Name: afm and microrheology in the zebrafish embryo yolk cell
Uploaded: 2017-11-29T12:30:00
Description: Watch this Scientific Journal Video about AFM and Microrheology in the Zebrafish Embryo Yolk Cell at JoVE.com

我们感谢 Amayra 埃尔南德斯和菲利普-亚历山大 Pouille 参与建立这些协议的基础。我们还感谢 IBMB 的分子成像平台, 泽维尔伊斯特班和实验室成员的持续支持。加泰罗尼亚自治区加泰罗尼亚和 dg 的联合小组方案以及西班牙经济部和竞争力向教统局和 DN 提供的 Consolider 赠款支持了这项工作。

下面所述的所有协议步骤都遵循我们机构的动物保育准则。
1. 斑马鱼文化
<ol><li>在标准条件下培育和维持成年斑马鱼。 注: 本研究采用 AB 型和铊类野生胚。</li>
	<li>收集胚胎并在28.5 ° c 的 E3 胚培养基上生长它们24。根据先前描述的19的形态学对它们进行阶段。</li>
</ol>2. 原子力显微镜
<ol><li>手动 dechorionate 分期斑马鱼...

<ol>
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本文通过 AFM 和纳米粒子 microrheology, 研究了斑马鱼卵黄细胞在外包中的物质特性和生物力学参数。
在生理条件下, afm 被用来检索细胞和组织的流变学特征4,5,25,28, 这里我们开发了一个应用 afm 的协议, 用于完整的开发我们使用的胚胎在外包期间测试斑马鱼胚胎的卵黄细胞表面。
...

生物力学控制形成过程的物理原理在很大程度上是没有定义的。虽然生物力学研究在分子和细胞水平正在积聚相当大的动量, 生物力学参数的探索在组织/有机体水平是在它的初期阶段。流体力学回归1或视频力显微镜2允许研究人员区分主动和被动的力量, 而激光显微手术3, 或较少侵入原子力显微镜 (AFM) 的离解细胞4或在体内5或纳米粒子 microrheology6允许对组织的机械性能和响应进行预测。
AFM 是一个三维的地形技术, 具有高的原子分辨率, 解决表面粗糙度和遵守从悬臂尖端的挠度后, 与探测表面7。采用 AFM 的不同方法来研究表面性质, 包括测量摩擦、附着力和不同材料的粘弹性特性。最近, AFM 已成为一个强大的工具, 以获取信息的机械性能的生物样品。特别是, AFM 可以性提取复杂的剪切模量从单一的细胞应用振荡凹痕与微尖端连接到一个已知的弯曲常数8。这让我们推断出产生的力。然而, AFM 不是唯一的, 不同的方法可以从单个细胞中提取流变数据和机械特性 (例如,微吸入9, 磁扭式细胞仪 (MTC)10, 或单轴拉伸测试11)。
然而, 这些新方法在复杂的形态发生过程中的应用并不简单。在确定胚胎组织的机械性能时所面临的主要挑战是小 (µm 至 mm), 软 (在 102 -104 Pa 范围) 和粘弹性 (引起时间依赖性现象) 的性质的材料12。因此, 重要的是要调整的方法, 以确定机械性能 (刚度, 粘度, 粘附) 的具体情况下, 胚胎和发展中的有机体。在分析研究发展的流变学方法时, 需要考虑两个重要的问题: 避免侵入性干扰, 并提供易于获取。在这种情况下, 微的愿望, MTC, 和拉伸试验展览的限制。在第一种情况下, 细胞遭受的高变形可能改变它的生理和机械特性9。在第二种情况下, 需要坚定地坚持实验组织的基板, 以确保施加的应力变形的骨架局部也可以引入影响细胞的活化状态, 因此, 在其机械特性10.最后, 单轴拉伸方法受发育生物体的几何和可达性11的限制。
AFM 似乎更适合于研究发展过程, 因为它允许研究生物样品直接在他们的自然环境, 没有任何繁琐的样品准备。此外, 由于胚胎组织的离解通常是困难的, 减少的原子力显微镜探头的尺寸提供了高度的通用性, 在选择介质 (水或非水滴), 样品温度或化学物质的类型上没有限制。样品的组成。AFM 具有足够的通用性, 可以应用于大的领域和时间尺度, 并被用来检索不同发育阶段或生理条件下组织的物质特性。整个原生组织, 如动脉13或骨骼的14已从地形角度进行了研究, 在某些情况下, 如巩膜, 机械性能也被检索15。在爪16的活体胚胎中也探索了地形, 允许可视化, 例如在形态发生过程中细胞重排。最后, 开发了全面的样品制备规程, 以确定不同发育过程中的力学参数。压的地图已经生成的小鸡胚胎消化道的本地无固定组织切片11;从 gastrulating 斑马鱼胚胎中分离出的单个外胚层、胚层和内胚层祖细胞的胶粘剂和力学性能4;禽胚外植体叶和原始条纹的刚度测量17;在爪5的胚脑中直接定义的一种明显的刚度梯度模式。
应用 AFM 探索胚胎发育的一个重要的质的飞跃可能来自于简单的容易接近的形态发生过程。斑马鱼外包是一个重要的和保守的事件, 其中不同的组织协调在一个限制的球形空间, 以指导扩大胚在几个小时。在斑马鱼外包 (球形阶段), 表面层的细胞, 包围层 (EVL), 涵盖了半帽的卵中心的动物极点坐在一个巨大的卵黄合胞细胞。外包包括 EVL 的皮质植物病房扩张, 胚的深细胞 (DCs), 和蛋黄周围的合胞卵黄细胞 (e-YSL) 的外层。外包结束与 EVL 和 DCs 在植物极点18,19,20。在外包期间如何以及在何处生成力以及它们是如何进行全局耦合的还不清楚1,21。
在这里, 我们详细地描述了如何应用 AFM 在斑马鱼卵黄细胞的外包进展中推断被动的机械组织特性在体内。为此, 我们采用了小微球连接到 AFM 悬臂作为探针。这允许在不均匀的卵黄细胞表面上检索精确的局部信息, 并研究拉伸梯度及其随时间变化的动态。替代 AFM 方法, 如那些使用楔形悬臂22, 将不会呈现足够精确的本地数据。楔形技术, 需要非常仔细的操作技巧, 以胶水楔形大于样本大小在悬臂底, 不太适合探测胚胎 (〜2倍大于悬臂的长度) 力学。
用定性和定量的方法对细胞的粘弹性特性进行建模和表征, 可以提高对其生物力学的认识。从生物力学的角度来看, 必须了解外包, 而不仅仅是要求知识的皮层张力测量和动力学, 可以提取的 AFM;因此需要关于组织的生物物理特性的信息。为了提取这些信息, 在不同的生理条件下, 为了刻画不同的细胞类型, 我们已经开发了各种细胞流变学技术23。它们包括 AFM、MTC 和光学镊子 (OT)。然而, 这些方法在胚胎或器官的尺度上被证明不足以进行介观分析。作为替代, 我们已经成功地适应了纳米粒子跟踪 microrheology6的体内流变测量。该技术分析了单个粒子的布朗位移, 推导了局部微机械特性。
原子力显微镜和纳米粒子 microrheology 允许定义的皮层张力动力学和内部力学性质的蛋黄在外包。这一信息完全赞同一个模型, 其中各向异性的应力发展的结果的差异, EVL 和卵黄细胞质层 (共青团) 的变形反应, 以各向同性动收缩的 e-YSL 皮层是必不可少的EVL 和外包进程的定向网移动1。

<table>
	<tbody>
		<tr>
			<td>Inverted optical microscope</td>
			<td>Nikon</td>
			<td>TE2000</td>
			<td>Visualization of the sample and AFM cantilever</td>
		</tr>
		<tr>
			<td>Atomic force microscope (AFM)</td>
			<td>Custom made</td>
			<td>-</td>
			<td>Any commercialized AFM could be employed</td>
		</tr>
		<tr>
			<td>Inverted Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM780</td>
			<td>Nanorheology data collection</td>
		</tr>
		<tr>
			<td>Agarose D1 Low EEO</td>
			<td>Conda</td>
			<td>8016.00</td>
			<td>Casting embryos</td>
		</tr>
		<tr>
			<td>Ultrapure LMP Agarose (low melting)</td>
			<td>Invitrogen</td>
			<td>16520-100</td>
			<td>Securing embryos</td>
		</tr>
		<tr>
			<td>Zebrafish Microinjection and Transplantation Molds</td>
			<td>World Precision Instruments</td>
			<td>Z-MOLDS</td>
			<td>Casting embryos. Custom made with the original dimensions can also be employed</td>
		</tr>
		<tr>
			<td>Dumont 5 Forceps</td>
			<td>FST</td>
			<td>11251-20</td>
			<td>1.5 mm diameter</td>
		</tr>
		<tr>
			<td>Si3N4 cantilever</td>
			<td>Novascan</td>
			<td>PT.GS</td>
			<td>Measuring the embryo by AFM. Spherical tip: 4.5 &#181;m diameter and k=0.01 N/m</td>
		</tr>
		<tr>
			<td>Fluorescent nanoparticles</td>
			<td>Life Technologies</td>
			<td>FluoSpheres F8811,</td>
			<td>For tracking nanorheology</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-2000</td>
			<td>Fabricating tailored microneedles</td>
		</tr>
		<tr>
			<td>Borosilicate capillary glass</td>
			<td>Warner Instruments</td>
			<td>G100TF-4</td>
			<td>Fabricating tailored microneedles</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td>Manipulating the micropipette</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Eppendorf</td>
			<td>FemtoJet Express</td>
			<td>Controlling injection time and pressure</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>DFC365FX</td>
			<td>Visualization of the embryos during injection</td>
		</tr>
		<tr>
			<td>Analysis Software</td>
			<td>MathWorks</td>
			<td>Matlab</td>
			<td>Analyzing AFM and particle tracking data</td>
		</tr>
		<tr>
			<td>Zebrafish</td>
			<td>AB and TL wild type</td>
			<td>-</td>
			<td>Strains employed for embryo collection</td>
		</tr>
		<tr>
			<td>Glass Bottom Plates</td>
			<td>Mat Tek</td>
			<td>P35G-0.170-14-C</td>
			<td>Mounting embryos for nanorheology data collection</td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>FST</td>
			<td>29025-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

afm and microrheology in the zebrafish embryo yolk cell

阐明影响组织演变的时空组织的因素是发展研究的主要目的之一。各种命题在不同的发育和形成过程中对其时空组织中细胞和组织的力学性质的理解有重要的贡献。然而, 由于缺乏可靠和容易接近的工具来测量材料的性质和张力参数在体内, 验证这些假设是困难的。在这里, 我们提出的方法采用原子力显微镜 (AFM) 和粒子跟踪的目的是量化的力学性能的完整斑马鱼的胚胎卵黄细胞在外包。外包是一个早期保守的发展过程, 其研究是由胚胎的透明度促进。这些方法是简单的实施, 可靠, 广泛适用, 因为他们克服侵入性干预, 可能影响组织力学。一个简单的策略, 用于安装标本, AFM 记录, 和纳米粒子注射和跟踪。这种方法可以很容易地适应其他发育时期或生物体。

皮质张力测量 对于每个测量点, 五力-位移 (F-z) 曲线获得了 AFM 通过斜向1赫兹的峰振幅为5µm (速度 = 10 µm/秒) 高达最大缩进〜2µm。这个程序是采取少于20分钟, 并没有受到外包进展。每个实验条件都在至少5胚胎中进行测试。
在胚胎卵黄细胞上记录的力-位移曲线, 与软的周围琼脂糖相对应, 呈现成正比线性关系 (R<s...

Watch this Scientific Journal Video about AFM and Microrheology in the Zebrafish Embryo Yolk Cell at JoVE.com

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

缺少用于测量材料属性和拉伸参数的工具在体内可防止在开发过程中验证它们的角色。我们使用原子力显微镜 (AFM) 和纳米粒子跟踪来量化外包期间完整的斑马鱼胚胎卵黄细胞的机械特性。这些方法是可靠的, 广泛适用于避免介入干预。

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

The overall goal of these procedures is to adapt specific methodologies for the determination of mechanical properties such as, stiffness, viscosity and adhesion to the specific case of embryos and developing organisms. The methods that we introduce in this paper can answer questions in the mechanobiology field, such as how their properties may respond to genetic or physical input during development or a pinch in the regulation of cellular activities. The many advantages of this technique is that they allow retrieval of crucial information with minimal manipulation, addressing questions that are inoperable by other procedures.These methods provide insight into understanding the role of the yolk cell during a and then they can be applied to different domains in the embryo to different developmental status and even to other model organisms. Visual demonstration of this methods is critical as embryo mounting and handling are difficult to learn. To begin, collect zebra fish embryos and grow them at 28 degrees Celsius in E3 embryo medium.Stage the embryos according to morphology. Manually decurionate stage zebra fish embryos at different ages, according to individual interest under a dissecting stereo microscope with an adjustable range in magnification between eight times and 50 times. Remove the chorions with two thin and sharp forceps.Grip the chorion using one set of forceps and make a tear in it with the other set of forceps, then, holding the chorion in a region opposite of that of the tear, push the embryo gently through the opening. To mount the embryos for AFM, first fill a 35 millimeter petri dish with 2%agarose and embryo medium and let it solidify. Then, make small holes of approximately 1.5 millimeter in diameter and approximately 350 microns depth with thin forceps in the agarose bed.Place the embryos in the pokes make in the agarose layer. Then, pour a solution of 0.5%low melting agarose and embryo medium, just around the embryos to secure them in the holes. Before the low melting point agarose solidifies at room temperature, rotate the embryos in such a way that the region of interest to be probed by AFM will face upwards.Examine the embryos. Locate and image them in the petri dish, employing a 20 times objective in an inverted optical microscope coupled with the atomic force microscope at room temperature. Probing the yolk cell surface at the right angle and at the right amplitude of oscillation and frequency is essential to retrieve those data.Different tests must be performed to optimize this parameters. Probe the yolk cell surface of the casted embryos, employing spherical polystyrene beads to 4.5 micron in diameter, attached to a cantilever with a nominal spring constant of 0.01 Newtons per meter. Set the peak to peak amplitude of cantilever oscillation to five microns and it's frequency to one Hertz.Collect data for each region to be tested in different positions and in several embryos as a routine for data averaging. To perform particle microinjection into the yolk cell fabricate tailored microneedles using a vertical micropipette puller in borosilicate capillary glass. Prepare microneedles that have an outer diameter or 1.00 millimeter, an inner diameter or 0.58 millimeters and a length of 10 centimeters.Prepare an injection petri dish plate by creating straight indentation lines in a 2%agarose and embryo medium bed employing custom made molds. Turn the molds upside down and place them on top of the liquid agarose gel. Remove the molds once the gel has solidified.Then, pipette the embryos into the grooves made by the molds in the agarose, under dissecting stereo microscope at 1.2 times magnification. Before injection, almost completely remove the medium. The surface tension prevents the embryo and chorion from sticking to the needle, when removing it after injection.Injection of the correct amount of piece is critical and will facilitate their dispersion within the yolk, afterwards and also, it's important for the retrieval of them in square displacement. Use florescent nano particles diluted in water for microinjection into the vegetal part of the embryo yolk cell. Adjust the micropipette with precision micromanipulators and use an automatic microinjector with time and pressure controls.Set the pressure between 10 and 20 psi. Employ a magnification of 1.6 times in the dissecting stereo microscope to visualize the embryos during the injections with are performed at room temperature. To assess the viscoelastic behavior of the yolk, first decurionate the microinjected embryos, as before.Then, embed the embryos in a 0.5%low melting point agarose and embryo medium solution at 30 degrees Celsius. While the agarose is still liquid, transfer the embryos to glass bottom plates. Orient and push them towards the cover slip.Place the embryos mounted in the glass bottom pates on the stage of a confocal inverted microscope two hours after microinjection. Capture images of the nano particles for 26 seconds at a sampling rate of 25 Hertz, with a 63 times objective at room temperature in an inverted confocal microscope, employing standard commercial microscope software. Force indentation curves were retrieved from individual AFM measurements at a distance of 10 microns from each other in different embryos.On average, force curves reflect a linear increase in deflection upon contact with the embryo yolk cell surface. And accurate fitting of the data with a liquid balloon model let's to infer cortical tension values, which increased gradually towards the vegetal pole and with time. Further, the observed differences between the approaching and retracting deflection values retrieved at AFM at each point in the force displacement curves, indicates the viscoelastic character of the cortex.This viscoelastic character of the yolk cell cortex can be characterized by multi-frequency oscillations AFM measurements to retrieve it's effective complex modulus in Pascals. The elastic modulus is at least four times higher than the viscous modulus at all tested frequencies. On the other hand, the yolk mechanics was probed with microparticle rhealogy.The mean squared displacement of individual nanoparticles injected in the yolk cell exhibited an approximately linear dependence with time lag. The ensemble average of the MSD's of the nanoparticle's population exhibits predominantly viscous character and diffusive properties. From these data, the viscosity of the yolk was calculated to be 129 millipascal seconds.Once mastered, AFM can be done in a few hours if it's performed properly. Following this procedure, additional questions, like the role of a specific genes or mechanical inputs and tensional parameters or material properties can be studied. After watching this video, you should have a good understanding of how to determine the mechanical properties of the yolk cell of the zebrafish embryo during epiboly When attempting a particle injection rheological data retrieval, it is important to remember to properly stage the embryo and be precise on the quantities of media to be injected as not to perturb embryo development.After this development, this technique will help exploration of the mechanobiology of model organisms in development and possibly on pathological conditions.

Research

JoVE Journal

Developmental Biology

Microbead Implantation in the Zebrafish Embryo

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

科研

发育生物学

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

日本鹌鹑胚胎的卵黄囊膜主内胚上皮细胞培养

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

两鲑鱼鱼、虹鳟 (虹鳟鱼虹鳟鱼) 和褐鳟 (鲑鲑形态石斑鱼) 的空气剥离效果

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...