Name: combining histochemical staining and image analysis to quantify starch in the ovary primordia of sweet cherry during winter dormancy
Uploaded: 2019-03-20T12:30:00
Description: Watch this Scientific Journal Video about Combining Histochemical Staining and Image Analysis to Quantify Starch in the Ovary Primordia of Sweet Cherry during Winter Dormancy at JoVE.com

作者感激地感谢玛丽亚·赫雷罗和埃利塞奥·里瓦斯的有益讨论和建议。这项工作得到了欧洲联盟欧洲区域发展基金----欧洲区域发展基金----的支持 [赠款编号 bes-2010-037992 至 E. f.];国家调查研究所 [赠款编号 Rfp2015-00015-00, RTA全85-00, RTA2017-00003-00];和 Gobierno de Aragón----欧洲社会基金, 欧洲联盟 [Grupo 格兰达·阿林塔多 a12-17r]。

1. 休眠测定和植物材料收集
<ol><li>在田野里的花蕾取样。休眠研究是长期的实验, 需要成年树足够大, 可以在整个冬天收集芽和芽, 而不会影响树木在明年春天的发育。根据培训系统, 可能需要特别的果园管理;因此, 修剪可能比水果生产的严重程度要小。<ol>
<li>每周, 从初秋到芽断裂开始, 收集和称重 1 0个花蕾。</li>
		<li>将花蕾放入带瓶盖的10毫升玻璃管中, 并在4°c 下将样品浸泡在乙醇/乙酸 (3:1) ?...

<ol>
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在气候变化的情况下, 木本多年生植物的休眠对水果生产和林业产生了明显的影响, 尽管休眠背后的生物过程仍不清楚。休眠研究可以从不同的角度来探讨, 但在过去几年里, 寻找冬季休眠生物标记的研究有所加强。然而, 大多数试图找到一个明确的指标, 当一个芽已经打破休眠的失败 3.本文所描述的方法,将组织化学技术18与分析图像相结合, 对研究某一特定组织?...

温带多年生植物通过调节生长发育来适应季节。虽然它们在春季和夏季发育, 但在秋天停止生长, 在冬季处于休眠状态。虽然休眠使他们能够在较低的冬季温度下生存, 寒意是一个先决条件, 在春天适当的芽2。休眠对温带水果生产和林业的重要影响, 导致了确定和预测休眠期的各种努力。在果树物种中, 将芽转移到强迫条件下的经验实验和基于开花数据的统计预测是目前确定休眠打破日期的方法, 这使得研究人员能够估计每个品种的冷却要求。然而, 如何根据生物过程确定休眠状态仍不清楚3。
在温带果树上开花, 如甜樱桃, 每年开花一次 , 持续约两周。然而, 花开始分化和发展大约10个月前, 在前一个夏天4。花原基在秋天停止生长, 在冬季在芽内保持休眠状态。在此期间, 每个品种需要积累适当开花的特定冷却要求 4.尽管在冬季芽缺乏物候变化, 但在休眠期间, 花原体在生理上是活跃的, 最近, 寒冷温度的积累与淀粉积累或减少的动态有关在卵巢原基细胞内, 为休眠测定提供了一种新的方法 5.然而, 卵巢原基的小尺寸和位置需要一种特殊的方法。
淀粉是木本植物中主要的碳水化合物.因此, 淀粉的变化与花组织的生理活性有关, 花组织需要碳水化合物来支持它们的发育7,8。不同的关键事件在生殖过程中也与不同花系中淀粉含量的变化有关, 如花药减数分裂9、花粉管的生长通过花粉室受精 10.组织化学技术可以在休眠期检测花原基每个特定组织中的淀粉。然而, 在量化淀粉, 使其随着时间的推移减少的模式或比较组织, 品种或年份之间的淀粉含量仍然是困难的。这是因为可用于分析技术的组织数量很少11。作为一种替代方法, 与显微镜12 相关联的图像分析允许对植物组织13的非常小的样本中的淀粉进行定量。
显微镜和图像分析相结合的方法已被用来量化植物组织中不同成分的含量, 如钙质14、微管15或淀粉16, 方法是测量按特定染色面积的大小。污渍。对于淀粉, 它可以很容易地检测到它使用碘钾 (i2ki) 反应17。此方法是非常具体的;i2 ki 在淀粉颗粒的层流结构内进行夹层, 并根据淀粉18的直链淀粉含量形成深蓝色或红棕色。染色 i2ki 染色的切片显示淀粉和背景组织之间的充分对比度, 允许明确的淀粉检测和随后的量化图像分析系统19。虽然这种染料不是化学计量的, 但碘的积累与淀粉分子的长度成正比, 淀粉分子的长度变化很大,变化很大。因此, 以像像像像素数表示的染色面积的大小可能无法准确地反映淀粉的含量, 因为在染色面积相似的字段之间可以发现淀粉含量的高差异。作为一种替代方法, 可以通过测量从显微镜获得的黑白图像上的染色颗粒的光学密度来评估淀粉含量, 因为在杏子8,13的不同组织中都有报道。,19, 鳄梨10,20和橄榄21.
本文介绍了一种将休眠状态的实验测定与秋季至春季甜樱桃卵巢原基组织淀粉含量的定量相结合的方法, 为理解和预测提供了一种新的工具。休眠的基础上研究的生物机制与休眠。

<table border="0" cellpadding="0" cellspacing="0" width="767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision scale</td>
			<td style="width:251px;">Sartorius</td>
			<td style="width:133px;">CP225D</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereoscopic microscope</td>
			<td style="width:251px;">Leica Microsystems</td>
			<td style="width:133px;">MZ-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Drying-stove</td>
			<td style="width:251px;">Memmert</td>
			<td style="width:133px;">U15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraffin Embedding station</td>
			<td style="width:251px;">Leica Microsystems</td>
			<td style="width:133px;">EG1140H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotatory microtome</td>
			<td style="width:251px;">Reichert-Jung</td>
			<td style="width:133px;">1130/Biocut</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtome blade</td>
			<td style="width:251px;">Feather</td>
			<td style="width:133px;">S35</td>
			<td>Stainless steel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bright field microscope</td>
			<td style="width:251px;">Leica Microsystems</td>
			<td style="width:133px;">DM2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital Camera</td>
			<td style="width:251px;">Leica Microsystems</td>
			<td style="width:133px;">DC-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Image Analysis System</td>
			<td style="width:251px;">Leica Microsystems</td>
			<td style="width:133px;">Quantiment Q550</td>
			<td></td>
		</tr>
	</tbody>
</table>

combining histochemical staining and image analysis to quantify starch in the ovary primordia of sweet cherry during winter dormancy

在几个植物发育过程中, 淀粉的变化与关键事件有关, 包括从授粉到受精的生殖阶段和果实的开始。然而, 淀粉在花分化过程中的变化尚不完全清楚, 主要是由于难以量化淀粉含量的特别小的结构的花原体。在这里, 我们描述了一种方法, 以定量淀粉在卵巢原基甜樱桃 (金银花) 使用图像分析系统附加到显微镜, 允许将淀粉含量的变化与不同阶段的关系从秋天到春天的休眠状态。为此, 通过评价在冬季不同时刻转移到可控条件下的芽芽生长, 确定花蕾的休眠状态。为了定量卵巢原基中的淀粉, 花蕾按顺序收集、固定、嵌入石蜡、切片, 并用 i2kl (碘化钾) 染色。在显微镜下观察制剂, 并通过图像分析仪进行分析, 该分析仪可将淀粉与背景明确区分开来。淀粉含量值是通过测量与染色淀粉相对应的图像的光学密度得到的, 同时考虑到每个像素的光学密度之和作为对所研究框架淀粉含量的估计。

休眠研究需要确定满足冷却要求的时刻。尽管在野外条件下冬季缺乏物候变化 (图 1a), 但樱桃树在低温下度过一定时期之前, 在适当的条件下无法恢复生长能力。在冬季, 芽定期转移到受控条件室 (图 1b), 从而可以评估花蕾的休眠状态。通过测定花蕾重量的增加, 对花蕾生长进行了评价。虽然在休眠期间, 在10天的适当条件下无法?...

Watch this Scientific Journal Video about Combining Histochemical Staining and Image Analysis to Quantify Starch in the Ovary Primordia of Sweet Cherry during Winter Dormancy at JoVE.com

Combining Histochemical Staining and Image Analysis to Quantify Starch in the Ovary Primordia of Sweet Cherry during Winter Dormancy

我们提出了一种方法, 以量化淀粉含量的卵巢原基在冬季休眠期间使用图像分析系统结合组织化学技术。

结合组织化学染色与图像分析对甜樱桃子房冬冬期淀粉的定量

Changes in starch in small structures are associated with key events during several plant developmental processes, including the reproductive phase from ...

这种方法可以帮助回答关于木本植物休眠的关键问题，例如为什么花蕾需要在冬季积累寒冷才能正常开花。该技术的主要优点是允许在非常小的花原样品中对淀粉进行定量。这种方法对于检测睡期间花香的生理活性非常有用，也可以应用于其他树种，如杏子或李子。虽然这种方法可以提供对休眠花蕾的洞察，它也可以应用于其他过程，如生殖阶段从授粉到开花的开始。首先，从现场收集五个拍摄。然后在10毫升玻璃管中称重并固定10个花蕾，用固定溶液在4摄氏度下至少24小时。在此之后，丢弃固定材料，并添加足够的75%乙醇覆盖样品。获得三个长15至30厘米、直径5毫米的芽，每个芽10个。然后将芽放在水浸花店的泡沫上，放在生长室中。在生长室10天后，从芽中去除花蕾并称重。每周从秋天开始到春天发芽，评估花蕾的生长。从每个样品中去除外部芽鳞片，然后将每个芽放在含有 75% 乙醇的制表师玻璃杯中。使用精密钳子和眼科手术刀解剖芽，并获得至少一个花的原芽从每个芽。将样品单独嵌入石蜡中，形成块状物，并将其分在旋转微原子上。分段样品后，在一节上涂抹一滴新鲜的卢戈尔碘。五分钟后，用印迹纸去除多余的污渍。接下来，涂抹一小滴合成安装介质，在样品上放置一个小盖玻璃，然后用硬按压。安装介质干燥后，在亮场显微镜下观察染色部分。首先根据文本协议调整光圈隔膜和亮度控制。然后确保滤光片支架中没有过滤器，并在显微镜中选择亮场条件。在此之后，调整相机设置的染色准备没有组织。将亮度固定为 50% 下一步激活过度曝光/曝光不足功能，并在过度曝光的极限下调整曝光时间。应用白平衡函数和着色校正。测量无组织无污迹制剂的黑白图像的灰色级别。然后应用 4N 滤镜，获取另一个黑白图像的准备并测量灰度。接下来获取没有光线的相同准备的图像，并测量生成的图像的灰度并进行校准。在此之后，获取分辨率至少为 300 dpi 的 TIFF 格式样本部分的颜色图像。创建与染色区域对应的二进制图像，然后设置三个颜色阈值，直到二进制图像准确反映染色淀粉颗粒。与其他制剂和组织一起重复此过程，以微调最终检测水平。使用图像分析系统，测量蒙版下每个像素的光学密度之和，以量化现场的淀粉含量。在该协议中，通过测量10天后在适当条件下的芽体重增加，评估花蕾的生长和休眠状态。在休眠期间，在适当的条件下，10天后未观察到任何变化。一旦休眠被克服，芽膨胀和爆裂在生长室。卵巢原膜中的淀粉含量通过每个微切分段的图像分析进行量化。一致地，在研究的两年中，早冬淀粉的含量达到4万分之一，最大含量达到12万到14万。考虑到休眠情况，淀粉的最大含量与这两年的寒流同时发生。重要的是要记住，图像分析器使用的检测级别直接取决于系统的校准。这包括光线条件、污渍强度和显微镜的放大倍率。必须确定此条件，以进行所有准备工作。将组化学技术与图像分析相结合，对研究花期不同阶段的碳水化合物储备非常有用。它也可以应用于其他物种和组织。使用这种方法在卵巢原体中释放与淀粉积累的关系为理解休眠和冷却需求的生物基础提供了良好的依据。今后的努力必须集中研究可靠的生物指标，这些指标可以很容易地表明树的休眠状况。同时，随着休眠的淀粉变异模式可用于进一步进行生理和遗传研究。

Research

JoVE Journal

Developmental Biology

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 ...

科研

发育生物学

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

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 ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

An In Vitro 3D Model and Computational Pipeline to Quantify the Vasculogenic Potential of iPSC-Derived Endothelial Progenitors

用于量化 iPSC 衍生内皮孕激素的血管生成潜力的体外 3D 模型和计算管道

Induced pluripotent stem cells (iPSCs) are a patient-specific, proliferative cell source that can differentiate into any somatic cell type. Bipotent ...

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture