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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#34;K14-H2B-mCherry&#34;</td>
		</tr>
		<tr>
			<td>Mouse: pTRE-H2B-GFP: STOCK 
			Tg(tetO-HIST1H2BJ/GFP)47Efu/J</td>
			<td>The Jackson Laboratory</td>
			<td>RRID: IMSR_JAX:005104</td>
			<td>&#160;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&#160;</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

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 Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

从长期来看，高解析度激光共聚焦时间流逝成像<em&gt;拟南芥子叶</em&gt;表皮萌发过程中

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

LarvaSPA，一种用于长期延时成像的安装果蝇幼虫的方法

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

急性小鼠胰腺组织切片钙动力学的共聚焦激光扫描显微镜

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

通过共聚焦显微镜成像分析进行细胞外囊泡摄取测定

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

在固体培养基上长期培养单个 秀丽隐杆线虫 ，用于纵向荧光监测和厌恶干预

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

多孔设备固体培养基上分离秀 丽隐杆线虫 的长期培养和监测

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

Methods Article

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