这项工作是由卫生部资助AR055686和AR060836和博士后研究全国学院公元法国肌肉萎缩症协会（AFM）的支持。在这项研究中所采用的细胞成像设备是由卫生赠款HD040677国家机构的支持。

1。成像细胞膜修复使用散装（玻璃珠）伤人该协议允许单独标记损伤的细胞和那些不能愈合。定量细胞的这些人群需要使用的三个条件：1。测试（C1）， -使细胞修复的Ca 2 +，2的存在下进行。控制1（无损伤C2） -孵育细胞中的Ca 2 +的存在下，但没有受伤，和3。控制2（无修复C 3） -允许细胞来修复在没有Ca 2 +的。 <ol><li>生长细胞&gt; 50％汇合三个无菌盖玻片，用CIM用PBS对硅胶O型圈洗C1和C2两次在37℃和C3在37°C和转移盖玻片。 </li><li>加入100μl对C1和C2或在PBS上C3在CIM预热FITC葡聚糖溶液。 </li><li>伤害在C1和C3质膜轻轻滚动40毫克玻璃珠在盖玻片在室温下通过手动倾斜盖玻片回来，列6-8次以30°的角度。 注意：对于轻度损伤，确保玻璃珠均匀地分散，从而最大限度地减少重复受伤。以提高样品之间损伤的再现，同时执行不同的样品进行比较的玻璃珠的伤害。 </li><li>避免了有孔玻璃珠进一步轧制，所有的盖玻片转移到37℃培养箱中在环境CO 2，并允许修继续进行5分钟。 </li><li>而不让珠滚动，通过与CIM（C1和C2）或PBS（C）在37℃下洗涤除去玻璃珠和FITC葡聚糖</li><li>放置盖玻片回O型圈，并添加对C1和C2或在PBS上C3 100微升的CIM预热赖氨酸可以解决TRITC葡聚糖溶液。 </li><li>在37℃下进行5分钟，在室温的CO 2。 </li><li>用预热的CIM两次洗涤盖玻片，用4％PFA中于室温10分钟，固定。 </li><li>用PBS洗两次后，于RT在赫希斯特染料2分钟。 </li><li>洗SAMPL上课两次，用PBS，安装使用的安装介质和细胞图像用落射荧光显微镜幻灯片。 </li><li>使用从C 2细胞，以确定背景的红色和绿色的染色强度，并使用该值来阈值的红色和绿色通道对于所有样品（C1-C3）。 </li><li>分数的细胞，一）绿色（受伤和修理）和b），红色或红色和绿色（受伤，但未能修复）C1和C3样品的总数。 </li><li>计数&gt; 100果岭细胞为每个条件和表达细胞不能修复的受伤的所有细胞（绿色和红色）的百分比的分数。 </li></ol> 2。细胞膜修复继激光损伤动力学的实时成像<ol><li>用预热CIM洗涤细胞，然后把盖玻片在CIM带FM染料。 </li><li>放置盖玻片在支架中保持在37℃的台顶培养箱</li><li>选择细胞膜的1-2毫米2区域，并照射于&lt;; 10毫秒的脉冲激光。通过软件衰减激光功率的峰值功率的40-50％。最佳功率允许一致，但非致命伤，这必须通过试验和错误使用每个单独的仪器和细胞系来确定。 </li><li>到显示器维修，图像每10秒在落射荧光和明，在开始之前，伤害和持续3-5分钟以下的伤害。 注：对于没有修复的控制，重复步骤2.1-2.4用在PBS受伤的细胞含有调频染料。 </li><li>量化修复的动力学，测量细胞调频染料的荧光，并绘制成像的过程中，在强度（ΔF/F0）的变化。此数据应在每种条件下进​​行平均超过10细胞和绘制为平均或个别单元格的值，如需要。 </li></ol> 3。成像散装（玻璃珠）损伤诱导溶酶体胞吐作用样本包括增长到&gt; 50％以下的细胞合流：1。测试（C1） -可以修复的Ca 2 +，2存在细胞。控制1（C2;没有受伤） - 细胞既不受伤也不孵育一抗，和3。控制2（C 3，无维修） -可以修复没有Ca 2 +的细胞。 <ol><li>洗盖玻片C1和C2两次CIM在37℃和C3用PBS在37℃，并转移他们对硅胶O形圈。 </li><li>加入100μl的CIM对C1和C2和在PBS预热赖氨酸可以解决TRITC右旋糖酐对C3的。 </li><li>伤在C1和C3质膜如在步骤1.3。 </li><li>避免了有孔玻璃珠进一步轧制，所有的盖玻片转移到37℃培养箱中在环境CO 2，并允许修继续进行5分钟。 </li><li>洗涤盖玻片在寒冷生长培养基中，从而也确保了没有轧制的细胞上的玻璃珠除去玻璃珠和TRITC葡聚糖。 </li><li>用冷水生长培养基冲洗盖玻片两次转移到O型圈。 </li><li>到C1和C3，加入100μl大鼠抗小鼠LAMP1抗体在寒冷的完全生长介质和冷，完全生长培养基添加到C2。 </li><li>以允许抗体结合孵蛋盖玻片30分钟，在4℃下</li><li>用冷水洗脸的CIM盖玻片三次并修复所有盖玻片，用4％多聚甲醛在室温10分钟，然后用清水冲洗3次与CIM。 </li><li>所有孵育盖玻片在100μl封闭液15分钟，在室温的</li><li>所有孵育盖玻片在100μl的Alexa Fluor 488的抗鼠抗体为15分钟，在4℃下</li><li>用PBS洗两次后，于RT在赫斯特2分钟。 </li><li>用PBS洗涤细胞两次，安装使用的安装介质和图像使用落射荧光显微镜幻灯片。 </li><li>使用C2细胞的图像，确定在红的非特异性背景染色（TRITC葡聚糖）和绿色（的Alexa Fluor 488抗体）通道，并利用这些背景染色的值的阈值的红色和绿色通道对于所有样品（C1-C3）。&lt;/ LI&gt; <li>使用&gt; 100红色标记的细胞从C1和C3来衡量LAMP1染色（绿色）在损伤细胞的强度。对于一个成功的实验中由C3细胞LAMP1染色将比从C1细胞显著更低。 </li></ol> 4。细胞膜损伤的实时成像触发亚细胞贩运<ol><li>荧光标记 利息转染适当的记者（ 如 CD63-GFP的溶酶体8），或通过使用荧光染料9车厢。 </li><li>对于标记溶酶体与荧光染料，孵化成长为在含FITC-葡聚糖的生长培养基50％汇合的细胞</li><li>允许（如需要的足够的内体标记的葡聚糖与由所关注的细胞系或更长的时间）在CO 2培养箱中的葡聚糖被内吞2小时。 </li><li>用预热的生长培养基洗涤细胞，并培育在其2小时，在CO 2培养箱中，以允许所有内吞葡聚糖积聚在溶酶体。 </li><li>前成像，冲洗预热的CIM盖玻片，在37°C安装在舞台上的顶级孵化盖玻片架</li><li>进行宽视场（对于整个细胞运动）或TIRF（在细胞表面和胞吐作用的运动）成像 - 细胞没有拥挤的FITC标记的右旋糖酐溶酶体是理想​​的成像单个溶酶体的运动和胞吐作用。 </li><li>对准激光TIRF成像显微镜：使用60X或100X物镜与&gt; 1.45 NA。设置角度为使用制造商的方法入射TIRF激光束。 </li><li>通过照射一个小的（1-2毫米2）区域为&lt;100毫秒，用脉冲激光在40-50％衰减损害细胞膜。 </li><li>以监测溶酶体的响应细胞损伤，细胞图像在4-6帧/秒，至少2分钟或更长的时间取决于胞吐作用的目的细胞的动力学。 </li></ol>

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作者什么都没有透露。

细胞膜损伤体内发生是由于多种生理应激源和几个实验方法已被开发来模仿这些。这些措施包括通过刮不送的菜，或者通过一个狭窄的孔注射器9,10打伤传代贴壁细胞的细胞膜。下面这种伤害的细胞愈合悬挂和不粘附到细胞外基质，因为他们通常做的组织。还有一些人，如使用成孔毒素通过提取脂质如胆固醇化学改变细胞膜，因此不模仿体内的机械性损伤5。因此，用?...

细胞膜保持细胞通过在细胞和胞外环境之间的屏障的完整性。化学，电或机械的刺激超过了正常的生理阈以及入侵的病原体的存在下可以将每个结果在伤到细胞膜，并触发随后的细胞反应来修复这种损伤。生存这些损伤细胞膜，细胞具有一个有效的机制进行维修。这种机制是钙依赖性，涉及蛋白质，如膜联 ​​蛋白和MG53细胞内贩卖（其中包括）以及亚细胞区室，如内涵体，溶酶体，高尔基体衍生的囊泡，线粒体的损伤细胞膜1-7。然而，参与修复受损的细胞膜分子和亚细胞的事件序列的细节仍然知之甚少。 细胞的修复反应可划分为耳LY和晚期反应。早期反应，在几秒钟内所发生到几分钟的时间尺度，是决定导致细胞成功修复或细胞死亡延迟响应的性质极为重要。基于大宗生物化学和细胞分析终点检测有助于建立维修分子和细胞过程的参与。但是，由于细胞修复反应的异质性和快速性，终点测定法不能提供导致修复事件序列的动能和空间细节。方法，使细胞膜的损伤控制，并允许监控细胞膜修复和高空间分辨率和时间分辨率相关的亚细胞的反应，非常适合这类研究。在这里，这种方法已被提出。两个协议的描述方法来监测细胞膜修复以下激光注入电流的实时动力学和活细胞的修复过程相关的细胞内反应URY。作为补充，这些活细胞成像的基础的分析，终点测定法也已被描述，提供了一个基于人口的措施为单个细胞和相关的细胞内反应的监测维修。证明其效用这些方法已被用于监测贩卖和溶酶体的胞吐响应于细胞膜损伤。

<table border="0" cellpadding="0" cellspacing="0" width="814" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>HBSS</td>
			<td>Sigma</td>
			<td>H1387</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP410</td>
			<td>Used to prepare CIM (HBSS/10 mM Hepes/2 mM Ca2+ pH 7.4)</td>
		</tr>
		<tr>
			<td>FITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1820</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC dextran (Lysine fixable)</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>2 mg/ml in CIM or PBS</td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>EMS</td>
			<td>15710</td>
			<td>4% in PBS</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>1/10 000 in PBS</td>
		</tr>
		<tr>
			<td>FM dye</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td>1 &#181;g/&#181;l in CIM or PBS</td>
		</tr>
		<tr>
			<td>FITC dextran</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>2 mg/ml in growth medium</td>
		</tr>
		<tr>
			<td>LAMP1&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-19992</td>
			<td>1/300 in growth medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 chicken anti-rat IgG</td>
			<td>Invitrogen</td>
			<td>A21470</td>
			<td>1/500 in blocking solution</td>
		</tr>
		<tr>
			<td>Mounting media</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-545-86</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petridish</td>
			<td>MatTek corporation</td>
			<td>P35G-1.0-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone O ring</td>
			<td>Bellco</td>
			<td>1943-33315</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip holder</td>
			<td>Bellco</td>
			<td>1943-11111</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen</td>
			<td>A-7816</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>12001-566</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>VWR</td>
			<td>MP1400-500H</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Penicillin/Sreptomycin</td>
			<td>VWR</td>
			<td>12001-350</td>
			<td>Used for growth medium: 10% FBS, 1% P/S in DMEM</td>
		</tr>
		<tr>
			<td>Chicken serum</td>
			<td>Sigma</td>
			<td>C5405</td>
			<td>5% chicken serum in CIM is used for blocking solution during immunostaining</td>
		</tr>
		<tr>
			<td>PBS (Ca2+ and Mg2+ free)</td>
			<td>VWR</td>
			<td>12001-664</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Olympus America, PA</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF illuminator&#160;</td>
			<td>Olympus America, PA</td>
			<td>Cell^TIRF (IEC60825-1:2007)</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence illuminator</td>
			<td>Sutter Instruments, Novato CA</td>
			<td>Lambda DG-4 (DG-4 30)</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Photometrics, Tucson, AZ</td>
			<td>Evolve 512 EMCCD&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and anaysis software</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Slidebook 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulsed laser</td>
			<td>Intelligent Imaging Innovations, Inc. Denver, CO</td>
			<td>Ablate (3iL13)</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage top incubator</td>
			<td>Tokaihit, Japan</td>
			<td>INUBG2ASFP-ZILCS</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cell membrane injury and subcellular processes involved in repair

损伤的细胞的愈合的能力是一个基本的细胞过程，而且参与愈合损伤的细胞的细胞和分子机制知之甚少。这里分析的描述来监测培养细胞的愈合下面的本地化损伤的能力和动力学。第一个协议描述终点为基础的方法来同时评估数百个细胞的细胞膜修复能力。第二协议描述了一种实时成像的方法来监测细胞膜修复中的单个细胞在与脉冲激光局部的损伤的动力学。由于愈合受伤的细胞涉及贩卖人口特异性蛋白和亚细胞对损伤的部位，第三协议描述了使用上述终点为基础的方法，以数百细胞的损伤评估这样一个拐卖事件（溶酶体胞吐作用），同时，最后协议介绍了利用脉冲激光损伤与全内反射荧光显微镜在一起复制并监控在损伤的细胞在高空间和时间分辨率个别亚细胞区室的动力学。而这里的协议描述了使用这些方法来研究之间细胞膜修复和培养的肌细胞溶酶体的胞吐作用的链接，他们可以作为这样的其他任何贴壁培养的细胞和所选择的亚细胞区室施加。

这里所描述的单细胞成像协议是监测细胞膜修复的能力和动力学（协议1和2）和修复过程中细胞内贩运和融合溶酶体（协议3和4）。 协议1显示了批量试验，允许标记所有受损细胞，并确定那些受伤的细胞，无法修复。在图1中显示的结果是，虽然未受伤的细胞（ 图1A）保持未标记的细胞通过玻璃珠中的FITC葡聚糖的存在受伤被标记为绿色（ 图1B</st...

Watch this Scientific Journal Video about Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair at JoVE.com

Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

愈合损伤的细胞的过程中涉及到贩卖特异性蛋白和亚细胞区室中的细胞膜损伤的部位。本协议描述分析，以监测这些进程。

成像生物膜损伤和参与亚细胞修复过程

The ability of injured cells to heal is a fundamental cellular process, but cellular and molecular mechanisms involved in healing injured cells are poorly ...

imaging-cell-membrane-injury-subcellular-processes-involved

Research

JoVE Journal

Biology

14.9K 次观看.  Children's National Medical Center. 愈合损伤的细胞的过程中涉及到贩卖特异性蛋白和亚细胞区室中的细胞膜损伤的部位。本协议描述分析，以监测这些进程。 

视频: Imaging Cell Membrane Injury and Subcellular Processes Involved in Repair

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

MG53介导的细胞膜使用维修的可视化在体内在体外系统

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

科研

生物学

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化：调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

实时成像检测方法的评估钙的作用<sup&gt; 2 +</sup&gt;和鞘磷脂酶在成孔毒素伤口的修复

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

亚细胞结构的超分辨率活细胞成像

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Photodiode-Based Optical Imaging for Recording Network Dynamics with Single-Neuron Resolution in Non-Transgenic Invertebrates

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light represents a revolutionary advance for studies of the neural basis of behavior. However, a downside of this development is its tendency to focus investigators on a very small number of &#8220;designer&#8221; organisms (e.g., C. elegans and Drosophila), potentially negatively impacting the pursuit of comparative studies across many species, which is needed for identifying general principles of network function. The present article illustrates how optical recording with voltage-sensitive dyes in the brains of non-transgenic gastropod species can be used to rapidly (i.e., within the time course of single experiments) reveal features of the functional organization of their neural networks with single-cell resolution. We outline in detail the dissection, staining, and recording methods used by our laboratory to obtain action potential traces from dozens to ~150 neurons during behaviorally relevant motor programs in the CNS of multiple gastropod species, including one new to neuroscience &#8211; the nudibranch Berghia stephanieae. Imaging is performed with absorbance voltage-sensitive dyes and a 464-element photodiode array that samples at 1,600 frames/second, fast enough to capture all action potentials generated by the recorded neurons. Multiple several-minute recordings can be obtained per preparation with little to no signal bleaching or phototoxicity. The raw optical data collected through the methods described can subsequently be analyzed through a variety of illustrated methods. Our optical recording approach can be readily used to probe network activity in a variety of non-transgenic species, making it well suited for comparative studies of how brains generate behavior.

This protocol presents a method for imaging neuronal population activity with single-cell resolution in non-transgenic invertebrate species using absorbance voltage-sensitive dyes and a photodiode array. This approach enables a rapid workflow, wherein imaging and analysis can be pursued over the course of a single day.

基于光二极管的光学成像，用于记录非转基因无脊椎动物的单神经元分辨率的网络动态

The development of transgenic invertebrate preparations in which the activity of specifiable sets of neurons can be recorded and manipulated with light ...

Isolating and Imaging Live, Intact Pacemaker Regions of Mouse Renal Pelvis by Vibratome Sectioning

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, propulsive contractions. Regular RP contractions rely on pacemaker activity, which originates from the most proximal region of the RP at the pelvis-kidney junction (PKJ). Due to the difficulty in accessing and preserving intact preparations of the PKJ, most investigations on RP pacemaking have focused on single-cell electrophysiology and Ca2+ imaging experiments. Although important revelations on RP pacemaking have emerged from such work, these experiments have several intrinsic limitations, including the inability to accurately determine cellular identity in mixed suspensions and the lack of in situ imaging of RP pacemaker activity. These factors have resulted in a limited understanding of the mechanisms that underlie normal rhythmic RP contractions. In this paper, a protocol is described to prepare intact segments of mouse PKJ using a vibratome sectioning technique. By combining this approach with mice expressing cell-specific reporters and genetically encoded Ca2+ indicators, investigators may be able to more accurately study the specific cell types and mechanisms responsible for peristaltic RP contractions that are vital for normal urine transport.

The goal of this protocol is to isolate intact pacemaker regions of the mouse renal pelvis using vibratome sectioning. These sections can then be used for in situ Ca2+ imaging to elucidate Ca2+ transient properties of pacemaker cells and other interstitial cells in vibratome slices.

隔离和成像现场， 通过颤音分割老鼠肾佩尔维斯的精致起搏器区域

The renal pelvis (RP) is a funnel-shaped, smooth muscle structure that facilitates normal urine transport from the kidney to the ureter by regular, ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

从成年、幼年、幼虫和胚胎斑马鱼中分离心脏和血管平滑肌细胞以进行电生理研究

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

对小鼠皮肤中的中性粒细胞迁移进行成像，以研究生理条件下的亚细胞膜重塑

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...