我们感谢 Valentina Greco 的 K14-mCherry-H2B 小鼠。我们感谢埃默里大学物理系机械车间生产玻璃盖玻片盘。这项工作由美国退伍军人事务部 BLRD 服务向 LS 颁发的职业发展奖 #IK2 BX005370 资助，向 MJMR 颁发 NIH 奖 RF1-AG079269 和 R56-AG072473，以及向 MJMR 颁发的 I3 Emory SOM/GT 计算和数据分析奖。

这项研究是按照埃默里大学和亚特兰大退伍军人事务医疗中心的动物护理和使用指南进行的，并已获得机构动物护理和使用委员会 （IACUC） 的批准。
1. 在倒置显微镜载物台上安装实时成像插件
<ol><li>使用 .stl 文件（补充文件 1、补充文件 2 和补充文件 3）构建插入物，指定 3D 尺寸和设计（参见图 1 和图 2A）、3D 打印机和聚乳酸 （PLA）。</li>
	<li>小心地将插入物（图2B，C）放入大显微镜载物台凹槽（图2C和图3A）中。使用螺钉通过位于插入物每个角的四个螺丝孔固定插入物（图 2B-C）。 注意：插入物是双向的，可以根据显微镜载物台和麻醉装置的方向旋转 180°。</li>
	<li>将加热板面朝下滑入插入插头（图 2D），使装置位于下部插入槽的顶部，插头端口穿过舞台下方（图 3B）。</li>
	<li>将插入物中的凹槽圆形开口与40倍硅油浸没物镜对齐（图3C），并将硅油涂在物镜的中心。<ol><li>如果成像时间较长（>1 小时），则在整个成像过程中，涂抹大量油，该油将持续存在于盖玻片/物镜界面处。不要让油溢出镜头的边缘。</li>
	</ol></li>
	<li>使用注射器沿带槽的圆形开口涂抹少量真空润滑脂，并将盖玻片放在顶部以密封在插入物上（图3D）。 注意：盖玻片是通过去除35 mm x 10 mm玻璃底细胞培养皿（由机械车间执行）的壁来创建的。</li>
	<li>抬起 40 倍物镜，直到油亲吻玻璃盖玻片底部。</li>
</ol>2. 异氟醚构型和小鼠制备
<ol><li>放置低流量电子汽化器组件（麻醉室，管道，汽化器，木炭罐），以使鼻锥和连接的管道到达插入物（图3A）。 注意：异氟醚是一种吸入麻醉剂，应小心处理，以避免溢出并尽量减少人体接触。</li>
	<li>使用前测量异氟醚瓶的重量并记录。将电子蒸发器的盖子连接到异氟烷瓶上。</li>
	<li>将电源线连接到电子蒸发器。关闭蓝色鼻锥夹并打开白色感应室夹，让气流通过腔室进入木炭罐。从机器左侧取下红色环境空气帽，让空气流通。</li>
	<li>让系统以 200 mL/min（低流量）和 2% 异氟醚预热 5 分钟。 注意： 虽然选择了鼻锥设置，但蓝色鼻锥线应保持关闭状态，白色感应室线应保持打开状态。</li>
	<li>一旦系统正确平衡，吹扫管路以从腔室中除去剩余的异氟烷。</li>
	<li>将鼠标放入感应室并选择高流量。在将动物的身体放在插入物上之前，完成所有小鼠准备工作（即脱毛、局部给药、眼药膏应用等）。使用脚趾捏反射法确认鼠标已完全麻醉。<ol><li>腿部伸展，用指甲用力捏住脚趾，而不会造成物理伤害。如果小鼠对刺激表现出积极反应（即腿部缩回、脚抽搐等），则继续在腔室内施用麻醉剂，直到没有观察到任何反应。一旦适当麻醉，小鼠呼吸频率应减慢至每分钟~55-65次呼吸18。</li>
	</ol></li>
	<li>当鼠标完全麻醉后，再次选择高流量以停止异氟烷输送。在打开之前吹扫腔室并调整夹子（蓝色：打开;白色：关闭）以通过鼻锥输送异氟醚。当鼻锥连接到鼠标时选择低流量以继续异氟醚输送。</li>
	<li>将带有连接的鼻锥的异氟醚管穿过插入物上的角管支架（图3A 和 图5）。</li>
</ol>3. 将鼠标放置在插入物上以进行活体成像
<ol><li>将加热板插入控制器（包含连接的肛门探头），打开电源，让加热板达到36°C（图4A）。</li>
	<li>将麻醉的小鼠从诱导室中取出并放在加热板上（图4B 和 图5A）。快速执行从腔室到插入物的转移，以最大限度地减少小鼠在没有吸入麻醉的情况下活动的时间。</li>
	<li>将鼻锥固定在鼠标上（图 4B、C 和 图 5）。如有必要，使用胶带进一步固定鼻锥的角度和位置。</li>
	<li>插入肛门探头并调节控制器温度，直到小鼠体温稳定在~36°C（图4A，B）。鼠标正确放置后，如果需要，使用塑料保鲜膜吸收热量，以进一步支持适当的体温（图4C）。</li>
	<li>放置鼠标，使头部与盖玻片对齐，并使用金属耳夹（图5A，B）或胶带（图5C）将耳朵固定在玻璃盖玻片的中心。 注意： 可以通过松开将夹子固定到插件上的螺钉来调节金属耳夹在耳朵上的压力。可以添加金属弹簧以增加夹子拧紧的灵活性。<ol><li>要更改耳夹位置，请用 2.5 毫米内六角扳手拧下螺栓并转移到辅助部位（图 2B、C）。重新组装耳夹时，将耳夹靠在插入物上，然后将垫圈放在顶部。使用螺栓用力牢固地固定夹子，以旋转夹子。</li>
	</ol></li>
	<li>调整物镜z定位，直到细胞位于焦点位置（图6A，D）。根据实验目标设置z-stack和延时参数，开始图像采集。 注意：如果实现正确的设置，则可以对小鼠耳朵进行至少连续3小时的成像（图6A'，D'）。<ol><li>设置z堆栈边界后，调整激光功率和增益，以确保没有z平面过饱和，以最大程度地减少光漂白。使用 z 堆栈的总厚度、步长数（建议使用奈奎斯特采样）和时间间隔确定延时参数。</li>
		<li>使用多种因素确定成像参数（时间间隔、总成像时间等），包括麻醉下的动物活力、激光诱导的光漂白/光毒性以及活体成像的目标（即细胞分裂动力学、细胞间相互作用等）。</li>
	</ol></li>
</ol>4. 成像终止
<ol><li>打开水循环加热垫，设置为 连续循环 和 35 °C 至少 30 分钟，然后终止图像采集。</li>
	<li>成像完成后，在电子蒸发器上选择 低流量 以停止异氟醚输送并将鼠标移动到加热垫上，直到可以走动。</li>
	<li>唤醒后，将鼠标移回转移容器，同时继续保持在加热垫上，直到完全可以走动。在加热垫上持续监视鼠标，直到它响应。</li>
	<li>在电子蒸发器上，单击 “选择菜单”>“麻醉控制”>“清空”。从系统中取出异氟醚瓶，然后将制造商的瓶盖放回瓶子上。</li>
	<li>测量异氟烷瓶和木炭罐的重量，并将重量输入日志。一旦木炭罐的重量超过基线测量值 50 克，请处理罐并更换新装置。将异氟醚放回密码箱。</li>
	<li>取下盖玻片，用镜头纸和镜头溶液擦拭干净。妥善存放以避免划伤以备重复使用。</li>
	<li>从插入支架上取下麻醉管，然后从显微镜载物台上拧下插入物。</li>
</ol>

使用倒置显微镜进行活体成像以跟踪皮肤细胞动力学

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作者没有要披露的利益冲突。

在这项研究中，我们提出了一种新工具，该工具有助于在倒置共聚焦显微镜上对完整的小鼠皮肤上皮进行稳定、长期的活体成像。本发明由PLA制成，PLA是最常见和最便宜的3D打印材料;该插件的所有内部 3D 打印成本为 < 美元。两个独立的插入件（图 1、补充文件 1 和补充文件 2）可以使用固定螺钉轻松组装（参见 材料表）。值得注意的是?...

活体显微镜是一种强大的工具，可以监测细胞在不受干扰的体内环境中的行为。这种独特的方法为复杂哺乳动物器官系统的内部运作提供了关键的见解，包括肺1、大脑2、肝脏3、乳腺4、肠5 和皮肤6。此外，这种方法还揭示了肿瘤发展过程中的细胞行为改变7、伤口愈合 8,9、炎症10 和其他不同的原位病理。在这项研究中，我们专注于增强活体显微镜对完整小鼠皮肤中实时上皮和基质动力学成像的可及性。由于哺乳动物皮肤具有显着的再生和致瘤能力，了解哺乳动物皮肤中的细胞行为具有很高的临床意义。
小鼠的活体成像主要使用正置多光子显微镜进行，因为它们能够在组织深度 >100 μm11,12 提供高分辨率成像。然而，这些仪器缺乏倒置共聚焦显微镜的主力多功能性和更普遍的可及性，后者更人性化、更具成本效益，能够对培养的细胞进行成像，在图像采集过程中不需要完全黑暗，并且通常更安全，以及其他显着优势13,14.在这项研究中，我们提出了一种新工具，通过将这种方法应用于倒置共聚焦显微镜，显着提高了活体成像的可及性。
在这里，我们展示了一种 3D 打印的定制载物台插入设计，该设计结合了几个关键功能，以促进在倒置共聚焦显微镜上对小鼠耳部皮肤进行稳定、长期的活体成像（图 1、图 2、图 3、图 4 和图 5）。这些特殊功能包括一个偏移物镜孔，允许成年小鼠的整个身体在成像过程中完全平放。这最大限度地减少了小鼠身体运动对成像的振动干扰，并且无需使用氯胺酮和甲苯噻嗪来抑制呼吸，这种做法通常与活体成像相结合6。此外，插入物上的角支架可正确定位异氟醚鼻锥以与鼠标正面对齐，金属耳夹将鼠标耳朵固定在定制的盖玻片上，可选的可拆卸闭环生物反馈加热板齐平放置在插入物内，以在长时间成像期间支持小鼠体温。定制的盖玻片盘为小鼠头部和耳朵平放提供了必要的平坦表面，它是在机械车间通过去除含有通用盖玻片的细胞培养皿的壁而产生的。将 40 倍硅油浸没透镜（1.25 数值孔径 [N.A.]，0.3 mm 工作距离）与盖玻片和定制载物台插件结合使用，可提供深入耳真皮> 50 μm 的高分辨率图像。
为了测试这种新的倒置显微镜载物台插入物的功能，我们在活的转基因K14-H2B-mCherry15成年小鼠的耳朵中捕获了跨越所有表皮上皮层的z-stacks（该小鼠系中的上皮细胞核含有红色荧光标记）（图6A-A'）。我们还在活转基因 Pdgfra-rtTA16 的耳朵中捕获了 3 小时内跨越皮肤真皮内多个成纤维细胞层的 z 堆栈;pTRE-H 2 B - GFP17成年小鼠（该小鼠系中的成纤维细胞核在多西环素诱导后含有绿色荧光标记）（图6B-D'）。我们的高分辨率数据显示，由于 x、y 和 z 平面没有漂移，因此具有一致的稳定性，从而证明了这种新型活体成像工具在倒置显微镜上使用的有效性。重要的是，这种3D打印载物台插件的尺寸可以调整，如补充文件1、补充文件2和补充文件3中所述，以适应任何倒置显微镜，并且物镜开口的位置可以移动到插件内的其他位置，以更好地适应感兴趣的特定组织和/或动物模型的成像。因此，这项发明可以使单个实验室或具有核心设施共聚焦访问能力的研究人员能够根据其独特的活体成像需求调整该工具，从而简化对各种体内细胞生物学的评估。

<table><tbody><tr><td>3D Printer</td><td>Qudi Tech</td><td>i-Fast</td><td>3D prints using PLA material</td></tr><tr><td>40x 1.25NA silicone objective lens</td><td>Nikon</td><td></td><td></td></tr><tr><td>AxR Laser Scanning Confocal Microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>Cotton Tipped Swab</td><td>VWR</td><td>76337-046</td><td>Cream/ointment application</td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891</td><td>Induces GFP labeling of fibroblast nuclei in Pdgfra-rtTA; pTRE-H2B-GFP mice</td></tr><tr><td>Flathead Screwdriver (2.5 mm)</td><td></td><td></td><td>Affiix insert to microscope stage</td></tr><tr><td>Flathead Screws x 4 (#6-32)</td><td>Nikon</td><td></td><td>Screw insert into microscope stage</td></tr><tr><td>Glass Bottom Culture Dish</td><td>chemglass Life Sciences</td><td>CLS-1811-002</td><td>Modified by removing walls of dish for use as coverslip disk compatible with live insert; 35 mm wide disk contains 20 mm wide glass coverslip; dish walls were removed by machine shop</td></tr><tr><td>Heat Plate controller</td><td>Physitemp</td><td>TCAT-2LV</td><td>Animal Temperature Controller - Low Voltage; anal prob attachment for mouse body temperature monitoring</td></tr><tr><td>Hex Wrench (1.5 mm)</td><td></td><td></td><td>For M3 setscrew adjustments</td></tr><tr><td>Hex Wrench (2.5 mm)</td><td></td><td></td><td>Adjust tension on metal ear clip</td></tr><tr><td>Intravital Imaging Insert</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Med-Vet International</td><td>HPA030782-100uL</td><td>Mouse anesthesia</td></tr><tr><td>Labeling Tape (or Scotch Tape)</td><td>VWR</td><td>10127-458</td><td>Alternative to metal ear clip to immobilize ear to coverslip</td></tr><tr><td>Metal fastener</td><td></td><td></td><td>used as ear clip</td></tr><tr><td>Mouse: C57BL/6-Pdgfraem1(rtTA)Xsun/J</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:034459</td><td>Fibrroblast-specific promoter driving doxycycline-inducible rtTA expression</td></tr><tr><td>Mouse: K14-H2BPAmCherry</td><td>Courtesy of Dr. Valentina Greco at Yale University</td><td></td><td>Labels epidermal epithelial cell nuclei with mCherry; referred to in text as &quot;K14-H2B-mCherry&quot;</td></tr><tr><td>Mouse: pTRE-H2B-GFP: STOCK&lt;br/&gt; Tg(tetO-HIST1H2BJ/GFP)47Efu/J</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:005104</td><td>&amp;nbsp;Labels fibroblast nuclei with GFP when combined with Pdgfra-rtTA and induced with doxycycline</td></tr><tr><td>Multipurpose Sealing Wrap</td><td>Glad</td><td></td><td>Enhance mouse warmth</td></tr><tr><td>Optixcare</td><td>VWR</td><td>MSPP-078932779</td><td>Eye lubricant</td></tr><tr><td>Set screws x 3 (M3; 6 mm)</td><td>Thorlabs</td><td>SS3M6</td><td>Attachment for heatplate module</td></tr><tr><td>Silicone Immersion Oil</td><td></td><td></td><td>Applied to 40x silicone objective</td></tr><tr><td>Small Animal Heating Plate</td><td>Physitemp</td><td>HP-4M</td><td>Provides heat to animal</td></tr><tr><td>Somnoflow Low-Flow Electronic Vaporizer</td><td>Kent Scientific</td><td>SF-01</td><td>Mouse anesthesia</td></tr><tr><td>Vacuum Grease</td><td>Flinn Scientific</td><td>AP1095</td><td>Seals coverslip disk to insert</td></tr><tr><td>Veet</td><td></td><td></td><td>hair removal&amp;nbsp;</td></tr><tr><td>Water circulating heat pad</td><td>Stryker Medical</td><td>TP700</td><td>for mouse revival post-imaging</td></tr></tbody></table>

streamlined intravital imaging approach for long-term monitoring of epithelial tissue dynamics on an inverted confocal microscope

了解正常和异常的 体内 细胞行为对于开发临床干预措施以阻止疾病的发生和进展是必要的。因此，优化成像方法至关重要，以促进原 位观察细胞动力学，其中组织结构和组成不受干扰。表皮是人体最外层的屏障，也是最普遍的人类癌症（即皮肤癌）的来源。皮肤组织的可及性为使用非侵入性活体显微镜监测完整动物的上皮和真皮细胞行为提供了独特的机会。然而，这种复杂的成像方法主要是使用正置多光子显微镜实现的，这对大多数研究人员来说是一个重要的进入壁垒。本研究提出了一种定制设计的 3D 打印显微镜载物台插件，适用于倒置共聚焦显微镜，简化了活转基因小鼠耳部皮肤的长期活体成像。我们相信，这项多功能发明可以定制以适应所选择的倒置显微镜品牌和型号，并适用于对其他器官系统进行成像，这将通过显着提高活体显微镜的可及性，为广大科学研究界证明是无价的。这种技术进步对于加强我们对正常和疾病背景下活细胞动力学的理解至关重要。

通过在≥1小时的时间过程中获取荧光标记的活耳组织的z堆栈来验证在倒置共聚焦显微镜上正确组装活成像插入物以及转基因小鼠在插入物顶部的适当方向，并且在x轴，y轴和z轴上漂移的证据最小。图像应以一致的间隔捕获（间隔时间将取决于生物学问题、荧光信号的强度等），以便可以随时间跟踪细胞动力学和图像漂移。在整个时间过程中，监测单个 z 平面以确保它们保持焦点，揭示动物运动?...

Watch this Scientific Journal Video about Developing a Tool for Using Inverted Confocal Microscopes for In Vivo Intravital Imaging at JoVE.com

作者聚焦：开发使用倒置共聚焦显微镜进行体内活体成像的工具 

该协议提供了一种新工具，可使用倒置共聚焦显微镜简化活体成像。

简化活体成像方法，用于在倒置共聚焦显微镜上长期监测上皮组织动力学

Understanding normal and aberrant in vivo cell behaviors is necessary to develop clinical interventions to thwart disease initiation and progression. It ...

我们开发了一种工具，使用倒置共聚焦显微镜对活体小鼠进行活体成像。这使我们能够跟踪整个稳态过程中的皮肤细胞动力学，并比较疾病发作和进展期间的细胞行为。多光子显微镜是实现活细胞追踪的主要方法。然而，这些显微镜的缺点包括日常成像多功能性有限、成本高和技术复杂性。我们的新工具支持使用多功能倒置共聚焦显微镜进行活体成像。通过修改设计文件，可以定制载物台插件尺寸，以适应任何品牌的倒置显微镜。物镜孔可以调整大小和重新定位，以适应任何选择的动物模型和组织。

streamlined-intravital-imaging-approach-for-long-term-monitoring

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Streamlined Intravital Imaging Approach for Long-Term Monitoring of Epithelial Tissue Dynamics on an Inverted Confocal Microscope

1.5K 次观看.  Emory University School of Medicine. 我们开发了一种工具，使用倒置共聚焦显微镜对活体小鼠进行活体成像。这使我们能够跟踪整个稳态过程中的皮肤细胞动力学，并比较疾病发作和进展期间的细胞行为。多光子显微镜是实现活细胞追踪的主要方法。然而，这些显微镜的缺点包括日常成像多功能性有限、成本高和技术复杂性。我们的新工具支持使用多功能倒置共聚焦显微镜进行活体成像。通过修改设计文?...

视频: 作者聚焦：开发使用倒置共聚焦显微镜进行体内活体成像的工具 

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

科研

生物工程

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

纵向，非侵入性的移植到眼睛的前房<em在体内</em&gt;单细胞分辨率的实时成像

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

医学

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

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 ...

癌症研究

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

神经科学

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

实时多细胞动力学延长时间推移活体成像在肿瘤微环境

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

应用先进的背皮窗室对转基因荧光小鼠肿瘤相关血管的活体显微镜观察

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Laser Ablation and Intravital Microscopy to Study Intestinal Remodeling

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into the cell and tissue scale dynamics that orchestrate intestinal regeneration. Here, we describe an intravital microscopy method that locally induces tissue damage at the single crypt scale and follows the regenerative response of the intestinal epithelium in living mice. Single crypts or larger intestinal fields were ablated by a high-intensity multiphoton infrared laser in a time- and space-controlled manner. Subsequent long-term repetitive intravital imaging enabled the tracking of the damaged areas over time and allowed for the monitoring of crypt dynamics during tissue recovery over a period of multiple weeks. Crypt remodeling events such as crypt fission, fusion, and disappearance were observed in the neighboring tissue upon laser-induced damage. This protocol enables the study of crypt dynamics both in homeostatic and pathophysiological settings, such as aging and tumor initiation.

Here, we present a method to obtain images of the intestine upon laser-induced wounding. By exposing the mouse intestine to a multiphoton laser, the loss of a single or multiple crypt(s) is induced locally. By repeatedly imaging the damaged area over months, the real-time dynamics of intestinal recovery is captured.

激光消融和活体显微镜研究肠道重塑

Investigating intestinal recovery in vivo is an exquisite technical challenge. A lack of longitudinal imaging protocols has prevented deeper insights into ...

简化活体成像方法，用于在倒置共聚焦显微镜上长期监测上皮组织动力学

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