Name: in vivo two-color 2-photon imaging of genetically-tagged reporter cells in the skin
Uploaded: 2019-07-11T13:30:00
Description: Watch this Scientific Journal Video about In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin at JoVE.com

此工作由授权 NS096030 (MDB) 支持。我们还要感谢成像核心经理维德·普拉卡什。我们还要感谢奥林巴斯发现中心/位于达拉斯大学成像中心设施提供设备和支持

动物程序由德克萨斯大学达拉斯机构动物护理和使用委员会批准,符合国家卫生研究院的指导方针。 所有实验均使用在C57BL/6背景下在内部培育的8-12周大雄性小鼠和雌性小鼠进行。由成纤维细胞特异性蛋白-1启动剂驱动的具有cre-重组酶的转基因小鼠被商业购买(Jackson,012641) (FSP1cre)+/-并与tdTomatolox-停止-lox小鼠交叉,也购买商业(杰克逊, 007914) 和 (FSP1cre)+/- ;tdTomatolox-停?...

<ol>
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可以说,体内2光子成像最重要的步骤是:1)为多光子设置和实验需求选择正确的基因报告小鼠和荧光标记蛋白21、22;2) 成像正确的焦平面,以准确表示组织中细胞群23;3) 尽量减少因动物不适当固定而的运动24;4)选择何时对数据进行定性分析,而定量分析数据为25、26、27。<s...

脂多糖(LPS)是在克阴性细菌1的外膜中发现的内毒素。LPS对收费受体4(TLR4)/CD14/MD2受体复合物2具有高结合亲和力。TLR4是一种模式识别受体,常见于广泛免疫细胞、中位细胞和感觉神经元3、4、5的外膜。在免疫细胞上表达的TLR4的激活导致MyD88依赖和独立的第二信使系统,最后以核因子卡帕β(NF+B)易位到细胞核结束。这导致原体免疫细胞产生和释放亲炎细胞因子,如白细胞介素(IL)-1+,IL-6和TNF-+6。然而,其他细胞类型对TLR4刺激的反应并不清楚。纤维细胞已涉及广泛的病理学,如癌症和囊性纤维化,最近已被证明起一种单细胞化疗吸引和促进炎症7,8,9的作用。 我们的实验室对成纤维细胞在急性和慢性疼痛发展中的作用感兴趣,因为早期证据表明,纤维细胞(基质金属蛋白酶(MmPs)、金属蛋白酶(TimPs)组织抑制剂和成纤维细胞释放的因素生长因子(FF)涉及神经病痛10。
细胞的活化状态可以由多种因素决定,包括:诱导立即早期基因,改变蛋白质表达,细胞增殖和形态变化11,12,13。有许多技术存在,以回答我们可能有问题,细胞的激活如何有助于病理,但他们都有其局限性。原型免疫组织化学使用荧光标记抗体在固定组织中标记感兴趣的蛋白质,这些蛋白质可能是非特异性的,并且往往需要大量的故障排除,才能产生丰硕的成果14。西方印迹是比较死后组织蛋白质表达水平的有用技术;然而,组织学成分缺乏这种技术,研究人员无法识别形态学的任何变化15。RNA-Seq使我们能够量化样本中信使RNA的存在,在许多情况下,该样品会产生有见地的数据;然而,转录和翻译之间的差距使得在刺激16之后很难进行时间解析。共聚焦成像有助于确定存在于组织横截面的蛋白质的表达和共定位17。通常,这不能代表整个组织样本。相比之下,多光子显微镜允许用户将大约1毫米深的图像成像到样品中,从而形成全面的三维表示。因此,我们选择专注于体内2光子成像制备,因为这些实验收集的数据更直接地与生物组织高度可塑性和相互连接的微环境相对应。
研究体内蛋白质-受体相互作用的一个优点是,我们可以清楚地捕捉细胞在原生环境中对刺激的反应,没有死后组织提取的有害和不可预知的影响。此外,可以进行纵向研究,以评估由于激活而可能发生的细胞可塑性和充注性。 使用2光子成像,我们保留了应用外部刺激时微环境的完整性。该协议提供了一种方法,以识别在体内几个小时内注射荧光标记LPS后成纤维细胞中分子的摄取量,以及TLR4在成纤维细胞活化中的作用。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:1131px;">10x PBS, 4 L</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:116px;">BP3994</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">700 Series MICROLITER Syringes, Hamilton, Model 705 LT Syringe</td>
			<td>Hamilton</td>
			<td>80501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Precision Glide Needle 30G</td>
			<td>VWR</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blue Pad</td>
			<td>Fisherbrand</td>
			<td>1420665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Cube: Green/Red (BP 495-540 DM570 BP 575-645)</td>
			<td>Olympus</td>
			<td>FV30-FGR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane, USP 250 mL</td>
			<td>Vedco</td>
			<td>50989-150-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipopolysaccharides from Escherichia Coli O111:B4 - FITC conjugate</td>
			<td>Sigma-Aldrich</td>
			<td>F3665-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Main scanner laser: Spectra Physics INSIGHT DS+ -OL pulsed IR LASER, tunable from 680 nm to 1300 nm, 120 fs pulse width at specimen plane</td>
			<td>Spectra Physics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Centrifuge Tubes, 1.5 mL</td>
			<td>VWR</td>
			<td>20170-333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiphoton Microscope: Olympus MPE-RS TWIN</td>
			<td>Olympus</td>
			<td>MPE-RS TWIN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective: Ultra 25x MPE water-immersion objective 1.05 NA, 2 mm WD</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Personal Lubricant Jelly (Gel)</td>
			<td>equate</td>
			<td>ZH727 2E F1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SGM-4 Stereotaxic Apparatus</td>
			<td>Narishige</td>
			<td>16030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SomnoSuite Low-Flow Anesthesia System</td>
			<td>Kent Scientific Corporation</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stimulation laser: Olympus-specific SPECTRA PHYSICS MAI TAI HP DEEP SEE-OL pulsed IR LASER, tunable from 690 nm to 1040 nm, 100 fs pulse width at specimen plane</td>
			<td>Spectra Physics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo two-color 2-photon imaging of genetically-tagged reporter cells in the skin

纤维细胞是中位细胞,在激活时改变其形态,最终影响它们所在的组织的微观环境。虽然传统的成像技术在识别固定组织中的蛋白质相互作用和形态方面很有用,但它们无法洞察细胞能够以多快的速度结合和摄取蛋白质,一旦激活了细胞的形态变化。体内。在本研究中,我们提出两个主要问题:1) 通过类似收费的受体-4(TLR4)和脂多糖(LPS)相互作用进行成纤维细胞活化的时间过程是什么？2) 这些细胞一旦激活后如何运作？利用2光子成像技术,我们开发了一种新技术,以评估LPS-FITC与基因报告小鼠线中外周成纤维细胞表达的共生受体TLR4结合的能力;FSP1cre;在体内的tdTomatolox-停止-洛克斯。这种独特的方法使研究人员能够创建深入、延时的视频和/或蛋白质与活细胞相互作用的图片,从而使一个人在了解蛋白质如何改变细胞行为方面拥有更高的粒度。

最初,我们向野生型小鼠的后爪注射LPS-FITC,以可视化LPS-FITC在爪子皮肤层中存在的所有细胞类型的接受。在野生型小鼠(视频图1,图2)中观察了后爪取影LPS的皮肤层中无数的细胞(视频图1,2),我们试图专门针对成纤维细胞,因为它们是我们研究的主要焦点。在成像注射LPS-FITC的动物的爪子之前,我们希望清楚,皮肤层中没有细胞固有的荧光。这是为了确保注射后,我们拍摄的图像是细胞与荧光标记...

Watch this Scientific Journal Video about In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin at JoVE.com

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

激活后免疫反应性成纤维细胞发生形态变化,促进细胞招募的改变。结合基因工程的纤维细胞特异性蛋白1(FSP1)-cre,利用2光子成像;tdTomato 絮凝停止絮凝(TB/TB)小鼠线和绿色荧光标记的脂多糖-FITC,我们可以说明在皮下成纤维细胞和体内形态变化中脂多糖的高度特异性吸收。

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

我们的协议使我们能够实时可视化荧光标记细胞对活体动物中炎症外周刺激的反应。2-光子成像允许对活标本的深层组织进行可视化，保持其细胞及其微环境的完整性，并提供生物系统的准确表示。呼吸会随时间移动爪子，导致模糊和焦点平面丢失。在成像之前，一定要用胶带将爪子牢牢地贴在稳定的表面上。在此，你必须能够找到合适的平面的兴趣，确保目标是接近爪子没有接触，并直接在注射部位上方。在开始该过程之前，打开多光子系统并选择 25 倍主观。将立体机械装置放在多光子显微镜的舞台上，然后将仪器连接到麻醉输送机。将一块哑光黑纸放在设备表面，作为鼠标爪的连接点。将谐振扫描仪设置为 512 到 512 微米的固定扫描区域。将激发激光器分别调谐为绿色和红色荧光蛋白信号的 930 和 1，100 纳米激发波长。使用 690 至 1，050 纳米的二色镜将两个激励激光器的光路径引导到单个目标，使 930 纳米调谐激发激光器反射到主扫描仪，1，100 纳米调谐激光器直接进入主扫描仪。然后将 FITC 的激光功率设置为 5%，将绿色荧光蛋白设置为 20%，并关闭顶灯。对于体内成像，麻醉鼠标并在文本协议中概述。确认麻醉鼠标对手趾捏没有反应，然后将鼠标放在可接触到鼻锥的立体仪器中。使用黑色胶带将后爪牢牢地贴在黑纸上，同时与感兴趣区域保持近向，确保爪子的植物表面畅通无阻，朝向目标。将大量水性润滑剂放在爪子的植物表面。触摸目标到润滑剂，在爪子和目标之间创建一列液体。使用 FITC 激发光对焦到爪子的皮层中，确认可可视化串联的二丁二暗番茄标记成纤维细胞。用两个激光成像位于后爪植物表面下方的细胞区域。以每片约 1 微米的速度获得大约 5 到 10 Z 切片的 15 分钟时间，以建立环境的基线表示。获得基线成像后，每 20 微升 PBS 装载 25 微升玻璃汉密尔顿注射器，并装载 5 微克 FITC 联结脂聚糖或 LPS。在不干扰爪子位置的情况下，通过植物内注射到实验后爪中来管理溶液。然后用两个激光成像位于后爪植物表面下方的细胞区域。获得 60 到 120 分钟的时间间隔约 5 至 10 Z 片，每片约 1 微米，以识别 LPS FITC 的细胞中培养中植物内吸收。由于皮肤层内的细胞没有固有荧光，因此在野生型小鼠的植物内注射后，可以观察到后爪皮肤层中的无数细胞服用荧光标记LPS。LPS FITC 注射后，只有成纤维细胞特异性蛋白质一个正成纤维细胞表达像受体四结合，并吸收注射的蛋白质与这些细胞表达的串联二丁番茄标签的高水平共定位。相比之下，像受体一样有4个的老鼠在注射后不会结合和吸收LPS。事实上，LPS FITC注射后，细胞轮廓可见，表明药物在细胞周围的间束流体中分散，但实际上并没有被受体所束缚。在此协议中要记住的最重要的事情是确保爪子的固定，以便视频中不会因运动或呼吸而失真。使用这个程序，可以跟踪在受伤后将荧光标记细胞的招募到一个区域，以及细胞对刺激反应的形态变化。因此，2-Photon成像允许研究人员将基因报告小鼠和荧光标记的化合物相结合，以评估注射后活体动物发生的情况。

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

三维光学分辨率的光声显微镜

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

科研

生物工程

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

多参数的Langendorff灌注兔心脏的光学测绘

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Viral Nanoparticles for In vivo Tumor Imaging

Viral Nanoparticles for In vivo Tumor Imaging

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

一个完整的皮肤缺损模型，以评估生物材料的血管化<em&gt;体内</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

使用多通道双电场微流控芯片肺癌细胞Electrotaxis研究

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...