Name: afm and microrheology in the zebrafish embryo yolk cell
Uploaded: 2017-11-29T12:30:00.000+00:00
Duration: 9 min 47 s
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 分期斑马鱼胚胎 (不同年龄根据个人利益)。用两个薄而锋利的镊子将 chorions 取出 (请参见材料表)。<ol>
<li>用一个钳子夹住绒毛膜, 用另一个钳子把它撕裂。然后, 把绒毛膜在一个与撕裂的区域相对的地方, 轻轻地通过开口推动胚胎。</li>
		<li>执行 dechorionation 下的解剖显微镜与可调范围的放大倍数之间的8X 和50X。</li>
	</ol></li>
	<li>为 AFM 安装胚胎
	<ol>
<li>在胚胎培养基中制备2% 琼脂糖溶液, 并用此填充 35 mm 的培养皿。让它凝固。制造1.5 毫米直径的小孔 (两倍于胚大小) 和大约350µm 深度 (一半胚胎大小) 与薄钳在琼脂糖床上。</li>
		<li>将胚胎放入琼脂糖层中, 并通过在胚胎培养基中0.5% 低熔点琼脂糖的溶液中传播, 确保它们到位。把这个溶液倒在胚胎上, 把它们固定在洞里。</li>
		<li>在低熔点琼脂糖凝固在室温下, 旋转的胚胎, 在这种方式, 该地区的利益将被探测的 AFM 将面临向上 (图 1A)。</li>
	</ol></li>
	<li>用 AFM 检查胚胎
	<ol>
<li>在室温下 (23-24 ° c) 中, 在一个倒置的原子力显微镜中找到并利用20X 目标的培养皿中的胚胎定位和图像 (参见材料表25)。 注意: 胚胎被保存在胚胎培养基中并附着在琼脂糖床上。</li>
		<li>用直径4.5 µm 的球形聚苯乙烯珠, 用公称弹簧常数为0.01 米/秒, 对铸造胚的卵黄细胞表面进行探针研究. 将悬臂振荡的峰振幅设置为5µm, 频率为1赫兹。</li>
		<li>为每个区域收集数据, 以便在不同的位置和在几个胚胎中进行测试, 以作为例行数据, 以进行统计。 注: 一个好的出发点是探测五位置位于中间和角落的一个10µm x 10 µm 广场通过控制悬臂位置与 XY 压电驱动器的 AFM 显微镜。成像和数据收集约20分钟每胚。在胚胎卵黄细胞表面的不同区域记录数据,例如, 植物极或靠近 EVL 细胞边缘的不同区域 (图 1B和1C), 只能通过使用不同的不同标本来完成。</li>
	</ol></li>
	<li>计算力
	<ol>
<li>测量 AFM 悬臂梁 (z) 与压电驱动器耦合的应变式传感器的垂直位移及其偏转 (d) 用光学杠杆法与显微镜控制软件的象限光电二极管 (参见材料表25)。</li>
		<li>使用从玻璃片的裸区域收集的数据得到的 d-z 曲线的斜率, 以校准光电二极管信号与悬臂偏转 (d) 之间的相关性。 注: 测量的垂直位移等于悬臂偏转, 斜率表示光学杠杆的偏转灵敏度26。</li>
		<li>从先前描述的27的热波动推断出悬臂弹簧常数 (k)。</li>
		<li>将示例 (h) 的缩进计算为: h = (z-zc) (d-d关闭) (1)           注意: 这里的zc 是触点的位置, doff是光电二极管的偏移。</li>
		<li>计算悬臂上的力 (F) 为: F = k · d (2)</li>
	</ol></li>
	<li>计算的张力, 从一个液体气球模型, 包括一个弹性层的皮质张力 Tc包围一个粘性液体。在这个模型中, 对于小凹痕 (与胚胎的大小相比), 力按比例增加4,28为: F = π Tc[(rb / re) + 1)] h (3) 注意: 这里的Rb 是珠的半径, re 是胚胎的半径。对于斑马鱼胚胎, 作为胚胎的半径 (400 µm) 是两个数量级大于那珠 (2.25 µm), 这个等式可以近似地是: F = π Tc h (4)<ol>
<li>计算每个胚胎卵黄细胞区的张力-压痕 (F-h) 曲线, 用于五不同点的测量。</li>
		<li>通过非线性最小二乘法拟合, 计算每个Fh曲线的Tc 。对于统计分析, 所计算的皮质张力Tc 必须从不同的Fh曲线中进行平均。</li>
	</ol></li>
	<li>采用低振幅 (100 nm) 多振荡的方法, 在不同频率的正弦波组成的原子力显微镜下测量皮层的粘弹性特性 (流变学)25。
	<ol>
<li>在频率域中从力压痕曲线计算一个有效的复模量g * (ƒ) , 如下所示: g *(f) = [f(f)/ h(f)]- ifb (5) 这里i是虚数单位, F(f) 和h(f) 是力和缩进的频率 (f) 频谱。b是对从表面上应用的振荡中提取的悬臂的粘性阻力的修正。</li>
		<li>将g * (ƒ)分离为真实的和虚构的部分, 如: g *(ƒ) =           g"(f) + ig" (f) (6) em > g "(f) 是弹性模量, 是每周期振荡所储存和恢复的弹性能量的量度。g"(f) 是描述耗散能量的粘性模量。</li>
		<li>计算损耗正切值, 该切线提供了材料的固体样 (< 1) 或类似液体 (> 1) 行为的指数, 如: g"(f)/ g" (f) (7)           注: 有了这种类型的测量, 可以提取参数, 表明如何粘性和如何弹性是被探测的材料。虽然b稍微依赖于悬臂的末端到曲面的距离, 但由于卵黄细胞所展示的g´´的值非常低 (见图 2D ), 所以在b中的细微变化对度量值29。</li>
	</ol></li>
</ol>3. 粒子跟踪 Microrheology
<ol><li>
对卵黄细胞进行微粒显微注射
	<ol>
<li>使用水平微拉拔器和硼硅酸盐毛细管玻璃制造定制的微 (请参见材料表)。<ol>
<li>准备微, 外径为1.00 毫米, 内径为 0.58 mm, 长度为10厘米, 使用拉拔器设置: 压力: 500;热量: 510;拉力:65;速度:25;时间:50。</li>
		</ol>
</li>
		<li>制作一个注射培养皿板通过创建一个1% 琼脂糖在胚胎中型床使用定制模具的直压痕车道 (请参阅材料表)。<ol>
<li>把模具倒置, 并把它们放在液体琼脂糖凝胶, 并删除模具一旦凝胶已固化。在1.2X 放大倍数下, 将胚胎移入琼脂糖中的模子所制成的凹槽中的显微镜。 注: 模具的宽度和设计使胚胎调整。</li>
		</ol>
</li>
		<li>注射前, 几乎完全清除培养基以方便注射 (表面张力防止胚/绒毛膜在注射后将其粘附在针头上)。</li>
		<li>Microinject 荧光纳米粒子 (半径 a = 100 nm, 见材料表) 稀释在水 (1:1, 000) 在植物部分的胚胎卵黄细胞 (图 3A)。</li>
		<li>调整微与精密动和注入的珠子自动微量的时间和压力控制 (请参见材料表)。设置10和 20 psi 之间的压力。</li>
		<li>在注入磁珠溶液之前, 通过测量微量带 1x 0.01 mm 级微米的液滴尺寸来校准体积以注入 (0.5 nL) (参见材料表)。</li>
		<li>在解剖显微镜中采用1.6X 的放大倍数, 在注射过程中对胚胎进行可视化, 在室温下进行。</li>
	</ol>
</li>
	<li>
通过记录热涨落来评估蛋黄的粘弹性行为
	<ol>
<li>Dechorionate 微量胚在步骤2.1 中, 并将其嵌入到30° c 的胚胎培养基溶液中的0.5% 低熔点琼脂糖中。然后, 将它们转移到玻璃底板 (请参阅材料表), 并将其定向至片, 当琼脂糖仍为液态时。 注意: 当琼脂糖在室温下凝固时, 胚胎就可以被成像。</li>
		<li>将胚胎放置在玻璃底板上, 在共聚焦倒置显微镜的舞台上注射后2小时。利用标准商用显微镜软件 (像素大小 = 166 nm, 每40毫秒捕获的图像), 以25赫兹的采样率在室温下的63X 目标下捕获纳米粒子的图像。</li>
		<li>计算粒子的质心的位置和定义他们的轨迹随着时间的 TrackMate 插件的开放源码 ImageJ 软件 (图 3B)。</li>
		<li>用 custom-made 软件6,30计算每个粒子的 two-dimensional 均方位移 (下): < Δr2 (τ) > = < [x(t + τ)- x(t)]2 + [y(t + τ)- y(t)2>          (8) 注意: 这里的t是经过的时间和滞后时间。在纯粘性液体中, 根据斯托克斯-爱因斯坦关系, 该流变液与粘度 (ν) 反向增加:           < Δr2 (τ) > = 4kB Tτ/6πνa, (9) 其中, T是绝对温度。</li>
	</ol>
</li>
</ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheological+analysis+and+measurement+of+neutrophil+indentation.">Rheological analysis and measurement of neutrophil indentation.</a> Biophys J. 87, 4246-4258 (2004).</li><li>Alcaraz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+Microrheological+Measurements+of+Soft+Samples+with+Atomic+Force+Microscopy+for+the+Hydrodynamic+Drag+on+the+Cantilever.">Correction of Microrheological Measurements of Soft Samples with Atomic Force Microscopy for the Hydrodynamic Drag on the Cantilever.</a> Langmuir. 18, 716-721 (2002).</li><li>Daniels, B. R., Masi, B. C., Wirtz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+single-cell+micromechanics+in+vivo:+the+microrheology+of+C.+elegans+developing+embryos.">Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos.</a> Biophys J. 90, 4712-4719 (2006).</li><li>Kalwarczyk, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viscosity+of+complex+liquids+and+cytoplasm+of+mammalian+cells+at+the+nanoscale.">Comparative analysis of viscosity of complex liquids and cytoplasm of mammalian cells at the nanoscale.</a> Nano Lett. 11, 2157-2163 (2011).</li><li>Soroldoni, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+oscillations.+A+Doppler+effect+in+embryonic+pattern+formation.">Genetic oscillations. A Doppler effect in embryonic pattern formation.</a> Science. 345, 222-225 (2014).</li><li>He, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apical+constriction+drives+tissue-scale+hydrodynamic+flow+to+mediate+cell+elongation.">Apical constriction drives tissue-scale hydrodynamic flow to mediate cell elongation.</a> Nature. 508, 392-396 (2014).</li><li>Johansen, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+micromanipulation+of+nanoparticles+and+cells+inside+living+zebrafish.">Optical micromanipulation of nanoparticles and cells inside living zebrafish.</a> Nat Commun. 7, 10974 (2016).</li></ol>

作者声明没有相互竞争的金融利益或其他利益冲突。

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>Si&lt;sub&gt;3&lt;/sub&gt;N&lt;sub&gt;4&lt;/sub&gt; cantilever</td><td>Novascan</td><td>PT.GS</td><td>Measuring the embryo by AFM. Spherical tip: 4.5 &amp;micro;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>Measuring injection volume</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. ...

afm-and-microrheology-in-the-zebrafish-embryo-yolk-cell

Research

JoVE Journal

Developmental Biology

8.2K 次观看.  Consejo Superior de Investigaciones Científicas. 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。

视频: AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

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 ...

科研

发育生物学

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

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 ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

生物学

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

神经科学

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

使用光学镊子测量禁闭中的亚细胞力学的直接力

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

<meta charset="utf8"></head><body>斑马鱼胚胎的精细多视图光片显微镜实验方案

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

<meta charset="utf8"></head><body>作者聚焦:用于高分辨率多视图光片显微镜的斑马鱼胚胎安装策略 — 成像和图像重建技术

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

早期斑马鱼胚胎的光片显微镜成像和安装策略

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

摘要

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此视频中的章节

在活斑马鱼胚胎成像的亚细胞结构

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

观察有丝分裂和动力学在现场斑马鱼胚胎

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

斑马鱼幼虫显微注射和成像的微装置

现场共聚焦显微镜对斑马鱼的胚胎脑成像

解剖和斑马鱼的胚胎横向安装：脊髓发展分析

使用光学镊子测量禁闭中的亚细胞力学的直接力

早期斑马鱼胚胎的光片显微镜成像和安装策略

早期斑马鱼胚胎的光片显微镜成像和安装策略