个小切口，露出下面的肌肉层，然后延伸初始切口，用钝性解剖暴露肋骨。使用止血钳，抓住解剖的上皮和脂肪组织，并清除手术区域。为了荧光标记白细胞和血流，通过尾静脉每公斤推注罗丹明6G和FITC白蛋白2.5毫升。使用齿形镊子，在末端吸气时立即抓住低于肺底位置的肋骨。稍微缩回镊子以将肋骨从肺部拉开，然后切开肋骨以诱发气胸。在两个方向上横向扩展切口，注意不要接触肺部。用钝镊子抓住下一个最高的肋骨，并稍微缩回，让肺从胸壁上脱落。如果肺没有脱离，将胸壁轻轻按压在肺上，使肺粘附在下面的胸膜上，从而更容易脱落。继续原来的切口，最终直到胸骨，颅骨直到肺的顶端暴露出来。使用涂抹棉花和纱布来减轻出现的任何出血。抬起肋骨笼，露出胸腔背侧的肋间血管，注意不要损伤肺，烧灼脊柱附近最下肋间血管，然后切开相邻的肋骨。颅骨移动，最终使用重复的烧灼和切割模式来切除大约一乘二厘米的胸腔部分。为了准备成像，将活体显微镜平台转移到显微镜载物台，并将成像窗口直接放置在暴露的肺部上方。使用微机械手小心地降低成像窗口，直到它粘附并稳定肺表面。要可视化肺部微循环，请使用装有20倍物镜和标准FITC和罗丹明滤光片组的宽视场荧光显微镜。使用黑白CCD相机以最佳对比度录制视频。使用FITC过滤器组，根据血流的收敛模式识别肺部场所，并在聚焦场地的情况下录制30秒的视频，然后使用罗丹明过滤器集在同一视野中重复录制以可视化白细胞。要定位肺动脉，请使用FITC过滤器组来识别具有不同血流模式的血管并录制30秒的视频。使用罗丹明过滤器组在同一视野中重复记录以显示白细胞。要定位感兴趣的毛细血管区域，请使用FITC过滤器集识别未与较大血管相交的肺泡和毛细血管区域，并录制30秒的视频。使用罗丹明过滤器组在同一视野中重复记录以显示白细胞。该视频描述了绿色箭头所示的肺部腔内血流的FITC成像。罗丹明成像允许在同一场所可视化白细胞，其中两个粘附的白细胞由红色箭头指示。这些方法还用于可视化相同的现象以及感兴趣的肺动脉和毛细血管区域。为了证明微循环参数的定量，用鼻内LPS治疗小鼠以诱导肺部炎症。这里显示了肺部腔室中的白细胞运输，其中轮廓区域代表幼稚和LPS治疗小鼠中分析的内皮区域。LPS处理的小鼠白细胞粘附和滚动增加。这里显示了肺动脉中的白细胞运输，其轮廓区域代表了在幼稚和LPS治疗的小鼠中分析的内皮区域。LPS处理的小鼠白细胞粘附性较高。该图显示了幼稚和LPS治疗小鼠肺毛细血管内的白细胞粘附。LPS治疗小鼠的白细胞粘附率高于幼稚小鼠。该图显示了幼稚和LPS治疗小鼠肺毛细血管的灌注。LPS治疗小鼠的功能毛细血管密度比幼稚小鼠低。该协议有很多具有挑战性的方面。优化鼠标在平台上的位置是一个简单的步骤，可以使手术和成像步骤更具可重复性。该程序适用于广泛的研究参数和显微镜方法。因此，它应该促进健康和疾病状态下肺部微循环的进一步研究。

Research

JoVE Journal

Immunology and Infection

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

科研

免疫与感染

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Pseudomonas aeruginosa Induced Lung Injury Model

Pseudomonas aeruginosa Induced Lung Injury Model

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Influenza Virus Propagation in Embryonated Chicken Eggs

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...