我们感谢道格拉斯·本森先生关于二次上颚成像的初步对话。我们还承认David Castaneda-Castellanos（Leica）和Chris Rieken（蔡司）帮助调整共聚焦显微镜中的成像条件。感谢Lynsey Hamilton（Bitplane）对使用Imaris软件进行定量图像分析的有用建议。这项工作由NIH / NIDCR R01 DE025887资助。

所有动物实验均按照加利福尼亚大学旧金山机构动物护理和使用委员会的协议进行。 活动影像制作准备<ol><li> 液体培养基的制备 <ol><li>向Dulbecco&#39;s Modified Eagle培养基（DMEM）/ F12中加入20％胎牛血清（FBS），2mM L-谷氨酰胺，100U / mL青霉素，100μg/ mL链霉素，200μg/ mL L-抗坏血酸，15mM HEPES媒体。 注意:在成像之前，实时成像介质是新鲜的。这种介质含有酚红，这可能导致长时间的活体成像过程中的光毒性。代替不含酚红的培养基可以改善长期成像。 </li></ol></li><li> 制备低熔点琼脂糖溶液 <ol><li>将350毫克低熔点琼脂糖溶解在10毫升高压灭菌的蒸馏水中以制备3.5％储备溶液。 </li><li>将琼脂糖溶液充分混合，并在70℃在水浴中孵育以完全溶解琼脂糖。 </li><li>在37℃水浴中冷却琼脂糖溶液，然后将其加入到活体成像介质中。 </li><li>将1 mL琼脂糖储备液加入5 mL液体活体成像培养基中，使其达到最终浓度（0.6％）。 </li><li>将培养基用琼脂糖保存在37°C水浴中，以防止过早凝固。 </li></ol></li></ol> 2.准备活体外植体文化<ol><li> 近期附着的E14.5二级腭架的解剖 注意:在我们的经验中，成像融合需要从轻柔的一对腭架开始;这种粘附防止在成像期间防止融合发生的外植体运动。 <ol><li>在1x磷酸缓冲盐水（PBS）中解剖E14.5期胚胎，并选择绿色荧光蛋白（GFP）表达（来自K14-CRE; ROSA26-mTmG flox ）或（红色荧光蛋白）表达RFP（来自Lifeact-mRFPruby ）的阳性胚胎，使用装备有荧光灯的解剖显微镜。将组织转移到没有琼脂糖的预热活体成像培养基中。 </li><li>切割胚胎头并使用两个5号镊子去除上脑区域（ 图 1A-C ）。使用5号镊子握住胚胎，而另外5号镊子切除腭架外的额外组织。 注意:精剪可用于切割胚胎头（第一步），而不是一个5号镊子。然而，两个5号细镊子将能够更好地控制精确切割。 </li><li>没有如图1（D）和图1E损坏腭小心取出下颌骨。为了减少腭裂的机会，保持舌头通过下颌骨的解剖，f通过仔细去除舌头。 </li><li>在去除下颌骨和舌头后，用立体显微镜用荧光检查二尖瓣的口腔侧面，以确认MES中的强报道信号（ 图1 M ）。 </li><li>在去除下颌骨和舌后解剖后部部分，包括后脑和脑干（ 图 1F ）。 </li><li>用粘附的腭架除去两个上颌骨（ 图1 G ）。 </li><li>切割前上​​颌组织保持原发性和次级腭部完整（ 图1 H和I ）。 </li><li>去除露出MES的外植体鼻侧的鼻中隔。 注意:这些以后的解剖步骤需要注意，不要破坏两个辅助腭架之间的接触。去除鼻中隔有助于减少其他区域的背景信号，并减少垂直组织移动，使稳定成像（ 图1 J-L ）。 </li><li>解剖腭架后，再次检查中线记者信号，确保组织之间上皮层无损伤。 </li></ol></li><li> 在活体成像介质的培养皿中设置腭外植体 <ol><li>将活体成像介质放入35毫米玻璃底盘中。 </li><li>将解剖的腭骨架面朝下，尽可能靠近玻璃底部，用倒置共聚焦显微镜进行成像（ 图 N1 ）。 </li><li>将干冰粒放在培养皿附近的导电表面上，以快速降温或将培养皿放置在4℃加速琼脂糖凝固。也可以使用冷板，如用于石蜡包埋的冷板踏歌。 注意:当使用干冰时，需要仔细监测琼脂糖介质的变化以防止冷冻介质。 </li></ol></li></ol>腭内窥镜的共焦延时成像<ol><li> 成像和数据采集 <ol><li>琼脂糖半固化后，将培养皿安装在装有37℃孵育室的倒置共焦显微镜中。使用白光共焦显微镜和细胞观察器旋转盘共聚焦显微镜。 注意:我们使用含有15mM的HEPES无CO 2的气体的修改的媒体。 </li><li>使用注射器将培养皿和盖子之间的果冻放入培养基中以防止培养基蒸发（ 图 N ）。 注意:过夜（O / N）培养发生后，培养皿盖顶部有一些冷凝，但介质的体积不会改变，这表明石油胶可以防止培养基蒸发。 </li><li>使用低放大倍数目标（10x）找到感兴趣的区域，并使用更高放大倍数的目标（20倍物镜，3倍数码变焦或40x物镜与Immersol W）进行延时实时成像。 注意:解剖后的腭骨架不平坦。二级腭是一个弯曲的结构，这使得难以在同一平面上对整个MES进行成像。低倍率（10倍）成像可以允许全腭成像，但40倍的目标将提供更好的图像分辨率，以检查更深的Z位置的细胞细胞运动。中腭区曲率高。与中腭相比，前后腭相对平坦。我们经常朝向二级腭中段前方，以避免曲率最大的区域。 </li><li>使用适当的激光激发（对于GFP为488nm，RFP为532/561nm）扫描多个Z堆叠，每步5μm为16-24小时（ 图2 ）。 注意:为了降低潜在的光毒性，请使用最小的激光功率和曝光时间。特别是在多色成像的情况下尤为重要。使用52nm激光功率的22％用于membraneTomato（mT），30％的495nm激光功率用于膜GFP（mG）和25％的561nm激光功率用于膜RFP。平均曝光时间为550 ms。 </li><li>使用图像分析软件执行细胞跟踪和定量分析。 </li></ol></li></ol>数据图像分析注意:这里使用了Imaris。也可以使用ImageJ或Volocity软件包执行类似的数据分析。 <ol><li> 从图像序列渲染三维（3D）曲面。 注意:本部分可以从使用膜GFP信号生成的影片中生成曲面。 <ol><li>单击编辑|显示显示调整并使用直方图滑块调整信号强度，使膜信号清晰出片的长度（ 图3 的A）。 </li><li>使用图像正确漂白的信号 处理|衰减校正 。调整强度前沿和强度返回值，以使膜信号在整个电影长度内清晰。设置这些值可能需要一些试验和错误。 注意:由于激光穿透受到限制，强度返回（更深的Z位置）值将小于强度前沿（表面Z位置）值。因此，这些值也可以改变为使Z系列中的信号强度正常化。可以调整这两个值以实现漂白校正。衰减校正也称为发射衰减校正19 。 </li><li>为了从膜信号产生表面，选择Surpass |表面 ，选择源通道和阈值类型（绝对强度），并在算法设置下LECT 仅划分兴趣区域 并完成整个图像的处理 ，然后点击向前箭头;继续按向前箭头（使用默认设置，或通过反复试验指定表面细节等级和阈值方法），检查生成的表面是否反映荧光信号。要指定兴趣区域，请选择通过裁剪X，Y，Z位置来呈现的区域和时间。 注意:选择 整个图像的过程最终将使整个电影的调整和阈值。 </li><li>再次单击向前箭头按钮生成曲面。然后通过更改滤波器中的直方图来调整阈值，以对应于膜信号。 </li><li>表面生成后，在"曲面/设置"中选择曲面，以便可视化表面信号，并通过推动双重打击来完成表面生成过程箭头按钮（ 图3 B ）。 </li><li>去Surface |编辑并单击蒙版 属性中的 全部 遮罩以生成蒙版通道图像。 </li><li>点击确定后，屏幕通道窗口将自动打开;选择GFP通道，将表面外部的体素设置为最大信号强度值（在编辑|显示调整中找到 ）和表面内的体素到最小GFP强度值（也见于 编辑|显示调整 ），在面罩设置中生成膜信号的掩码。 </li><li>由于膜信号的掩模将在体视图中显示，请使用图像处理|生成反转图像对比变化|反转 （ 图3 C ）。 </li></ol></li><li>从反面图像中检测斑点。 注意:此部分允许每个单元格的中心标志着一个独特的地点，跟踪了太空和时间。 <ol><li>选择超越场景|斑点|创建 ;在算法设置中，选择仅划分兴趣区域， 最终完成整个图像的处理和跟踪点（随着时间的推移） 。 </li><li>点击向前箭头并指定感兴趣的区域;选择区域和时间跟踪斑点;继续按向前箭头，选择屏蔽通道作为源通道，并确定点的估计XY直径。 </li><li>点击向前箭头按钮将自动打开一个过滤器窗口;通过改变直方图中的值来调整点检测阈值，以在每个单元格的中心产生单个点（ 图3 D ）。 </li><li>再次点击向前箭头按钮将打开算法窗口，选择自回归运动跟踪模型 20，并指定最大跟踪di立场和最大间隙大小，以连接在短时间内变得不连续的轨道。 </li><li>再次点击向前箭头按钮将打开"过滤器"窗口，通过调整直方图来设置跟踪持续时间阈值，以便跟踪每个单元格。单击双向向前箭头按钮将完成点检测。 </li><li>为了纠正组织漂移，请点击Spots |轨道编辑器，并选择正确的漂移 。 算法窗口将自动打开;选择平移漂移，包括整个结果和正确的对象位置 。 </li></ol></li><li> 生成有利位置进行定量分析。 注意:程序的Scatterplot是用于生成具有多个对象的多维（X，Y，Z，颜色和缩放）图形的工具。这些图可用于查看许多单元格移动随时间变化的趋势。 <ol><li>使用"2D"或"3D"绘制单元格跟踪数据Vantage-Add New Vantage绘图菜单（ 图3 E和3F ）。 </li><li>选择卷或点，然后选择创建/类别和绘图类型 </li><li>调整绘图值以生成不同的有利图。 </li><li>使用快照功能导出图形。 注意:统计数据也可以作为电子表格导出，以进一步进行定量分析，方法是点击绘图数字区域保存 。 </li></ol></li></ol>

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作者没有什么可以披露的。

使用3D器官外植体培养的组织形态发生的活体成像可以提供关于固定组织切片的常规染色分析中不能显示的细胞过程的详细信息。使用小鼠胚胎中性腭裂的体外外植体培养，我们观察到几个有趣的细胞行为，导致我们提出了一种涉及上皮收敛和细胞挤出的腭裂融合的新机制。 在这种研究中的一个常见挑战是通过长时间的连续激光激发进行光漂白。在我们的研究中，使用...

组织融合是多器官发育的重要一步。主要的人类出生缺陷如唇腭裂，脊柱裂和心脏畸形可能会导致从在组织融合1缺陷。小鼠二级腭融合已被广泛研究，以确定细胞和分子机制发展2，3，4控制组织融合。在小鼠中，二尖瓣发育开始于E11.5附近，其中每个双侧上颌过程均产生二级腭骨架。腭架的初始生长沿着舌头垂直发生，直到大约E14.0，此时腭架在舌头上方水平上升。中度指导的生长导致两个腭搁架的上皮的物理接触，形成中线上皮（MES）在E14.5。必须从中间腭架之间移除插入的MES，以允许间充质融合，并通过E15.5 3开发完整的，完全融合的中腭。 如何在两个单独的腭架之间形成共享的上皮MES细胞层，然后移除以实现间充质融合，一直是腭发育的中心问题。基于小鼠组织学和电子显微镜（EM）研究，外植体培养研究和功能小鼠遗传学实验，几个基本细胞行为已经涉及到这个过程。从每个货架腭的内侧边缘上皮MEE丝状伪足状突起便于初始接触5， 图6，随后将这些上皮细胞的嵌入到共享单个MES 6,7。删除生成的共享MES已被提议通过三个非排他性机制进行。采用组织学观察和离体谱系用活体染料示踪的早期研究表明，MES可能通过上皮被除去以MES细胞的间充质转换（EMT）8,9，虽然最近，上皮细胞的遗传谱系追踪已升高的不确定性，以上皮细胞的长期贡献腭间质架10，11，12。凋亡细胞的数量显著，并在他们的人数在某些突变体不能进行适当的味觉融合的减少，导致了想法，凋亡可能是MES溶解2,3的主要驱动力。最后，最初基于涉及上皮标记和进行时间点静态观察的研究MES细胞提出在口鼻及前后尺寸11，13迁移，但这样的动态细胞行为最初未经证实的由于无法观察他们在活组织腭。最近，我们能够通过开发一种新的活体成像方法直接观察这些行为，该方法结合了荧光标记的小鼠遗传方法与外植腭架的共聚焦活检。 首先，腭融合过程中可视化腭上皮细胞动态细胞行为，我们通过杂交ROSA26-mTmG FLOX小鼠Keratin14-CRE小鼠14,15生成的上皮特异性报道小鼠。所产生的胚胎的腭外植体培养物的共聚焦活体成像证实了一些先前提出的细胞行为，并在融合过程中鉴定了新的事件6 </SUP&gt;。上皮细胞膜突起先于初始细胞 - 细胞接触，然后通过细胞插入和鼻孔细胞置换上皮收敛。值得注意的是，我们还发现，电池挤出，报道在上皮内环境稳定中发挥重要作用的过程，是一个重大的机构驱动鼠标继发腭融合6,16。该成像方法可与其他记者行一起使用;我们利用Lifeact-mRFPruby转基因小鼠17，18在融合过程来检查肌动蛋白细胞骨架的动态。其他记者也可以用来观察腭部融合的其他具体方面，这种方法可以适用于激光扫描共焦显微镜或旋转盘共焦显微镜，这取决于成像需要和显微镜可用性。现场影像越来越成为发育生物学中的基石方法。班驳轻微地，颅面形态发生是复杂的，影响面部的人类出生缺陷是常见的。这种共焦现场成像方法将有助于更好地了解潜在的基本发育机制以及人类颅面异常的起源。

<table border="0" cellpadding="0" cellspacing="0" width="1193">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Reagents</td>
			<td style="width:360px;"></td>
			<td style="width:225px;"></td>
			<td style="width:260px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DMEM/F12</td>
			<td>Life technology</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Fetal Bovine Serum</td>
			<td>Life technology</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-glutamine</td>
			<td>Life technology</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">L-Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544-100G</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Pennicillin/Streptomycin</td>
			<td>Life technology</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Low melting agarose</td>
			<td>BioExpress</td>
			<td>E-3111-125</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">35mm glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Petrolieum Jelly (Vaseline)</td>
			<td>Sigma</td>
			<td>16415-1Kg</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Keratin14-cre</td>
			<td></td>
			<td>MGI: J:65294</td>
			<td>Allele = Tg(KRT14-cre)1Amc</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">ROSA26mTmG</td>
			<td></td>
			<td>MGI: J:124702</td>
			<td>Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Lifeact-mRFPruby</td>
			<td></td>
			<td>MGI: J:164274</td>
			<td>Allele = Tg(CAG-mRuby)#Rows</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">White Light SP5 confocal microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging of mouse secondary palate fusion

二级腭架融合形成完整的二级腭是哺乳动物发育的关键过程，其破坏可导致腭裂性腭裂，这是人类常见的先天性异常现象。二次腭融合已被广泛研究，导致几个可能介导该过程的细胞机制。然而，这些研究主要在开发期间的进行时间点或在静态时间点分析的固定外植体培养物中对固定的胚胎组织进行。静态分析限于动态形态发生过程的分析，如腭裂融合，以及什么类型的动态细胞行为介导腭融合不完全了解。这里我们描述了一种用于小鼠胚胎体外二次腭融合的活体成像方案。为了检查腭裂融合的细胞行为，上皮特异性角蛋白14 -cre用于标记ROS中的腭上皮细胞A26-mTmG flox记录胚胎。为了可视化丝状肌动蛋白，使用Lifeact-mRFPruby报道小鼠。通过解剖最近粘附的胚胎期（E）14.5期胚胎的次级腭架并在玻璃底盘上的含琼脂糖的培养基中培养以使用倒置的共聚焦显微镜成像来进行中度腭融合的活体成像。使用这种方法，我们检测到在二次腭融合期间的各种新型细胞行为。对不同细胞行为在空间和时间上的协调的欣赏大大有助于我们对这种动态形态发生过程的理解。该方案可应用于突变小鼠系，或用药理学抑制剂处理的培养物，以进一步提高对二次腭融合如何控制的理解。

腭外植体活体成像显示多种细胞过程介导腭裂融合6 。两个上皮细胞之间的初始接触由膜突起（ 图 2B-2E ）制成。当两个上皮层相遇时，它们形成多细胞分层MES，然后插入以通过上皮收敛形成整合的单细胞层MES（ 图 2A-2E和图 2K-2O ）。较深Z水平的上皮...

Watch this Scientific Journal Video about Live Imaging of Mouse Secondary Palate Fusion at JoVE.com

Live Imaging of Mouse Secondary Palate Fusion

在这里，我们提出了使用共聚焦显微镜的小鼠二次腭融合的活体成像方案。该方案可以与各种荧光报告小鼠系列结合使用，并可与途径抑制剂一起用于机械性的洞察。该协议可适用于其他开发系统中的实时成像。

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

live-imaging-of-mouse-secondary-palate-fusion

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Research

JoVE Journal

Developmental Biology

7.5K 次观看.  University of California San Francisco. 在这里，我们提出了使用共聚焦显微镜的小鼠二次腭融合的活体成像方案。该方案可以与各种荧光报告小鼠系列结合使用，并可与途径抑制剂一起用于机械性的洞察。该协议可适用于其他开发系统中的实时成像。 

视频: Live Imaging of Mouse Secondary Palate Fusion

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

In Vitro Differentiation of Mature Myofibers for Live Imaging

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation.
Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.

In Vitro Differentiation of Mature Myofibers for Live Imaging

Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages.

在成熟的肌纤维诱导分化实时成像

Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

用于微CT成像的成人和早期产后小鼠肺的血管铸造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

晚期胚胎 果蝇 性腺的解剖和活体成像

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...