Name: photostimulation by femtosecond laser activates extracellular-signal-regulated kinase (erk) signaling or mitochondrial events in target cells
Uploaded: 2019-07-06T14:00:00
Description: Watch this Scientific Journal Video about Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells at JoVE.com

这项工作得到了国家自然科学基金(81571719和81661168014、81673014、81870055)、上海市科学技术委员会(18QA1402300和16XD1403100)和国家重点研发计划(2017YFA01046000)的支持。

警告:下面介绍的协议涉及使用近红外飞秒激光和有毒化学品。请注意实验程序引起的所有可能的损害。使用前请阅读所有相关化学品或其他材料的安全数据表。在操作激光源之前,请遵循激光设施的安全说明或咨询专业人士的指导。
1. 实验准备
<ol><li>设置光刺激系统 注:该系统由飞秒激光和激光(荧光激发的可见范围)扫描共聚焦显微镜...

<ol>
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我们将飞秒激光与激光扫描共聚焦显微镜系统相结合,演示了光刺激策略。光刺激可以通过相应地定义Stim-B直接作为双光子显微镜。我们提供了一个详细的协议,利用短闪光的飞秒激光触发ERK信号或线闪光的目标细胞。不同的刺激模式可以根据不同的实验目的和系统进行。基于共聚焦显微镜可轻松设置 Stim-A。飞秒激光可以在FOV中心的衍射极限水平聚焦。亚细胞目标需要手动移动到飞秒激光对焦位置,...

控制细胞信号分子的技术是生命科学发展的重要组成部分。传统上,最常用的方法是用药物或生物材料1、2、3进行生化处理。在过去的十年里,光遗传学的发明开启了细胞分子信号调制的新时代。基因工程对光敏感蛋白的转染使光成为调节靶细胞中各种蛋白质活性的有力工具。该技术取得了令人鼓舞的进展,如激发和抑制神经信号,促进基因表达,操纵细胞信号模式,导致不同的细胞命运和病理调查4,5 ,6,7,8,9.然而,光只能通过用光遗传蛋白转染细胞来工作。在现阶段,除了光遗传学之外,还有一种罕见的使光能够直接控制细胞分子的方法。
飞秒激光在保持良好生物安全性的同时,通过提供高效的多光子激发,在生物学方面具有先进的生物学研究。通过部署多种光处理策略,它取得了多光子显微镜、微手术和多光子光遗传学应用10、11、12、13等多项成果。 ,14,15,16.近期的研究表明,飞秒激光刺激已被证明是直接诱发分子信号事件的一种高效光学方法。研究发现,对内质视网膜(ER)的紧聚焦飞秒激光照射能够耗尽ER中的钙,并激活钙释放活性钙(CRAC)通道,在细胞17中形成钙信号。这种光激活的钙信号可以在多种类型的细胞之间传播18,19,20。此外,它还能够激活细胞信号通路,如激活T细胞(NFAT)的核因子和ERK信号通路21,22。通过调整细胞中飞秒激光照射的强度和定位,例如,将激光聚焦在线粒体上,可以影响线粒体形态和分子事件23、24 25.具体来说,线粒体ROS的突发可以通过光刺激激发,光刺激被称为线粒体(线粒体)中的荧光闪烁。
因此,光刺激技术在相关生物研究中具有广泛的应用潜力。这也是一个扩大飞秒激光应用在控制细胞信号分子和功能,除了显微镜的好机会。在这里,我们提供光刺激的技术细节。光刺激是通过将飞秒激光耦合到共聚焦显微镜来实现的,从而提供具有短闪光光刺激的单靶细胞。它可以在细胞中启动高效且可控的 ERK 激活。如果光刺激位于线粒体管状结构上,线粒体膜电位、形态、ROS和渗透性过渡孔隙,都可以由光刺激控制。基于该光刺激方案,我们提供了激活ERK信号通路和影响Hela细胞中多个线粒体事件的详细方法。该协议阐明了向目标细胞提供飞秒激光刺激的过程。
光刺激系统建立在共聚焦显微镜上,带有飞秒激光耦合,可同时进行刺激和连续显微镜。飞秒激光(波长:1,040 nm,重复率:50 MHz,脉冲宽度:120 fs,最大输出平均功率:1 W)在耦合前被分成两个光束。其中一个通过由一对透镜组成的中继望远镜进行引导。然后,它直接反射到目标的后孔径(60x,N.A. = 1.2,水浸)中,形成衍射极限聚焦(Stim-A)。另一种被反射到共聚焦显微镜的扫描光学路径上,作为双光子扫描模式(Stim-B)工作。Stim-A 在视场中心 (FOV) 中显示一个固定焦点。Stim-B 是 FOV 中预先设计的部分共聚焦扫描区域。如图1A所示,显示了 Stim-A 和 Stim-B。二色镜 (DM) 下的 CCD 摄像机提供明场成像,用于监控飞秒激光对焦。
以下实验有一些关键要点。在此协议中,使用飞秒光纤激光源(1040 nm、50 MHz、120 fs)作为示例。在实践中,大多数商用飞秒振荡器可以使用,只要脉冲宽度短于200fs,峰值功率密度应高于10 11~1012 W/cm 2的水平。例如,通常用于多光子显微镜的 Ti: 蓝宝石激光能够取代图 1B所示的飞秒激光。激光功率和其他一些光刺激参数需要调整,因为光学参数(脉冲宽度、波长和重复率)在不同的飞秒激光器中变化很大,从而产生不同的多光子激发效率。
与飞秒激光刺激一起,共聚焦显微镜提供连续细胞成像,以Stim-A和Stim-B模式实时监测分子动力学。两种光刺激方案(Stim-A 和 Stim-B)均由具有毫秒分辨率的机械快门控制(图 1)。
在Stim-A模式下,激光对焦的位置固定在FOV的中心。继电器望远镜用于通过调整垂直方向的两个透镜之间的距离(激光传播方向,垂直到共聚焦成像平面,如图 1.通过CCD相机的明场成像,可以测量激光对焦的直径(+2μm,图2B)。刺激持续时间和曝光时间在共聚焦成像过程中由快门控制。
在 Stim-B 模式下,刺激区域可以在共聚焦成像控制软件中手动预分配给任何形式,如直线、多边形或圆。快门与共聚焦扫描过程同步。它在通过共聚焦成像控制软件设置的预设计时间打开。然后,用飞秒激光扫描刺激区域作为共聚焦显微镜。因此,当共聚焦扫描过程进入给定的成像帧时,只有飞秒激光刺激样品。
根据实验对象,可在倒置和直立冶金显微镜上建立光刺激系统。培养在培养皿中的体外细胞更适合倒置显微镜。动物,特别是活体动物的大脑,更适合使用直立显微镜。在这项研究中,我们以倒置显微镜为例。需要注意的是,在整个实验过程中,培养皿的盖子没有打开。

<table>
	<tbody>
		<tr>
			<td>inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>femtosecond laser</td>
			<td>Fianium</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubation system</td>
			<td>Olympus</td>
			<td>MIU-IBC</td>
			<td></td>
		</tr>
		<tr>
			<td>petri dish</td>
			<td>NEST</td>
			<td>801002</td>
			<td></td>
		</tr>
		<tr>
			<td>petri dish with imprinted grid</td>
			<td>Ibidi</td>
			<td>81148</td>
			<td></td>
		</tr>
		<tr>
			<td>ERK-GFP</td>
			<td>addgene</td>
			<td>37145</td>
			<td>A gift from Rony Seger&#39;s lab</td>
		</tr>
		<tr>
			<td>mt-cpYFP</td>
			<td></td>
			<td></td>
			<td>A gift from Heping Cheng&#39;s lab</td>
		</tr>
		<tr>
			<td>mito-GFP</td>
			<td>Invitrogen</td>
			<td>C10508</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine (TMRM)</td>
			<td>Invitrogen</td>
			<td>T668</td>
			<td>Dilute in DMSO</td>
		</tr>
		<tr>
			<td>polyethylenimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>9002-98-6</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Solarbio</td>
			<td>P8430</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>Dilute in PBS</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Sigma-Aldrich</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-eIF4E antibody</td>
			<td>abcam</td>
			<td>ab76256</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Bax antibody</td>
			<td>abcam</td>
			<td>ab53154</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-cytochrome C antibody</td>
			<td>abcam</td>
			<td>ab90529</td>
			<td></td>
		</tr>
		<tr>
			<td>secondary antibody (anti-Rabbit IgG H&#38;L, Alexa Fluor 488)</td>
			<td>abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
	</tbody>
</table>

photostimulation by femtosecond laser activates extracellular-signal-regulated kinase (erk) signaling or mitochondrial events in target cells

直接控制细胞定义的分子事件对生命科学很重要。最近,研究表明,飞秒激光刺激可以同时激活多个细胞分子信号通路。在此协议中,我们表明,通过将飞秒激光耦合到共聚焦显微镜中,通过紧密聚焦的激光可以精确刺激细胞。一些可以同时观察到的分子过程随后被激活。我们提出了光刺激的详细方案,以激活Hela细胞中的细胞外信号调节激酶(ERK)信号通路。如果将飞秒激光脉冲聚焦到特定的线粒体管状结构上,也可以刺激活性氧物种 (ROS) 和其他线粒体事件的线粒体闪光。该协议包括光刺激前对细胞进行预处理,通过飞秒激光照射到靶上进行光刺激,以及观察/识别随后的分子变化。该协议是相关生物研究的全光学工具。

光刺激可以同时进行连续共聚焦扫描显微镜。光刺激可以从延时共聚焦显微镜序列中的任何预定义的时隙开始。共聚焦显微镜可以通过荧光成像监测细胞分子。光刺激和其他动力学的分子反应可以这样识别。从理论上讲,如果ERK被激活,它将从细胞质磷酸化移动到细胞核27。特定的细胞命运可以由这个ERK信号28的某些模式调节。在最近的研?...

Watch this Scientific Journal Video about Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells at JoVE.com

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

紧密聚焦的飞秒激光可以结合到共聚焦显微镜中,从而对细胞提供精确的刺激,从而实现实时观察和光刺激。光刺激可以激活细胞分子事件,包括ERK信号通路和反应氧物种的线粒体闪光。

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

光遗传学等光学方法可以提供细胞分子信号的精确调制。我们的协议演示了通过飞秒激光刺激对细胞信号分子进行口头光学操作。通过将我们的飞秒激光耦合到共和显微镜中，我们的技术可以通过飞秒激光刺激和通过共和显微镜进行实时分子动态监测来提供单细胞或亚细胞水平操作。该方法可应用于任何荧光显微镜系统，以适当的方式将飞秒激光耦合到系统中。对于任何首次尝试我们的方法的人，请按照激光设施的单独说明操作，或在操作实验室飞秒激光源时寻求专业人士的指导。首先，打开飞秒激光器和共合显微镜。使用反射镜通过机械快门引导飞秒激光束。接下来，设置一个由一对透镜组成的中继望远镜，以扩大传输光束宽度。这应该与目标的后孔径一致。现在，引导反射镜将展开的光束与显微镜对齐。调整两个反射镜，这里指出，将飞秒激光的焦点调整到视场的中心。最后，测量和调整激光，如随附的文本协议中所述。使用标准技术培养希拉细胞。当准备执行实验时，使用随附的文本协议中描述的 ERK2-GFP 转染细胞。接下来，打开激光扫描共声显微镜和飞秒激光。确保激光快门已关闭。然后，打开显微镜软件。将激励激光器设置为 488 纳米，将其功率级别设置为 0.1 毫瓦。将成像大小设置为 512 x 512 像素，将每个像素的间隔时间设置为 2.4 微秒。然后，将两帧之间的间隔时间设置为六秒，以尽量减少照片漂白和照片对细胞的伤害。此外，将总成像帧设置为大约 300 帧，在单个实验中提供约 30 分钟的连续显微镜。从培养箱中取出含有与ERK2-GFP一起转染的希拉细胞的盘子，将该培养皿放在显微镜舞台上。在软件中，启动快速扫描模式，并调整目标，以获得清晰的荧光图像的细胞。然后，停止快速扫描并使用 CCD 摄像机切换到亮场成像模式。打开快门并调整继电器望远镜的两个镜头之间的距离，以确保飞秒激光对焦的直径约为 2 微米。然后，关闭快门以完成调整。将 810 纳米激光的飞秒激光功率设置为 15 至 40 毫瓦，1040 纳米激光的功率设置为 20 至 60 毫瓦。然后，将快门打开时间设置为 05 至 0.2 秒。使用快速扫描模式选择强烈表达 ERK2-GFP 的目标单元。找到后，移动舞台以定位选定目标细胞的细胞索区域，以便在亮场成像下处于视场的中心。居中后，单击"开始"按钮开始连续成像。在任何预定义时隙打开快门，将飞秒激光刺激传递到目标单元。成像过程完成后，保存成像数据以进行进一步分析。将飞秒激光的功率设置为 15 至 40 毫瓦和 810 纳米，在试样时设置为 20 至 60 毫瓦和 1040 纳米。然后，从培养箱中取出含有与ERK2-GFP转染的细胞的培养皿，并放在显微镜的舞台上。使用快速扫描模式选择表达 ERK2-GFP 的目标单元。然后，设置共声成像过程，并定义一个特殊的扫描帧作为成像过程的刺激帧。定义刺激帧的参数。在靠近目标细胞核的细胞索尔区域设置2到3平方微米的扫描区域。将总扫描时间设置为 0.1 到 0.2 秒。将两帧之间的间隔时间设置为 6 秒，将总成像帧设置为大约 300 帧，以提供大约 30 分钟的连续显微镜。保存成像数据以进行进一步数据分析。根据预定义的照片刺激区域，将飞秒激光的快门与共声扫描的快门同步，只有在激光扫描在刺激帧中下降时，该光秒激光的快门才打开。单击"开始"按钮开始连续显微镜成像过程。为了观察线粒体形态动力学，用米托-GFP转染希拉细胞以荧光指示线粒体，或用线托-cpYFP来观察线粒体的基座。转染后，打开飞秒激光，确保快门关闭。然后，打开激光扫描共声显微镜。将激励激光设置为 488 纳米。然后，将 488 纳米激光器的功率级别设置为 0.1 毫瓦。此外，将成像大小设置为 512 x 512 像素，将每帧的总成像时间设置为 2.2 秒。然后，将两帧之间的间隔时间设置为零秒，将总成像帧设置为 200 帧，以在单个实验中提供约 440 秒的连续显微镜。从培养箱中取出含有与线托 GFP 或线粒体靶向的圆形黄色荧光蛋白的培养皿，将培养皿放在显微镜舞台上。检查飞秒激光器的状态，确保飞秒激光器的焦点位于视场的中心。然后，在荧光成像窗口的中心设置一个参考箭头，以指示焦点的位置。接下来，将飞秒激光的功率设置为 5 到 30 毫瓦，激光功率为 810 纳米，激光功率为 10 至 40 毫瓦，功率为 1040 毫米。将快门的打开时间设置为 05 至 0.1 秒。使用快速扫描模式选择表达良好的线托 GFP 目标细胞或线粒体靶向圆形黄色荧光蛋白。在靶细胞中随机选择一个线粒体作为实验对象。接下来，移动显微镜阶段，以便通过参考箭头所示的快速扫描模式，在视场中心定位目标线粒体管状结构。单击"开始"按钮开始连续成像。最后，在任何预定义时间打开快门，将飞秒激光刺激传递到目标线粒体管状结构中。等待成像过程完成，然后保存成像数据以进行进一步的数据分析。使用本协议中介绍的方法，ERK2在经过短速的飞秒激光处理后，会进入核。ERK2-GFP荧光在经过几分钟的飞秒激光刺激后达到最大值。ERK2 分子在激活核中的下游基质后将被脱磷化，然后 ERK2 回到核 GFP 荧光减少所指示的细胞质。ERK2 可以通过多次照片刺激多次激活。因此，可以通过控制多个刺激之间的间隔时间来精确操作 ERK2 信号模式。此外，ERK2偶尔可以在刺激细胞周围的相邻细胞中激活。此观察表明，使用飞秒激光处理的细胞可能释放一些可扩散分子，以激活相邻细胞中的 ERK2。ERK2下游蛋白eIF4E的磷酸化可以通过免疫荧光显微镜进行确认。结果表明，飞秒激光刺激能成功激活ERK信号通路。线粒体氧化闪光或三叶草是线粒体的氧化爆发，根系于复杂的分子动力学。实施此照片刺激方案，成功进行了三闪性激发。此照片刺激还显示各种线粒体分子动力学，包括线粒体形态的碎片和恢复，以及线粒体膜电位振荡。与照片激活的三毛线相似，这些线粒体事件具有不同的功率强度的不同性能。最重要的是确保飞秒激光器的焦点、大小和精度是细胞刺激的一个属性。近红外飞秒激光器可能危害用户。遵循此协议时，请佩戴适当的保护，防止激光从光学表扩散出来。

Research

JoVE Journal

Biology

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

科研

生物学

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

脂筏的测定分区在活细胞中的荧光标记探针的荧光相关光谱（FCS）

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

的细胞内信号与相邻小区发生细胞凋亡通过互动活细胞诱导活动标识

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...