Name: a flexible chamber for time-lapse live-cell imaging with stimulated raman scattering microscopy
Uploaded: 2022-08-31T15:00:00
Description: Watch this Scientific Journal Video about A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy at JoVE.com

我们要感谢宾汉姆顿大学2019年本科生高级设计团队（Suk Chul Yoon，Ian Foxton，Louis Mazza和James Walsh）对显微镜外壳盒的设计，制造和测试。我们感谢宾汉姆顿大学的Scott Hancock，Olga Petrova和Fabiola Moreno Olivas的有益讨论。这项研究得到了美国国立卫生研究院的支持，奖励号为R15GM140444。

1.构建显微镜环境外壳
注意：这个大型显微镜环境外壳用于控制显微镜主体的温度和要稳定在37°C的成像环境（图1A）。
<ol><li>使用光学台上的记号笔标记SRS显微镜架和电动载物台的支脚位置。在显微镜的振镜扫描仪前面安装两个虹膜，并进行调整以使泵浦和斯托克斯激光束穿过虹膜光阑的中心。</li>
	<li>从光学台上取下显微镜架和载物台。</li>
	<li>将?...

<ol>
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延时活细胞SRS显微镜是一种以无标记方式进行分子追踪的替代成像技术。与荧光标记相比，SRS成像没有光漂白，可以对分子进行长期监测。然而，迄今为止，正立式SRS显微镜上的活细胞成像系统尚未商业化。在这项工作中，开发了一种具有稳定隔热显微镜外壳盒和柔性内部软室的活细胞成像系统，以实现透射SRS延时成像。在此设置中，大型外壳盒将温度稳定性保持在37°C，而内部软室提供加湿空?...

光学显微镜用于观察样品的微观结构。与其他技术相比，光学成像速度快、侵入性更小、破坏性更小1。使用光学显微镜进行活细胞成像是为了长时间捕获培养活细胞的动态2。不同类型的光学对比度提供有关生物样品的不同信息。例如，光学相位显微镜显示了样品3中折射率的细微差异。荧光显微镜广泛用于对特定的生物分子或细胞器进行成像。然而，当进行多色成像时，荧光的宽带激发和发射光谱通常会导致光谱重叠4。荧光分子对光敏感，可以在长期、周期性的光照后漂白。此外，荧光标记可以改变细胞中分子的生物分布5。SRS显微镜是一种无标记的化学成像技术6。SRS的对比度依赖于特定化学键的振动跃迁。化学键的振动频率通常表现出较窄的光谱带宽，因此可以对同一样品中的多个拉曼波段进行成像7。SRS显微镜是一种独特的活细胞成像工具，以无标记的方式提供多种化学对比8。
虽然未染色细胞的SRS成像已被用于许多研究，但活细胞的长期延时SRS成像尚未被广泛采用。一个原因是商业开放室不能直接用于SRS成像，因为它们的厚度为9，10，11，12。这些带玻璃盖的腔室大多设计用于明场或荧光成像，使用具有反向检测方案的单个高数值孔径物镜。然而，SRS成像更喜欢同时使用高数值孔径物镜和高数值孔径聚光镜的透射检测，这在物镜和聚光镜之间只留下非常短的间隙（通常为几毫米）。为了克服这个问题，我们设计了一个使用软材料的柔性腔室，以便使用正置显微镜框架对活细胞进行延时SRS成像。在这种设计中，水浸物镜被封闭在软室中，可以在三维空间中自由移动，以进行聚焦和成像。
培养大多数哺乳动物细胞的最佳温度为37°C，而室温始终比这低10°。高于或低于37°C的温度对细胞生长速率有显着影响13。因此，活细胞成像系统需要对细胞培养环境进行温度控制。众所周知，温度不稳定会导致长期成像过程中的散焦问题14。为了实现稳定的37°C环境，我们建造了一个大型封闭室来覆盖整个显微镜框架，包括显微镜下方的隔热层（图1）。在相当大的温度控制室中，小型柔性室有助于通过补充 5% CO 2 的调节气流准确保持生理湿度和 pH 值（图 2）。测量腔室的温度和湿度，以确认双腔室设计为长期周期性SRS成像下的细胞生长提供了最佳的细胞培养条件（图3）。然后，我们展示了该系统在延时成像和跟踪SKOV3癌细胞中的脂滴（LD）中的应用（图4，图5和图6）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A lab-built SRS microscope</td>
			<td></td>
			<td></td>
			<td>https://rdcu.be/cP6ve</td>
		</tr>
		<tr>
			<td>HF2LI 50 MHz lock-in amplifer</td>
			<td>Zurich Instruments</td>
			<td>HF2LI</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris diaphragm</td>
			<td>Thorlabs Inc</td>
			<td>SM1D12</td>
			<td></td>
		</tr>
		<tr>
			<td>Kinematic mirror mount</td>
			<td>Thorlabs Inc</td>
			<td>KM100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope frame</td>
			<td>Nikon Inc</td>
			<td>FN-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Motorized microscopy stage</td>
			<td>Prior Scientific</td>
			<td>Z-Deck</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil-immersion condenser (C-AA Achromat/Aplanat, NA 1.4)</td>
			<td>Nikon Inc</td>
			<td>MBL71405</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-immersion objective (CFI75 Apo 25XC W 1300)</td>
			<td>Nikon Inc</td>
			<td>MRD77225</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and parts for the microscope enclosure (31&#39;&#39; x 29&#39;&#39; x 28&#39;&#39; L x W x H)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Airtherm heater module</td>
			<td>World Precision Instruments (WPI)</td>
			<td>AIRTHERM-SAT-1W</td>
			<td></td>
		</tr>
		<tr>
			<td>Airtherm heater controller, CO2 and humidity monitor</td>
			<td>World Precision Instruments (WPI)</td>
			<td>AIRTHERM-SMT-1W</td>
			<td></td>
		</tr>
		<tr>
			<td>Air/CO2 mixer module</td>
			<td>World Precision Instruments (WPI)</td>
			<td>ECU-HOC-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Flexible duct hose (2-1/2&#39;&#39; ID, 2-3/4&#39;&#39; OD)</td>
			<td>McMaster-Carr</td>
			<td>56675K71</td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature glass-mica ceramic, easy-to-machine (6&#39;&#39; x 6&#39;&#39;, 1/4&#39;&#39; thickness)</td>
			<td>McMaster-Carr</td>
			<td>8489K62</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate sheets (thickness 0.25&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>8574K286</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber sheets (36&#39;&#39; x 36&#39;&#39;, thickness 1/8&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>5827T43</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and parts for the Flexible chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>McMaster-Carr</td>
			<td>31745K11</td>
			<td></td>
		</tr>
		<tr>
			<td>High-purity inline filter, 1/4 NPT</td>
			<td>McMaster-Carr</td>
			<td>6645T18</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw (cutting diameter 1-7/8 inch)</td>
			<td>McMaster-Carr</td>
			<td>4066A34</td>
			<td></td>
		</tr>
		<tr>
			<td>Hole saw (cutting diameter 50 mm)</td>
			<td>McMaster-Carr</td>
			<td>4556A19</td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature silicone rubber tubing, soft, 2 mm ID, 5 mm OD</td>
			<td>McMaster-Carr</td>
			<td>5054K313</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline filter (1/4 NPT, 40 micron)</td>
			<td>McMaster-Carr</td>
			<td>98385K843</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum round tube (1/8&#39;&#39; wall thickness, 4&#39;&#39; OD)</td>
			<td>McMaster-Carr</td>
			<td>9056K42</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum round tube (3/4&#39;&#39; wall thickness, 3-3/4&#39;&#39; OD)</td>
			<td>McMaster-Carr</td>
			<td>9056K47</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum bar (12&#39;&#39; x 12&#39;&#39;, thickness 1/4&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>8975K142</td>
			<td></td>
		</tr>
		<tr>
			<td>Multipurpose 6061 Aluminum bar (8&#39;&#39; x 8&#39;&#39;, thickness 3/8&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>9246K21</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective nosepiece (single)</td>
			<td>Nikon Inc</td>
			<td>FN-MN-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample holder (modified)</td>
			<td>Prior Scientific</td>
			<td>HZ202</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-thin natural rubber film (thickness 0.01&#39;&#39;)</td>
			<td>McMaster-Carr</td>
			<td>8611K13</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum-sealable glass jar</td>
			<td>McMaster-Carr</td>
			<td>3231T44</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ (Fiji)</td>
			<td></td>
			<td>imagej.net</td>
			<td></td>
		</tr>
		<tr>
			<td>ScanImage</td>
			<td>Vidrio Technologies, LLC</td>
			<td></td>
			<td>SRS imaging software</td>
		</tr>
		<tr>
			<td>Materials for live-cell imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass bottom sterile culture dishes (Dia.x H, 50 x 7 mm)</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>70674-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM cell culture medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoSensor fluorescent dye DND-189</td>
			<td>ThermoFisher Scientific</td>
			<td>L7535 (Invitrogen)</td>
			<td></td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>MilliporeSigma</td>
			<td>364525</td>
			<td></td>
		</tr>
		<tr>
			<td>SKOV3 cell line</td>
			<td>ATCC</td>
			<td>HTB-77</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flexible chamber for time-lapse live-cell imaging with stimulated raman scattering microscopy

受激拉曼散射（SRS）显微镜是一种无标记化学成像技术。SRS活细胞成像已被证明可用于许多生物和生物医学应用。然而，活细胞的长期延时SRS成像尚未被广泛采用。SRS显微镜通常使用高数值孔径（NA）水浸物镜和高数值孔径油浸式冷凝器来实现高分辨率成像。在这种情况下，物镜和聚光镜之间的间隙只有几毫米。因此，大多数商用载物台环境箱不能用于SRS成像，因为它们的厚度很大，带有刚性玻璃盖。本文描述了一种柔性腔室的设计和制造，该腔室可用于延时活细胞成像，并在正置显微镜架上进行透射SRS信号检测。腔室的柔韧性是通过使用柔软的材料 - 薄的天然橡胶膜来实现的。新的外壳和腔室设计可以轻松添加到现有的SRS成像设置中。测试和初步结果表明，灵活的腔室系统能够对活细胞进行稳定、长期、延时SRS成像，可用于未来的各种生物成像应用。

我们制造并组装了用于延时SRS成像的柔性腔室系统（图1 和 图2），然后评估了系统的性能。显微镜环境外壳内的温度在1小时内达到预期的37°C，这没有显着影响室温（图3A）。柔性室中的温度在1.5 h内达到37 °C，并且在37 °C稳定保持至少24 h（图3B）。柔性室中的相对湿度可在1小时内达到85%，然后保持至少24?...

Watch this Scientific Journal Video about A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy at JoVE.com

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

我们报告了一种台顶柔性环境室，用于使用具有透射信号检测的正置受激拉曼散射显微镜对活细胞进行延时成像。脂质液滴在用油酸处理的SKOV3细胞中成像长达24小时，时间间隔为3分钟。

用于使用受激拉曼散射显微镜进行延时活细胞成像的柔性腔室

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

灵活的环境室技术可实现延时摄影，如SRS成像，并在手术显微镜架上进行透射信号检测。SRS显微镜使用高数值孔径物镜和高端聚光镜，仅留下几毫米的间隙，而传统腔室无法容纳。灵活的腔室解决了这个问题。使用SRS显微镜的研究人员可以很容易地采用这种技术。柔性室也可用于解剖或无菌显微镜，以将标本保持在最佳生理条件下。首先，使用光学台上的记号笔标记SRS显微镜架和电动载物台的支脚位置。从光学台上取下显微镜架和载物台后，将硅橡胶片放在工作台上。用刀沿着标记切割硅橡胶。取出小橡胶片，然后将方形云母陶瓷片放入相同的位置。然后将显微镜架和载物台移回光学台，并沿着标记线小心地将脚对准云母陶瓷片。重新对准SRS光路后，使用五块大型聚碳酸酯板将环境外壳组装在隔热基础的顶部，以覆盖整个显微镜框架。使用铝箔胶带密封盒子的边缘和接口。将柔性管道软管连接到外壳盒的入口和出口，以允许由加热器模块泵送和控制的循环、温暖的气流。使用三个固定螺钉将机加工的空心圆柱形铝片1安装到物镜的鼻片上。接下来，使用四个螺钉将机加工的空心圆柱形铝片 2 安装到样品架上。将带有铝片 2 的样品架安装到电动载物台上，并使用螺钉安装。将天然橡胶膜套放在两个机加工铝片之间，并在两端使用橡皮筋安装。使用适当的管道和连接器将压缩的 CO2 气瓶连接到气体混配器模块。将 CO2 输入压力设置为 20 至 25 psi。使用内置的 CO2 传感器和控制器，确保空气混合器模块可以调节 5% 的 CO2 并将其混合到气流中。使用在线过滤器清理气流。使用适当的管道和连接器，将混合空气引导到放置在加热板上的预灭菌水瓶，然后将加湿的空气引导到柔性腔室。将加热板设置为 37 摄氏度。在温水中冒泡气流以增加气流的湿度。通过卸下螺钉将机器铝片 2 与样品架断开连接。用70%乙醇擦拭柔性室的所有部分，包括鼻梁、样品架和水浸物镜。使用放置在机柜中的紫外线灯对整个机柜系统进行 20 到 30 分钟的净化。然后装入细胞培养皿。取下盘子的盖子并使用夹子将其固定。将物镜降低到细胞培养基中进行粗聚焦。降低铝片2以封闭柔性室，并使用螺钉将其安装到样品架上。然后插入传感器。将空气供应设置为5%CO 2和19%O 2，用于正常细胞培养，气流速率为每分钟200立方厘米，温度为37摄氏度。将激光波长调谐到 805 纳米，以瞄准 2， 854 厘米的逆拉曼位移，这归因于 CH2 化学键的振动。使用低激光功率来减少对细胞的光损伤。使用软件主控制面板上的对焦按钮调整和聚焦物镜，以实现细胞的良好SRS成像。要执行快速对焦，请在配置面板上将像素数设置为 512 x 512 像素，像素停留时间为 4.8 微秒。实现良好对焦后，将成像分辨率设置为 2048 x 2048 像素，以获得 175 微米的平方视场，以采集高质量图像。检查主控制面板上的保存功能，并在通道面板上检查SRS通道。在主控制通道上将两帧之间的间隔时间设置为 180 秒。将采集编号设置为 480，以便在 24 小时内进行延时成像。使用主控制面板上的循环功能开始自动成像采集。评估了用于延时SRS成像的柔性腔室系统的性能。显微镜环境外壳内的温度达到预期的37摄氏度，对室温没有显着影响。柔性腔室内的温度在1.5小时内达到37摄氏度，并稳定保持至少24小时。柔性腔室的相对湿度在一小时内达到85%，并且可以保持至少24小时。活的SKOV3细胞的延时SRS成像显示细胞内脂滴的快速和活跃运动，时间分辨率为三分钟。在24小时的成像结束时，细胞仍然显示出正常的形态和密度，表明细胞是健康的。接下来，对用油酸处理的SKOV3细胞进行成像，并在10小时内跟踪脂滴积累。使用脂滴与细胞体面积比和脂滴的总SRS强度来量化脂滴量。结果表明，脂滴的量在10小时内持续增加。还展示了脂质液滴的同时正向SRS成像和用荧光染料DND-189标记的溶酶体的后向两个光子荧光成像。观察到脂滴和溶酶体的共定位程度非常低。尝试此协议时，请记住，焦点漂移是活细胞成像中的常见问题。在大约两个小时的延时成像后，可能需要重新聚焦。延时SRS成像在其开发后已被用于各种研究，以跟踪活细胞中无标记分子或带有拉曼文本的标记分子。

Research

JoVE Journal

Bioengineering

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

刀口扫描显微镜的样品制备，图像和分析协议

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

科研

生物工程

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

时间推移荧光成像拟南芥根系生长的根环境的快速操作使用RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

样品漂移校正继4D共焦时间推移成像

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

从快速荧光成像在分子扩散法对活细胞的膜在商业显微镜

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

荧光淬一个脂质体包封的近红外荧光团的作为一个工具<em&gt;在体内</em&gt;光学成像

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Live Cell Imaging during Mechanical Stretch

在机械拉伸活细胞成像

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

活细胞层的快速消减带图案微流控探头

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

利用先进的纺额圆盘全内反射荧光显微镜观察细胞粘附形成

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

使用延时显微镜连续测量 大肠杆菌 中的生物噪声

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...