Name: simultaneous live imaging of multiple insect embryos in sample chamber-based light sheet fluorescence microscopes
Uploaded: 2020-09-09T15:00:00
Description: Watch this Scientific Journal Video about Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes at JoVE.com

我们感谢安永 H. K. Stelzer 有机会利用他的资源，以及他对手稿的宝贵评论，安妮塔·安德尔对 Tribolium 现场成像的支持，斯文·普拉斯提供技术支持，以及伊兰·戴维斯、妮可·格里德和杰罗德·舒比格通过布卢明顿股票中心分享他们的转基因 Drosophila 生产线。

1. 筹备工作
<ol><li>为适合科学问题的 LSFM 选择照明镜头/检测镜头/相机组合，并设置显微镜。视场的大小是相机芯片大小的商量和检测镜头的放大倍数。照明透镜应选择，以便整个视野被大致平面光片34覆盖。 表1中列出了三个推荐组合。</li>
	<li>要准备阿加罗斯别名和检索菜肴，请将 2 克低熔性蔗糖加入 200 mL 高压自来水中，并在 600-800 W 的微波炉中加热混合物，直...

LSFM 的独家应用领域之一是发育生物学。在这个学科中，观察活的标本很重要，否则无法动态地描述形态遗传过程。此处描述的基于样品室 LSFM 的同步实时成像实验框架很方便，原因有二。
环境方差是连续实时成像中不可避免的，可以主动解决。在与昆虫胚胎相关的活成像中，我们看到以下易感性。在胚胎采集时，卵子收集培养可能有不同的年龄。在 德罗索菲拉，已?...

荧光显微镜是生命科学中最重要的成像技术之一，特别是在细胞和发育生物学方面。在共焦荧光显微镜1中，这是自20世纪90年代中期以来最先进的三维荧光成像技术，同一镜头用于荧光激发和发射光检测。照明激光束激发照明/检测轴沿线的所有荧光素，在通过针孔检测之前，对各自的对焦信号进行区分。因此，对于每个二维图像，整个标本都会被照亮。因此，对于每个三维图像，即一叠空间连续的二维图像，整个标本被照亮几十到几百倍2，这促进光漂白和光毒性3。
近二十年前，光片技术4 成为三维荧光成像的有前途的替代品，成为发展生物学5的宝贵工具。在这种方法中，照明和检测是脱钩的。照明透镜用于在垂直排列的检测透镜的焦平面内生成深度只有几微米的光片。因此，对于每个二维图像，只有焦平面周围的细平面体积被照亮。因此，对于每个三维图像，整个标本只被照亮一次，这强烈地降低了光漂白和光毒性6。因此，光片荧光显微镜（LSFMs）为研究多个生物相关尺度的复杂过程提供了有效的解决方案，因此，在发育生物学中具有特殊价值，在亚细胞水平上必须分析高达几毫米的标本。
从历史上看，LSFM一直以样品室为基础，即7、8。在这些设置中，照明 （x） 和检测 （z） 轴通常垂直于重力轴 （y） 排列。样品室提供充足的实验自由。首先，它们提供大型成像缓冲能力，这反过来又便于使用灌注系统控制环境，例如维持特定温度9或应用生化应激器。此外，他们支持定制安装方法10，根据各自的实验需要量身定做，同时保持三维，在某些情况下动态11，标本的完整性。此外，基于样品室的设置通常配备了旋转功能，用于将标本围绕 y 轴旋转，从而沿两个、四个甚至多个方向对标本进行成像。由于常用模型生物的胚胎在显微镜中，沿腹腔多面、横向和/或前后体轴的连续成像规模相对较大，具有更全面的表示性。这允许长期跟踪沿着复杂的三维迁移路径12，13移动的细胞。
光片荧光显微镜已广泛应用于研究甲流的胚胎形态，系统地研究14、15，并特别注重生物物理方面的发展。例如，它被用来收集高分辨率的形态遗传学数据，以检测在细菌带拉长16期间内皮体入侵和轴延伸之间的生物力学联系，并进一步将复杂的细胞流与气喘17期间的力生成模式联系起来。它还与其他最先进的技术相结合，例如光遗传学，以研究在表皮18前后模式期间对Wnt信号的调节。
然而，只研究一个物种并不能提供对发展演变的见解。为了了解植物学背景下的胚胎生成，对替代昆虫模型生物进行了深入的研究。其中一个最全面调查的物种是红面粉甲虫三宝座，一种经济上相关的储存谷物害虫19，其胚胎形态也已经系统地与LSFM20成像。这两个物种的胚胎形态在几个方面差异显著，如分割模式21，以及胚胎外膜22的形成和降解。使用 LSFM 对后一个方面进行了广泛的分析。例如，已经证明，血清，一种胚胎外组织，包围和保护三叶草胚胎免受各种危害，其胚胎生成的更好部分23，24，也作为致命的遗传"驱动因素"，为自己的提取过程在回旋关闭25。此外，已经证明，在气喘期间，爆炸体的一个特定区域仍然锚定在玻璃膜上，以产生不对称的组织运动26，并在此观察之后，区域化组织流化允许细胞在塞罗萨窗口关闭27期间按顺序离开血清边缘。
在上述所有与嗜血杆菌和三叶草相关的研究中，都使用了基于样品室的LSFM。在大多数情况下，胚胎是沿着多个方向记录的，使用样品旋转功能。虽然没有明确说明，但可以假定它们是单独记录的，因此在连续的实时成像测定中彼此独立，类似于我们之前在Tribolium20，28上的工作。在某些情况下，这种方法是可以接受的，但在定量比较方法中，环境差异可能会扭曲结果。例如，人们早就知道昆虫的发育速度取决于温度，但最近的一项研究进一步表明，在德罗索菲拉，温度也可能影响30型摩尔磷的浓度。因此，如果胚胎生成的某些特征，例如细胞的动态比例、分裂率和迁移速度，应精确量化，则需要充分重复，而不需要环境方差。这最大限度地减少了标准偏差和标准错误，这反过来又有利于与其他，甚至只是略有不同的实验条件并列。
但是，基于样品室的 LSFM 主要针对高含量而非高吞吐量检测而设计。与共焦显微镜不同，该显微镜通常配备了用于显微镜滑梯、培养皿和井板的标准化夹紧机制，几乎所有基于样品室的 LSFM 都使用基于圆柱形的夹紧机制。这些机制是针对定制样品持有人，是旋转兼容的，以及非侵入性的10，但通常不设计为多个标本20，31，32。两个或两个以上胚胎同时进行活成像的框架，其中基于样品的室设置的优点不会受到损害，解决了环境方差问题，从而增加了LSFM用于比较研究的价值。
在我们的协议中，我们提出了一个实验框架，用于在基于样品室的LSFM（图1A）中进行比较活成像，其中y轴被用作"堆叠"胚胎的选项。首先，我们为基于样品室的 LSFM 提供荧光微球校准指南，对于缺乏校准辅助的仪器来说，这一点尤为重要。其次，我们描述了基于蜘蛛网支架28（图1B）的多个胚胎的安装方法，该方法与样本旋转兼容，从而允许沿多个方向同时成像多个样本（图1C）。几个胚胎在薄的糖膜上对齐，在插入样品室后，通过光片连续移动以获取三维图像。第三，我们为德罗索菲拉和三宝莲提供三个示范性的实时成像数据集。对于前者，我们将转基因线与荧光标记的核并列。对于后者，我们比较携带相同转基因的转基因子线的性能，但在不同的基因组位置。最后，我们讨论了平行化对比较活成像和环境方差33的重要性，讨论了我们的实验框架的吞吐量限制，并评估了我们对其他模型生物体方法的适应情况。

<table>
	<tbody>
		<tr>
			<td>6-well plate</td>
			<td>Orange Scientific</td>
			<td>4430500</td>
			<td>&#160;</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Orange Scientific</td>
			<td>4430300</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>35-mm &#216; Petri dish</td>
			<td>Fisher Scientific</td>
			<td>153066</td>
			<td>Only for live imaging involving Drosophila.</td>
		</tr>
		<tr>
			<td>90-mm &#216; Petri dish</td>
			<td>Fisher Scientific</td>
			<td>L9004575</td>
			<td></td>
		</tr>
		<tr>
			<td>100-&#181;m mesh size cell strainer</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>250-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-038</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>300-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-040</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>710-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-050</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>800-&#181;m mesh size sieve</td>
			<td>VWR International</td>
			<td>200.025.222-051</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>405 fine wheat flour</td>
			<td>Demeter e.V.</td>
			<td>SP061006</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>commercially available Drosophila medium</td>
			<td>Genesee Scientific</td>
			<td>66-115</td>
			<td>Only for live imaging involving Drosophila / Custom-made Drosophila medium may also be used</td>
		</tr>
		<tr>
			<td>fluorescent microspheres, 1.0 &#181;m &#216;</td>
			<td>Thermo Fisher Scientific</td>
			<td>T7282</td>
			<td></td>
		</tr>
		<tr>
			<td>inactive dry yeast</td>
			<td>Genesee Scientific</td>
			<td>62-108</td>
			<td>Only for live imaging involving Tribolium</td>
		</tr>
		<tr>
			<td>low-melt agarose</td>
			<td>Carl Roth</td>
			<td>6351.2</td>
			<td></td>
		</tr>
		<tr>
			<td>narrow vials</td>
			<td>Genesee Scientific</td>
			<td>32-109</td>
			<td>Only for live imaging involving Drosophila</td>
		</tr>
		<tr>
			<td>small paint brush</td>
			<td>VWR International</td>
			<td>149-2121</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hypochlorite (NaOCl), ~12% active Cl</td>
			<td>Carl Roth</td>
			<td>9062.3</td>
			<td>Caution: sodium hypochlorite is corrosive</td>
		</tr>
		<tr>
			<td>whole wheat flour</td>
			<td>Demeter e.V.</td>
			<td>SP061036</td>
			<td>Only for live imaging involving Tribolium / United Kingdom: wholemeal flour</td>
		</tr>
		<tr>
			<td>wide vials</td>
			<td>Genesee Scientific</td>
			<td>32-110</td>
			<td>Only for live imaging involving Drosophila</td>
		</tr>
	</tbody>
</table>

simultaneous live imaging of multiple insect embryos in sample chamber-based light sheet fluorescence microscopes

基于光片的荧光显微镜为研究多个生物相关尺度上的复杂过程提供了有效的解决方案。样品室设置是发育生物学的最佳选择，这些设置是专门为保持标本的三维完整性而设计的，通常具有样品旋转功能。例如，它们被用来记录果蝇 德罗索菲拉褪黑麦加斯特 和红面粉甲虫 三宝座的整个胚胎形态。然而，许多可用的活成像协议只为单个胚胎提供实验框架。特别是对于比较研究，这种方法不方便，因为顺序成像标本受环境方差的影响。此外，这限制了在给定时间内可以检测的标本数量。我们为同步实时成像提供一个实验框架，以增加基于样品室的设置的吞吐量，从而确保所有标本的类似环境条件。首先，我们为光片荧光显微镜提供校准指南。其次，我们提出了一种与样本旋转相容的多个胚胎的安装方法。第三，我们提供示范性的三维D 罗索菲拉活成像数据集，为此，我们将三条转基因线与荧光标记的核以及 三聚氰胺并列，为此我们比较了携带相同转基因但在不同基因组位置的三条转基因子系的性能。我们的协议是专门为比较研究设计的，因为它主动解决环境方差，这总是存在于连续实时成像中。这对于定量分析和异常表型的定性尤其重要，例如，从淘汰实验中产生的表型。此外，它增加了总吞吐量，当光片荧光显微镜的获取有限时，这非常方便。最后，建议的安装方法可以适应其他昆虫物种和进一步的模型生物，如斑马鱼，基本上没有优化的努力。

我们的协议描述了基于样品室 LSFMs 的比较荧光实时成像的实验框架。例如，该框架可用于并列两个或两个以上物种的胚胎，（ii） 一个或多个基因被击倒的线状胚胎加上野生型对照组：（iii） 同一转基因系的多个胚胎：（iv） 来自不同转基因系的胚胎，或（v） 携带相同转基因基因的子线胚胎，但在不同的基因组位置。在本节中，我们为最后两个方案提供示例。
在我们的第?...

Watch this Scientific Journal Video about Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes at JoVE.com

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

光片荧光显微镜是发育生物学中最有价值的工具。比较研究中的一个主要问题是环境差异。我们的协议描述了多个标本同时进行实时成像的实验框架，因此，主动解决这个问题。

样品室光片荧光显微镜中多个昆虫胚胎的同步实时成像

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Sample Chamber-Based Light Sheet Florescence Microscopes are designed for high content rather than high throughput. Live imaging assays must therefore be performed sequentially and thus less affected by ambient variance. Our cobweb holder allow stacking and simultaneous imaging of multiple embryos, eliminating ambient variance and increasing throughput.Mounting embryos in the cobweb holder requires a certain finesse. An explainatory video is ideal for illustrating sequence of the many short conduct gestures. For calibration of the Sample Chamber-Based of Light Sheet Fluorescence Microscope.First, reliquify an agarose aliquot in a dry block heater mixer at 80 degrees Celsius. Allow the melted agarose to cool to 35 degrees Celsius and transfer 50 microliters of the agarose to a 1.5 milliliter reaction tube. Mix 0.5 microliters of fluorescent microsphere solution with the agarose at 1, 400 revolutions per minute for one minute, and fill the slotted hole of the cobweb holder with 10 microliters of the agarose fluorescent microsphere solution mixture.Aspirate as much agarose as possible until only a thin agarose film remains and allow the agarose to solidify for 30 to 60 seconds. Fill the sample chamber with autoclaved tap water, and slowly insert the cobweb holder into the sample chamber. Move the slotted hole in front of the detection lens and rotate the holder to a 45 degree position relative to the illumination and detection axes.The cobweb holder should not be visible in the transmission light channel. Switch to the appropriate fluorescence channel and adjust the laser power and exposure time, so that the fluorescent microspheres provide a proper signal. Specify a volume of view that covers the now transversely oriented agarose film completely and calculate the maximum possible axial resolution for the respective illumination and detection lens combination to define the Z spacing.Then, record a three-dimensional test Z stack of the fluorescent microspheres and compare the X, Y, and Z maximum projections to the calibration chart. If the microspheres appear blurry, fuzzy, and or distorted. Adjust the positions of the illumination and or detection lenses.For embryo dechorionation, add eight milliliters of autoclaved tap water into the A1, A2, A3 and B3 wells of one-six-well plate per line. Then, add seven milliliters of autoclaved tap water and one milliliter of sodium hypochlorite solution into the B1 and B2 wells. Position the first plate under a stereo microscope and insert first embryo containing cell strainer into the A1 well.Wash the embryos under gentle agitation for 30 to 60 seconds, before moving the strainer to the B1 well. Shake the plate vigorously for 30 seconds, before transferring the strainer to the A2 well. Wash the embryos for one minute under gentle agitation and move the strainer into the B2 Well for vigorous shaking until most of the embryos are completely dechorionation.Then, transfer the strainer to the A3 well for one minute of gentle washing, before storing the strainer of dechorionated embryos in the B3 well until the embryos of other lines have been treated as demonstrated. To Mount the embryos onto the cobweb holder, reliquify another aliquot of agarose as demonstrated. When the agarose has cooled, place the cobweb holder under the stereo microscope and add 10 microliter of the agarose onto the slotted hole.Use the pipette tip to spread the agarose over the slotted hole, before aspirating as much agarose as possible until only a thin agarose film remains. When the agarose has solidified, use a paintbrush to carefully pick and transfer the embryos onto the agarose film. Arrange them along the long axis of the slotted hole with their anterior-posterior axes aligned with the long axis of the slotted hole.To stabilize the embryos, carefully pipette one to two microliters of agarose into the gap between the embryos and the agarose film. When the agarose has solidified, slowly insert the cobweb holder with the mounted embryos into the image buffer filled sample chamber. To image the embryos, confirm that the cobweb holder is in a 45 degree position relative to the illumination and detection axes and in the transmission light channel, move the embryo into the center of the field of view.The cobweb holder should not be visible. Next, move the embryo with the micro translation stage in Z until the mid plane of the embryo overlaps with the focal plane and the outline appear sharp. Without switching to the fluorescence channel, move the embryo 250 microns in both directions to specify the volume of view.If imaging along multiple directions is required, rotate the embryo appropriately, bring the embryo into focus and specify the volume of view as just demonstrated. Then, repeat this procedure for all of the other mounted embryos. When the volume of view has been specified for all of the embryos, define the fluorescence channel and time-lapse parameters and start the imaging process.For assays that lasts several days, consider covering the sample chamber opening at least partially to reduce evaporation. When the imaging assay has ended, carefully remove the cobweb holder from the sample chamber and use a small paintbrush to detach the embryos from the agarose film and to place the embryos onto an appropriately labeled microscope slide. Place the slide into a retrieval dish for incubation under the appropriate standard rearing conditions.As the hatching time point approaches, check the retrieval dishes frequently and transfer any hatched larvae to individual wells of a 24-well plate. Fill the Wells halfway with the appropriate growth medium, then incubate the larvae under the appropriate standard rearing conditions. When the observed individuals reach adulthood, provide suitable mating partners and check for progeny after several days.In this video, the fluorescent signal dynamics of three embryos derived from different transgenic Drosophila lines over a period of about one day can be observed. A similar fluorescence pattern was expected since all of the lines expressed nuclear localized EGFP under the control of presumably ubiquitous and constituently active promoters. Comparative live imaging results, however, revealed strong spatial temporal differences in the expression patterns that were not secondary effects due to ambient variance.In this analysis of three Tribolium embryos carrying the same piggyback based transgene. Comparative live imaging results of the serosa window closure process during gastrulation suggest that the second sub-line provides a remarkably stronger overall signal than the other two sublines. As these data indicate, the genomic context may also have a strong influence on the expression level of transgenes in Tribolium.Our approach is well suited for analyzing knockdown and knockout phenotypes, as it allows wild type control embryos to be imaged simultaneously. Our protocol can also be used to compare the morphogenesis of two or more insect species. And thus, gain deeper insights into the evolution of development.

Research

JoVE Journal

Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

设置简单的轻表为显微镜<em&gt;在托托</em的&gt;成像<em&gt; C。线虫</em&gt;发展

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...