Name: confocal live imaging of shoot apical meristems from different plant species
Uploaded: 2019-03-29T15:00:00
Description: Watch this Scientific Journal Video about Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species at JoVE.com

作者感谢 Purdue Bindley 生物科学中心成像设施访问激光扫描共聚焦显微镜和技术支持, 并感谢 Andy Schaber 在 Purdue Bindley 成像设备中提供的帮助。这项活动由普渡大学资助, 作为 AgSEED 十字路口资金的一部分, 以支持印第安纳州的农业和农村发展。

1. 介质和成像盘的准备
<ol><li>MS 板: 添加 0.5倍 Murashige & Skoog ms 培养基, 1% 琼脂进入去离子水, 然后使用氢氧化钾溶液将 pH 值调整到 5.8 (可选: 为长期植物生长添加1% 的蔗糖)。高压灭菌器和倒板。</li>
	<li>成像盘: 将塑料培养皿 (6 厘米宽, 1.5 厘米深) 灌满 0.5-0.8 厘米, 并有1.5% 的熔融琼脂糖。</li>
</ol>2. 植物生长
<ol><li>
拟南芥生长<ol>
<li>将经过消毒的种子播种在 MS 板上, 并?...

<ol>
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本文介绍了一种简单的成像方法, 该方法可应用于不同植物的茎尖分生组织的研究, 并进行轻微的修饰, 为研究模型植物植物和生殖阶段的分生组织调控开辟了新的途径。作物。与扫描电镜和组织学染色方法不同, 该协议可以帮助揭示 Sam 的表面视图和内部细胞结构, 而不需要劳动密集型样品固定和组织切片步骤。该协议还与 Sam 中基于荧光的记者的既定成像方法兼容, 有可能提供良好的细胞分辨率, ?...

植物分生组织包含一个未分化的干细胞池, 并不断维持植物器官的生长和发育1。在胚胎后发育过程中, 植物的几乎所有地上组织都来源于茎顶端分生组织 (SAM)。在作物中, SAM 及其衍生花卉分型的活性和大小与许多农艺性状密切相关, 如芽结构、果实产量和种子产量。例如, 在番茄中, 扩大的 SAM 会导致芽和花序分枝的增加, 从而产生额外的花和水果器官2。在玉米中, sam 大小的增加导致种子数量和总产量增加3,4。在大豆中, 分生组织的不确定性也与芽的结构和产量密切相关。
Sam 的形态和解剖结构可以用几种不同的方法来表征, 包括组织学切片染色和扫描电镜 (SEM)6, 这两种方法都通过提供了分生组织的研究, 大大推进了分生组织的研究。截面视图或 sam 的三维 (3D) 表面视图。然而, 这两种方法都很耗时, 涉及从样品制备到数据采集的几个实验步骤, 这些方法主要依赖于固定样品。激光扫描共聚焦显微镜技术的最新进展克服了这些局限性, 为我们研究植物组织和器官的细胞结构和发育过程提供了一个强大的工具 7,8。通过光学而不是物理组织切片, 共聚焦显微镜允许收集一系列 z 堆栈图像, 并随后通过图像分析软件对样本进行3D 重建。
在这里, 我们描述了一个有效的程序, 用于利用激光扫描共聚焦显微镜研究不同植物物种的活体 Sam 的内部和表面结构, 这可能使研究人员能够完成所有的实验在短短20分钟内处理。不同于其他已出版的拟南芥花序 sam 9,10,11, 12,13,14的活共聚焦成像方法, 15和拟南芥花12,13, 这里我们证明了这个协议是高效的, 不仅研究了模型物种的花序分生子, 而且植物芽来自不同作物的顶端分生组织, 如西红柿和大豆。该方法不依赖转基因荧光标记, 有可能应用于研究来自许多不同种类和品种的幼苗分生组织。此外, 我们还介绍了在3D 视图中查看和分析不同 sam 的简单图像处理步骤。总之, 这种简单的方法将有助于研究人员更好地了解从模型生物和作物的分生组织的结构和发育过程。

<table border="0" cellpadding="0" cellspacing="0" style="width:968px;" width="968">
	<tbody>
		<tr height="32">
			<td height="32" style="height:32px;width:283px;">Agar Phyto</td>
			<td style="width:176px;">Dot Scientific Inc.</td>
			<td style="width:119px;">DSA20300-1000</td>
			<td style="width:391px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:283px;">Agarose</td>
			<td style="width:176px;">Dot Scientific Inc.</td>
			<td style="width:119px;">AGLE-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">Forceps&#160;</td>
			<td style="width:176px;">ROBOZ</td>
			<td style="width:119px;">RS-4955</td>
			<td>Dumont #5SF Super Fine Forceps Inox Tip Size .025 X .005mm, for dissecting shoot apices.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">LSM 880 Upright Confocal Microscope&#160;</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Murashige &#38; Skoog MS medium</td>
			<td style="width:176px;">Dot Scientific Inc.</td>
			<td style="width:119px;">DSM10200-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Plan APO 20x/1.1 water dipping lens</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:283px;">Plastic petri dishes 100 mm X 15 mm</td>
			<td style="width:176px;">CELLTREAT Scientific Products</td>
			<td style="width:119px;">229694</td>
			<td style="width:391px;">Use as making MS plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Plastic petri dishes60 mm X 15</td>
			<td style="width:176px;">CELLTREAT Scientific Products</td>
			<td style="width:119px;">229665</td>
			<td>Use as imaging dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Propagation Mix</td>
			<td style="width:176px;">Sungro Horticulture</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Propidium iodide</td>
			<td style="width:176px;">Acros Organics</td>
			<td style="width:119px;">440300250</td>
			<td>1 mg/mL solution in water, to stain the cell walls</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Razor blade</td>
			<td style="width:176px;">PERSONNA&#160;</td>
			<td style="width:119px;">62-0179</td>
			<td style="width:391px;">For cutting shoot apex from plants</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:35px;width:283px;">Stereomicroscope</td>
			<td style="width:176px;">Nikon</td>
			<td style="width:119px;">SMZ1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tissue</td>
			<td style="width:176px;">VWR</td>
			<td style="width:119px;">82003-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Zen black</td>
			<td style="width:176px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Image acquisition software</td>
		</tr>
	</tbody>
</table>

confocal live imaging of shoot apical meristems from different plant species

芽顶端分生组织 (SAM) 作为一个保守的干细胞库发挥作用, 它在胚胎发育过程中产生几乎所有的地上组织。Sam 的活性和形态决定了重要的农艺性状, 如芽结构、生殖器官的大小和数量, 最重要的是粮食产量。在这里, 我们提供了一个详细的协议, 用于分析表面形态和内部细胞结构的生活 Sam 从不同的物种通过激光扫描共聚焦显微镜。从样品制备到获取高分辨率三维 (3D) 图像的整个过程可以在短短20分钟内完成。我们证明, 该方案不仅可以很有效地研究模型种的花序 sam, 还可以研究不同作物的营养分生, 为研究模型的组织和发展提供了一个简单而有力的工具。不同植物物种的分生组织。

为了评价我们的协议的效率, 并探索来自不同物种的分生组织的形态, 我们对拟南芥的花序分生子和植物分生组织进行了共聚焦现场成像实验。西红柿和大豆。本研究以拟南芥生态型兰塞伯格、 番茄品种微汤姆和大豆品种威廉姆斯82为例。
从正交部分到拟南芥sam 中间, 很明显, pi 能够从多个细胞层的?...

Watch this Scientific Journal Video about Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species at JoVE.com

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

不同植物种的射击顶端共聚焦活图

该协议介绍了如何利用激光扫描共聚焦显微镜对不同植物种类的芽顶端分生组织进行成像和分析。

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

拍摄控制植物生长和繁殖的人工美士。这种方法不仅有助于我们研究模型物种，而且在不同的作物中，研究射向的形态和内部结构。这种技术是一个非常有效的过程。拍摄脂肪可直接以高细胞分辨率可视化，无需任何劳动密集型样品固定和组织分割步骤。首先，种植阿拉伯植物、番茄和大豆植物，详见文本协议。从螺栓的阿拉伯植物中切开花序射尖。握住主茎的基底部分，用钳子从主茎上去除尽可能多的旧花器官。继续去除立体显微镜领域中其余的花朵，直到从目镜中查看拍摄效果。要查看来自番茄或大豆的植物性射生，请解剖出植物中的青菜、叶子和根部。在立体显微镜下保存植物的低叶，用钳子进一步解剖覆盖植物的叶子和叶原体。在干净空的培养皿中，移液 50 微升的碘化溶液。将整个解剖的射点浸入染料中两分钟。在染色过程中，将整个花序拍摄成花膜或植物性花样，浸入碘化溶液中，实现均匀染色。在无菌、去维化的水中冲洗染色的射顶两次。现在，用钳子在准备好的成像盘的中心刺穿一个孔，并在介质中将染色的拍摄顶点直立。将成像盘充满无菌、去化的水，以完全浸入样品。通过立体显微镜查看样品。上下移液，去除夹在梅里斯特姆周围的气泡。然后，调整果糖中茎的角度，以确保从正上方完全可见射射。现在，将成像盘放在共合显微镜的样品阶段。降低浸水透镜，提高显微镜样品阶段，让透镜尖端浸入水中。打开共聚焦显微镜软件，使用目镜定位并聚焦在明亮领域的拍摄中。在共合显微镜软件中操作采集功能。启动实时模式，从计算机屏幕查看样本，并设置激光扫描实验的所有参数。调整参数时，使用范围指示器功能定义信号是否饱和。用于成像采集的相同软件可用于可视化三维透明投影。打开原始共和文件，单击 3D 菜单，然后选择"透明"以生成 3D 投影视图。选择"透明度"可调整投影的三个参数，包括阈值、"渐变"和"最大值"，以提升 3D 图像的透明度。现在，选择"浅色"可调整 3D 图像的亮度。导出投影图像，并将它们保存为 TIFF 文件。要可视化 3D 图像的深度编码视图，请使用同一软件。再次，单击 3D 菜单，然后选择"外观"。选择"特殊"，然后选择深度编码。导出投影图像，并将它们保存为 TIFF 文件。这张从正交部分到阿拉伯细胞中间拍摄的面壁图像显示，在多个细胞层中几乎所有的细胞中，水平壁都沾满了碘化二甲二醇。通过花序拍摄球体内的一个横向部分的视图显示，来自 XY 平面的细胞也清晰成像。3D投影视图显示，花序梅里斯特姆形成一个圆顶式结构，周围是正在发育的花香。这张从正交部分到番茄拍摄中间的图像也表明，水平墙壁上沾满了多个细胞层的碘化丙二，尽管来自深室内区域的碘化丙二信号略低。一张从一个横向部分穿过植物性射片的深层的图像揭示了来自XY平面的细胞和植物性梅里斯特姆和叶原体之间形成的边界。3D投影视图可以进一步全面查看番茄植物形状和组织。这里显示的是一个正交视图，通过大豆拍摄阿普西姆和同一拍摄阿普里斯特梅里斯特姆的3D投影中间。圆顶一样植物性梅里斯特姆及其衍生的新叶原始也可观察到。碘化丙二很容易被染色受损或死细胞，这将极大地影响图像的质量，因此在准备拍摄异体物质样本时，避免任何物理损伤非常重要。

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

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

In Vitro Differentiation of Mature Myofibers for Live Imaging

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

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. ...

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

长期共焦成像根和下胚轴发育的简单室

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Long-term Live Imaging of Drosophila Eye Disc

Long-term Live Imaging of Drosophila Eye Disc

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 ...

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

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

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 Imaging of Mouse Secondary Palate Fusion

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 Cell Imaging of Chromosome Segregation During Mitosis

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

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

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 ...

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

细胞细胞成像的微管细胞和微机械操作的 阿拉比多普西斯拍摄 阿皮克·梅里斯特姆

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in ...