Name: label-free imaging of lipid storage dynamics in caenorhabditis elegans using stimulated raman scattering microscopy
Uploaded: 2021-05-28T14:00:00
Description: Watch this Scientific Journal Video about Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy at JoVE.com

这项工作得到了NIH赠款R01AG045183（M.C.W.）、R01AT009050（M.C.W.）、R01AG062257（M.C.W.） 的支持。 DP1DK113644（M.C.W.），迪姆斯基金会（M.C.W.）的3月，韦尔奇基金会（M.C.W.），以及HHMI调查员（M.C.W.）。我们感谢卡诺哈布迪炎遗传学中心 （CGC） 的 C. elegans 菌株。

1. 刺激拉曼散射显微镜的仪器设置
注：SRS显微镜系统采用皮秒激光器，泵集成光学参数振荡器和共聚焦激光扫描显微镜。振荡器提供两个象形脉冲列车，包括1，064 nm的斯托克斯光束和700-990纳米之间的泵束。两束光束的时空重叠在激光内部实现。内置的光电调制器 （EOM） 专为 SRS 显微镜设计。该协议将侧重于将激光与显微镜耦合，以及该系统的日常运行（图1）...

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为了预防肥胖及其相关的代谢紊乱，已经开展了重要的研究工作，以更好地了解脂质平衡的调控机制。对于生物样本中脂质分子的定量检测，SRS显微镜的无标签成像已被证明是生化检测和其他染色方法的可靠替代品。我们小组和其他人通过结合使用C.elegans和SRS显微镜12、18、28、29、30、31，揭示了脂质代谢调节?...

肥胖已成为威胁全球三分之一人口的全球健康问题，并提出了严重的医疗问题，因为它与不良的心理健康1 和致命的疾病，包括糖尿病2，心血管疾病3 和某些类型的癌症4。脂质代谢研究对于更好地了解肥胖背后的生物学问题至关重要。脂质储存的快速和具体量化需要检测脂肪酸及其衍生物，以及含有固醇的代谢物，具有高灵敏度，最好是空间信息。脂质是具有挑战性的图像目标，因为它们缺乏内在荧光，不容易被荧光标记。荧光标签通常大于脂质分子，因此，在体内应用中，可能具有化学侵入性和不切实际性。无标签或最小标签策略是必要的，以保持脂质分子5的疏水结构。成像技术的最新发展为活细胞、组织和生物体中脂质的无标签成像创造了令人兴奋的机会。
生物样本中脂质储存分析的传统方法包括生化检测和用脂质染料染色协议。涉及质谱仪 （MS） 的生化定量测定在其分子可解性方面是无可比拟的，但它们需要非常大的样本量，样品制备通常需要几个小时，从而限制了它们对生活系统5的实时成像应用。这些测定的另一个主要限制是缺乏空间信息。另一方面，脂质染料，如油红O和苏丹黑提供组织和细胞分布的脂质储存细胞器，与MS技术相比，这些染色方法成本低，易于执行。然而，这些染色协议需要固定，这可以影响脂质液滴的疏水性质，产生人为的变化，其结构，并导致实验之间的不一致6。与生化和染色技术相关的技术困难导致寻找无标签方法来成像脂质分子，并导致在脂质成像中使用连贯的拉曼散射 （CRS） 显微镜的迅速增加。
拉曼效应首先被拉曼和克里希南所识别，他们报告说，在与光子相互作用时，分子可以产生散射光，波长（称为雷利散射）没有变化，或者很少改变波长（称为拉曼散射），波长的变化是分子7内功能化学组的特征。当分子内部的化学键被一种称为泵光子的事故光子激发到更高的振动能量水平时，称为斯托克斯光子的分散光子的能量就会变低。否则，如果化学键最初处于较高水平，则可达到较低的振动能量水平，而分散的光子获得能量，成为抗斯托克斯光子。事件与分散光子之间的频率差异称为拉曼移位。分子内的每个化学键都有一个特征和可量化的拉曼移位。例如，CH2 键具有2，845厘米-1的拉曼移位，富集脂肪酸链8。这种自发的拉曼信号通常非常微弱，这极大地限制了传统自发拉曼显微镜的成像速度。多年来，已经开发出各种方法，以提高自发拉曼显微镜的成像速度和灵敏度。连贯拉曼散射显微镜，包括连贯的反斯托克斯拉曼散射（CARS）显微镜和刺激拉曼散射（SRS）显微镜，是最新的进展。CARS 和 SRS 的工作原理略有不同，但两者都是无标签技术，具有实时成像能力，可以生成脂质存储动态的空间和时间信息，并且只需要少量样本。CARS显微镜具有非共振背景，来自各种非线性过程，而CARS信号也与分子浓度有非线性关系，共同使定量过程复杂化。与 CARS 显微镜不同，SRS 显微镜不会生成非共振背景信号，并且对感兴趣分子的浓度提供线性依赖。因此，目前SRS显微镜更广泛地用于脂质成像。
在 SRS 显微镜中，当兴奋时，弱自发拉曼信号可以放大两个同步激光束，其频率差异与化学键振动频率相匹配。由于连贯的兴奋，分子将经历向兴奋状态的增强过渡。因此，斯托克斯光子发电的速度得到提高。因此，传输的"斯托克斯"光束的强度增加（刺激拉曼增益，SRG），传输的"泵"光束的强度降低（刺激拉曼损失，SRL）。SRG或SRL信号的检测是刺激拉曼散射（SRS）显微镜成像分子的基础与特定化学键10。如果两个激光束之间的频率差异与感兴趣分子内化学键的振动频率不匹配，则不会生成 SRG 或 SRL 信号。SRS显微镜的成像速度约为每像素2微秒或每帧1秒，这比自发拉曼显微镜11快得多。SRS显微镜的典型横向分辨率有限，约为300纳米。此外，SRS显微镜的双光子光学过程允许对相对厚的组织样本进行体积3D成像，成像深度可达300-500 μm。总体而言，SRS显微镜提供了一种高效、无标签的成像技术，可检测特定的生物分子，尤其是脂质。
脂质液滴是单膜细胞，是中性脂质的主要细胞储存部位，包括三氯丁二醇 （TAGs） 和胆固醇酯 （CEs）。这些脂质分子脂肪酸链中的CH2键在兴奋8时产生2，845厘米-1的强烈SRS信号，从而能够检测和量化完整细胞、组织部分甚至整个生物体12、13、14、15中的储血脂水平。特别是，C. elegans由于其透明度，对脂质成像研究很有用。与哺乳动物一样，C.elegans也在脂质液滴中储存脂质，脂质分子的合成和降解途径被高度保存。在此协议中，我们将提供SRS显微镜的工作原理，其基本设置，并描述其在C.elegans脂质成像中使用的方法。

<table>
	<tbody>
		<tr>
			<td>A/D converter</td>
			<td>Olympus</td>
			<td>Analog Unit</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>GeneMate</td>
			<td>3119</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Alignment tool - adapter</td>
			<td>Thorlabs</td>
			<td>SM1A4</td>
			<td>For mounting the tool on scope</td>
		</tr>
		<tr>
			<td>Alignment tool - target</td>
			<td>Thorlabs</td>
			<td>VRC2SM1</td>
			<td>For viewing IR laser</td>
		</tr>
		<tr>
			<td>Alignment tool - tube</td>
			<td>Thorlabs</td>
			<td>SM1L40</td>
			<td>Length can vary</td>
		</tr>
		<tr>
			<td>Autocorrelator (Optional)</td>
			<td>APE</td>
			<td>pulseCheck</td>
			<td></td>
		</tr>
		<tr>
			<td>Bandpass filter</td>
			<td>minicircuits</td>
			<td>BBP-21.4+ if modulated at 20MHz or KR Electronics 2724 if modulated at 8 MHz</td>
			<td>For signal with modulation frequency filtering</td>
		</tr>
		<tr>
			<td>BNC cables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>For handling and picking worms for imaging</td>
		</tr>
		<tr>
			<td>Dodecane</td>
			<td>Sigma-Aldrich</td>
			<td>44010</td>
			<td>Used for calibration of the SRS signal</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Chroma Technology</td>
			<td>890/220 CARS</td>
			<td>For removing Stokes beam</td>
		</tr>
		<tr>
			<td>General purpose laboratory labeling tape</td>
			<td>VWR</td>
			<td>89097</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>VWR</td>
			<td>48393-106</td>
			<td>For covering worms for imaging</td>
		</tr>
		<tr>
			<td>Glass microscope slide</td>
			<td>VWR</td>
			<td>16004-422</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Laser scanning microscope</td>
			<td>Olympus</td>
			<td>FV3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens</td>
			<td>Thorlabs</td>
			<td>L1: AC254-050-B 
			L2: AC254-075-B</td>
			<td>For beam expander</td>
		</tr>
		<tr>
			<td>Lock-in amplifier</td>
			<td>Zurich</td>
			<td>HF2LI</td>
			<td></td>
		</tr>
		<tr>
			<td>Lowpass filter</td>
			<td>minicircuits</td>
			<td>BLP-1.9+</td>
			<td>For power supply noise suppression</td>
		</tr>
		<tr>
			<td>Mirrors</td>
			<td>Thorlabs</td>
			<td>BB1-E03</td>
			<td>For relay and periscope</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UPlanSAPO 20x 0.75, UPlanSAPO 60XW 1.20</td>
			<td></td>
		</tr>
		<tr>
			<td>Photodiode</td>
			<td>Thorlabs</td>
			<td>FDS1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Picosecond laser source</td>
			<td>APE</td>
			<td>picoEmerald</td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>TEKPOWER</td>
			<td>TP1342U</td>
			<td>For photodiode, reversed 50V voltage</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td>For anaesthesizing the worms</td>
		</tr>
		<tr>
			<td>Worm picker</td>
			<td>WormStuff</td>
			<td>59-AWP</td>
			<td></td>
		</tr>
	</tbody>
</table>

label-free imaging of lipid storage dynamics in caenorhabditis elegans using stimulated raman scattering microscopy

脂质代谢是细胞和生物体健康必需的基本生理过程。脂质代谢的调节障碍经常导致肥胖和许多相关疾病，包括心血管疾病、II型糖尿病和癌症。为了推进目前对脂质代谢调节的认识，精确测量体内脂质储存水平的定量方法在时间和空间上变得越来越重要和有用。分析脂质储存的传统方法是半定量进行微观评估或缺乏生化测量的时空信息。刺激拉曼散射（SRS）显微镜是一种无标签的化学成像技术，能够快速和定量地检测活细胞中的脂质，具有亚细胞分辨率。由于对比度利用了内在分子振动，SRS显微镜还允许对活体动物的脂质进行四维跟踪。近十年来，SRS显微镜在生物医学研究中广泛应用于小分子成像，克服了传统荧光染色和脂质提取方法的主要局限性。在实验室中，我们结合了SRS显微镜与强大的模型生物体 Caenorhabditis电子人 可用的遗传和生化工具，以研究脂质液滴在不同细胞和组织中的分布和异质性，并最终发现调节脂质代谢的新型保守信号通路。在这里，我们介绍了SRS显微镜的工作原理和详细设置，并提供了方法，用于量化脂质储存在野生类型和胰岛素信号不足突变 C.elegans的不同发展时间点。

胰岛素信号是影响发育、生殖、寿命和新陈代谢的重要内分泌途径。在蠕虫中，胰岛素信号包括大约40个胰岛素样肽配体、胰岛素样生长因子受体正牙DAF-2、下游PI3K/AKT激酶级联和FoxO转录因子正词，DAF-1620。缺乏胰岛素受体的daf-2突变体，其肠道中脂质液滴较多，蠕虫脂质储存组织21，22。使用 SRS 显微镜，我们量化了成年后年龄同...

Watch this Scientific Journal Video about Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy at JoVE.com

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

刺激拉曼散射（SRS）显微镜允许选择性，无标签成像的特定化学莫伊蒂，它已被有效地用于图像脂质分子在体内。在这里，我们简要介绍了SRS显微镜原理，并描述了用于 在卡诺哈布迪炎电子群中成像脂质储存的方法。

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

刺激拉曼散射显微镜是一种化学成像技术，能够快速和定量地检测脂质，并允许以无标签的方式跟踪活体动物中的脂质。SRS显微镜比传统脂质检测技术最重要的优点是，它无标签，因此，与其他荧光和染色物质一样，照片漂白不可持续。使用SRS显微镜与可用于模型生物体的遗传和生化工具相结合，特别是卡诺哈布迪炎电子人，为研究脂质生物学和新陈代谢的正常调节器提供了一个强有力的框架。要将激光束送入 SRS 显微镜，首先，设置潜望镜，将光束从皮秒光源出口提升到显微镜扫描仪上的红外激光输入端口，并打开激光控制软件启动激光。将泵激光功率降低到 50 毫瓦，并将泵信号设置为 750 纳米。使用光束膨胀器调整光束直径，以适应显微镜目标的后孔径。使用两个继电器镜，M1和M2，引导激光束到潜望镜，并将潜望镜的点头设置在调谐范围的中心。选择每个镜的初始位置和角度，使激光束大致击中镜中中心，将光束发射到显微镜中，并在显微镜的客观炮塔上使用空端口执行路线对齐。将功率计探头放置在目标端口，以测量传输光的功率，并将显微镜扫描仪设置为最高变焦 50 倍。用聚焦激光束点测量传输光的功率，并反复优化反射镜 M1 和 M2 的旋钮，实现最高的传输激光功率。使用对齐工具执行传输光的精细对齐，以确保光线通过目标中心，并将荧光目标对齐盖放在空目标座椅上。调整第一个转向镜 M1 的旋钮，以激光束点为中心，并将另一端的扩展管与对齐帽引入空目标座椅，用于监控激光束点。然后，调整第二个转向镜 M2 的旋钮，再次将激光束点中心放在扩展管的另一端。要设置 SRS 检测模块，请在凝结器后放置一个光束分流器立方体，将传输的激光引导到照片二极管模块。要设置电子连接，请在 picosed 可调谐激光器内使用 20 兆赫的内置电光调节器来调节斯托克斯光束的强度，并将照片二极管的输出信号输入锁定放大器，以降解刺激的 Raman 损失。然后，将锁定放大器输出输入显微镜的模拟盒，将电模拟信号转换为数字信号。为了优化成像条件，在迷你显微镜滑动室中加入五微升油酸，并盖上盖片覆盖样品。将样品放在显微镜阶段，并使用垫的边缘定位并聚焦于液滴。调整凝结器以进行科勒照明，并使用红外传感器卡和红外查看器检查泵束路径是否仍然正确。确认延迟阶段设置为零股骨秒，并将泵束波长设置为 795.8 纳米。将泵束的功率设置为 50 毫瓦，并将斯托克光束设置为 100 毫瓦。打开光束的快门以及激光的主快门。扫描样本并检查计算机屏幕上的图像。将显示模式更改为高低，并调整范围以查看大约 50% 的饱和度。检查饱和度是否以图像为中心，仔细调整 picosed 可调谐激光系统内的转向镜，以优化泵和斯托克斯的重叠，以最大限度地提高图像强度，并在必要时将峰值强度集中。每次成像会话之前，在带两层实验室胶带的支撑滑梯之间放置的干净玻璃滑梯上加入 100 微升的暖 2%agarose，并迅速将第二张幻灯片放在第一张幻灯片的顶部。轻轻按下，创建一个薄，均匀，蔗糖垫，让阿加罗斯冷却。要安装蠕虫进行成像，请将每 10 到 20 种蠕虫的麻醉剂滴 4 到 5 微升放在盖片上，然后使用解剖显微镜挑选要成像的蠕虫，将蠕虫放在麻醉液滴上收集。当所有的蠕虫都收集起来时，用玻璃滑梯用糖垫盖住蠕虫。用于成像，将蠕虫样本与盖片对面对着目标透镜，并将明亮的场光源引导到目镜中以定位蠕虫。将蠕虫集中到焦点中，并相应地调整凝结器位置。通过将泵激光功率设置为 200 毫瓦，将 IR 功率设置为 400 毫瓦来调整激光功率。设置锁定放大器，以演示蠕虫 SRS 信号。开始以快速扫描速率扫描第一个蠕虫，调整精细对焦以找到感兴趣的区域。优化信号后，切换到较慢的扫描速率和更高的像素分辨率，以获取 SRS 图像并以能够实现高分辨率的格式保存图像。当所有样品都成像后，将激光源置于待机状态，并关闭所有相关设备。要分析图像，请打开 ImageJ 中的文件，然后选择分析并设置测量结果，以选择要分析的属性。使用多边形选择工具勾勒出蠕虫肠道的区域进行量化，然后单击分析并再次测量。测量结果将显示在结果窗口中。当所有蠕虫都已勾勒出来时，将给定基因型中每个蠕虫的测量结果复制并粘贴到电子表格中。对于背景测量值，选择没有任何 SRS 信号的蠕虫附近区域，并从每个蠕虫的测量中减去背景平均灰色值，以计算背景的平均和标准偏差减去给定基因型或测试组中所有蠕虫的平均和标准偏差。然后，这些值可以标准化到对照组的平均值。在这项具有代表性的分析中，在成年期间对年龄同步的野生型蠕虫和缺乏胰岛素受体的daf-2突变体的肠道脂质水平进行了量化。不出所料，野生类型蠕虫的脂质水平下降，从第一天开始，一直持续到成年后的第九天。然而，daf-2突变体的脂质水平增加，直到它们五天大，此时它们保持恒定，比野生类型高2.5至3倍。Daf-16灭活抑制了dab-2突变体脂质水平的增加。daf-16a a 或 daf-16b 等形在 daf-2 中的表达，daf-16 双突变体不能恢复脂质水平。然而，daf-16d/f/h等形的表达足以将 daf-2、daf-16 双突变体中的脂质水平恢复到 daf-2 单突变体中观察到的水平。这些结果表明，daf-16d/f/h等形体特别调节了dab-2突变蠕虫中的脂质代谢。在整个成像过程中，使用相同的激光功率用于泵和斯托克斯光束，并且每六和四次一致的量化中包括一个对照组，这一点很重要。除了量化脂质储存外，SRS显微镜还可以与生物动脉标记（如宫内膜）相结合，并实现脂质结合、合成和降解的动态可视化。SRS显微镜允许多路复用，可实现在不同的子细胞隔间中对多个生物分子进行成像。所谓的超光谱SRS显微镜在探索代谢动力学方面有着美好的未来。

Research

JoVE Journal

Biology

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Label-free in situ Imaging of Lignification in Plant Cell Walls

Label-free in situ Imaging of Lignification in Plant Cell Walls

无标签原位成像在植物细胞壁的木质化

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

科研

生物学

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)

Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering CARS

差分成像生物结构与双共振相干反斯托克斯拉曼散射（CARS）

Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

尼罗红与油红 O 染色法测定秀丽线虫的脂质丰度及脂质分布评价

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

通过总内部反射荧光显微镜与数量和亮度分析相结合，活细胞中细胞表面受体的显微化动力学

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

使用刺激拉曼散射显微镜对卡诺哈布迪炎电子人中的脂质存储动力学进行无标签成像