Name: analysis of lipid droplet content in fission and budding yeasts using automated image processing
Uploaded: 2019-07-17T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing at JoVE.com

这项工作得到了查尔斯大学资助PRIMUS/MED/26、GAUK 1308217和SVV 260310的支持。我们感谢翁代伊·塞贝塔在显微镜和图像分析管道开发方面的帮助。我们感谢ReGenEx实验室的S.cerevisae菌株,以及JapoNet和HironoriNiki的S.japonicus菌株实验室。ppc1-88菌株由日本酵母遗传资源中心提供。显微镜在由欧洲区域发展基金和捷克共和国国家预算共同资助的共和荧光显微镜实验室进行(项目No.CZ.1.05/4.1.00/16.0347 和 CZ.2.16/3.1.00/21515)。

1. 解决方案和媒体的准备
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准备脂质染色溶液。
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<li>准备库存脂质染色溶液,在10mL无水DMSO(最终浓度1mg/mL)中溶解10毫克BODIPY 493/503。溶解 10 mg BODIPY 493/503 小瓶的全部含量,以防止在称重过程中材料丢失。 警告:DMSO 可能穿过皮肤。穿戴适当的个人防护装备。</li>
		<li>通过混合 1 mg/mL 493/503 库存溶液的 100 μL 和 900 μL 无水 DMSO(最终浓度 0.1 mg/mL)来制备工作脂质染色溶...

<ol>
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U., 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=Analysis+of+a+genome-wide+set+of+gene+deletions+in+the+fission+yeast+Schizosaccharomyces+pombe.">Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.</a> Nature Biotechnology. 28 (6), 1628-1629 (2010).</li><li>Giaever, G., Nislow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+deletion+collection:+a+decade+of+functional+genomics.">The yeast deletion collection: a decade of functional genomics.</a> Genetics. 197 (2), 451-465 (2014).</li><li>Meyers, A., 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=The+protein+and+neutral+lipid+composition+of+lipid+droplets+isolated+from+the+fission+yeast,+Schizosaccharomyces+pombe.">The protein and neutral lipid composition of lipid droplets isolated from the fission yeast, Schizosaccharomyces pombe.</a> Journal of Microbiology (Seoul, Korea). 55 (2), 112-122 (2017).</li><li>Meyers, A., 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=Lipid+Droplets+Form+from+Distinct+Regions+of+the+Cell+in+the+Fission+Yeast+Schizosaccharomyces+pombe.">Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.</a> Traffic (Copenhagen, Denmark). 17 (6), 657-659 (2016).</li><li>Long, A. 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对脂质代谢及其调控的理解对于基础生物学以及临床和生物技术应用都很重要。LD含量代表细胞脂质代谢状态的方便读出,荧光显微镜是LD含量测定的主要方法之一。提出的协议允许自动检测和定量描述三个不同和形态上不同的酵母物种的单个LD。据我们所知,裂变酵母没有类似的工具。图像处理所需的 MATLAB 脚本作为补充文件包含在内,也可从 Figshare 存储库 (DOI 10.6084/m9.figshare.7745738) 以及本手稿中的?...

脂质在细胞能量和碳代谢、膜组分合成和生物活性物质的生产中起着至关重要的作用。脂质代谢根据环境条件、营养供应和细胞周期第1阶段进行微调。在人类中,脂质代谢与肥胖、II型糖尿病和癌症2等疾病有关。在工业中,由微生物(如酵母)生产的脂质是可再生柴油的一个有前途的来源3。细胞在所谓的脂滴 (LDs) 中储存中性脂质。这些进化保存的身体由三甲苯甘油、固基酯、外磷脂单层和相关蛋白质1组成。LD起源于内质视网膜,施加细胞周期或生长阶段动力学,对细胞脂质平衡1非常重要。当检测各种生长条件下的脂质代谢或筛选一组突变体时,LD数和形态可作为方便的代理。鉴于其动态性质,能够分析单个LD特性的技术对脂质代谢的研究特别感兴趣。
各种酵母品种已被用来描述与脂质相关的代谢途径及其调节,或用于生物技术生产有趣的化合物或燃料1。此外,对于模型酵母,如萌芽酵母糖精或远亲裂变酵母Schizoscharmys pombe,全基因组的缺失菌株库可用于高通量屏幕4,5.最近LD成分和动力学在S.pombe6,7,8,9中被描述,与脂质代谢有关的突变体在新兴的模型酵母中被分离出来希佐萨卡莫西斯贾波尼库斯10。
有许多技术可用于研究 LD 内容和动力学。大多数使用某种染色的LD与亲脂染料,如尼罗河红或BODIPY 493/503。后者显示更窄的激发和发射光谱,并增加对中性脂质(LDs)的特异性,而不是磷脂(膜)11。荧光和流式细胞测定方法已成功地应用于各种真菌物种,以发现影响储存脂质含量12、13、14、15的基因和生长条件。虽然这些方法适用于高通量应用,但它们无法测量细胞中单个 LD 的数量和形态,这些在生长条件和基因型之间可能有很大差异。相干拉曼散射或数字全息显微镜是无标签的方法,产生LD级数据,但需要专门的昂贵的设备16,17,18。另一方面,荧光显微镜可以提供LD含量的详细数据,同时利用常用仪器和图像分析软件工具。存在多个分析工作流,这些工作流在图像数据的单元/LD 检测中具有不同程度的复杂性和自动化,并且针对不同的细胞类型进行了优化,例如具有大 LD 的元群细胞19、20,21,或萌芽酵母17,22,23。其中一些方法仅在 2D 中工作(例如,在最大投影图像上),可能无法可靠地描述蜂窝 LD 内容。据我们所知,没有工具从裂变酵母微观数据中确定LD含量和形态。开发自动化和强大的LD级分析将带来更高的灵敏度和增强的统计能力,并提供丰富的中性脂质含量信息,最好是在多个酵母品种中。
我们开发了一个工作流程,用于从酵母细胞的3D荧光显微镜图像进行LD含量分析。活细胞分别被BODIPY 493/503和级联蓝色dextran染色,以可视化LD和确定细胞边界。细胞固定在玻璃玻片上,并使用标准的荧光显微镜进行 z 堆栈成像。然后,图像由在 MATLAB 中实现的自动化管道进行处理,MATLAB 是一个广泛使用的统计分析(商业)包。管道执行图像预处理、分段(细胞与背景、删除死细胞)和 LD 标识。然后,以与主要电子表格软件工具兼容的表格格式提供丰富的 LD 级数据,如 LD 大小和荧光强度。该工作流程成功地用于确定氮源可用性对S.pombe24的脂质代谢的影响。现在,我们使用生长条件或影响细胞LD含量的突变体,在S.pombe、S.japonicus和S.cerevisae中演示工作流程的功能。

<table>
<tbody>
<tr>
<td>12-bit monochromatic CCD camera Hamamatsu ORCA C4742-80-12AG</td>
<td>Hamamatsu</td>
<td>&nbsp;</td>
<td>or equivalent</td>
</tr>
<tr>
<td>Adenine hemisulfate salt, &ge;99%</td>
<td>Merck</td>
<td>A9126-25G</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene)</td>
<td>Thermo Fisher Scientific</td>
<td>D3922</td>
<td>for neutral lipid staining</td>
</tr>
<tr>
<td>D-(+) - Glucose, &ge;99.5%</td>
<td>Merck</td>
<td>G7021</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable</td>
<td>Thermo Fisher Scientific</td>
<td>D1976</td>
<td>for negative staining of cells</td>
</tr>
<tr>
<td>Dimethyl sulfoxide, &ge;99.5%</td>
<td>Merck</td>
<td>D4540</td>
<td>or higher purity, keep anhydrous on molecular sieves</td>
</tr>
<tr>
<td>EMM broth without dextrose</td>
<td>Formedium</td>
<td>PMD0405</td>
<td>medium may also be prepared from individual components</td>
</tr>
<tr>
<td>Fiji/ImageJ software</td>
<td>NIH</td>
<td>&nbsp;</td>
<td>or equivalent; for visual inspection of microscopic data</td>
</tr>
<tr>
<td>High precision cover glasses, 22x22 mm, No 1.5</td>
<td>VWR</td>
<td>630-2186</td>
<td>use any # 1.5 cover glass</td>
</tr>
<tr>
<td>Image Processing Toolbox for MATLAB, version 10.0</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Lectin from Glycine max (soybean)</td>
<td>Merck</td>
<td>L1395</td>
<td>for cell immobilization on slides</td>
</tr>
<tr>
<td>MATLAB software, version 9.2</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Microscope slide, 26 x 76 mm, 1 mm thickness</td>
<td>Knittel Glass</td>
<td>L762601.2</td>
<td>use any microscope slide fitting your microscope stage, clean thoroughly before loading cells</td>
</tr>
<tr>
<td>Olympus CellR microscope with automatic z-axis objective movement</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>or equivalent</td>
</tr>
<tr>
<td>pentaband filter set </td>
<td>Semrock</td>
<td>F66-985</td>
<td>brightfield, green and blue channels are sufficient</td>
</tr>
<tr>
<td>Signal Processing Toolbox for MATLAB, version 7.4</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>SP supplements</td>
<td>Formedium</td>
<td>PSU0101</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>standard office computer capable of running MATLAB</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Statistics and Machine Learning Toolbox for MATLAB, version 11.1</td>
<td>Mathworks</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Universal peptone M66 for microbiology</td>
<td>Merck</td>
<td>1070431000</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>UPLSAPO 60XO objective</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>or equivalent</td>
</tr>
<tr>
<td>Yeast extract</td>
<td>Formedium</td>
<td>YEA03</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Yeast nitrogen base without amino acids</td>
<td>Formedium</td>
<td>CYN0405</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

analysis of lipid droplet content in fission and budding yeasts using automated image processing

脂质代谢及其调节对基础科学和应用生命科学和生物技术都十分有益。在这方面,各种酵母品种被用作脂质代谢研究或工业脂质生产的模型。脂液滴是高度动态的存储体,其细胞含量代表脂质代谢状态的方便读出。荧光显微镜是细胞脂滴定量分析的首选方法,因为它依赖于广泛可用的设备,并允许分析单个脂液滴。此外,微观图像分析可以自动化,大大提高了整体分析吞吐量。在这里,我们描述了一个实验和分析工作流程,用于自动检测和定量描述三个不同模型酵母物种中的单个脂液滴:裂变酵母schizoscharis pombe和希佐萨卡莫西斯贾波尼丘斯,和萌芽的酵母糖精塞雷维西亚。脂液滴通过BODIPY 493/503进行可视化,并且细胞不透水荧光dextran被添加到培养基中,以帮助识别细胞边界。细胞在绿色和蓝色通道中接受3D荧光显微镜,产生的z堆栈图像由MATLAB管道自动处理。该程序以表格格式输出丰富的细胞脂滴含量和单个脂滴特征的定量数据,适用于主要电子表格或统计包中的下游分析。我们提供影响细胞脂质代谢的各种条件下脂滴含量的实例分析。

图1对裂变酵母(萌芽酵母工作流程类似)总结了整个过程,下面我们提供了如何在已知各种条件下使用该工作流研究三个不同酵母物种的LD含量的示例影响蜂窝 LD 内容。每个示例代表一个生物实验。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59889/59889fig01v2.jpg"> 图1:实验和分析工作流程的原理图。裂变酵母的工作流程...

Watch this Scientific Journal Video about Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing at JoVE.com

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

在这里,我们介绍了在裂变和萌芽酵母细胞的荧光显微镜图像中自动检测和脂液滴定量描述的MATLAB实现。

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

该协议有助于对各种酵母物种的细胞储存脂质含量进行定量的小规模到大规模分析，并以公正和标准化的方式进行。该技术允许快速处理微观图像，并提供详细的定量输出，以便轻松比较样品，如各种突变体和在各种条件下生长的细胞。首先，请按照随附的文本协议准备染色解决方案、培养媒体和细胞。在运行实验之前，确保细胞健康，处于所需的生长阶段。接下来，为要成像的每个样品准备显微镜盖玻片。使用水平定位移液器尖端的长侧将一个微升的滑动涂层溶液铺到干净的盖玻片上。让涂层溶液完全干燥，然后将盖玻片存放在无尘环境中。接下来，测量细胞培养物的光学密度，并在同一生长阶段分析培养物，尝试在所有测试培养物之间达到类似的值，以确保类似的实验条件。然后，移液器将一毫升的细胞培养成1.5毫升的微离心管。仅对于 S.cerevisiae 细胞，在管中添加五微升的滑动涂层溶液。然后，短暂地旋转所有的微离心管，并在30摄氏度下孵育，摇晃5分钟。接下来，将脂质染色溶液的微升添加到每个培养的等分，并短暂地涡旋它们。然后，添加 10 微升的单元边界可视化解决方案，并再短暂旋转它们。在室温下，在1000倍重力下离心，收集细胞，收集细胞3分钟。旋转完成后，从上流液中去除950微升，并使用移液器将剩余的上流液中的细胞重新暂停。接下来，移液器的两微升的密集细胞悬浮到一个莱金涂层盖玻片。然后，将盖玻片放在干净的显微镜幻灯片上，形成细胞单层，并尽快进行显微镜检查，以最大限度地减少成像中的伪影。设置显微镜需要长时间接触强光源，这些光源可能会损坏样品并扭曲结果。因此，使用专用样品幻灯片设置成像条件，该幻灯片不会进一步用于脂液滴定量。将专用样品放在相位对比度或差分干扰对比度显微镜设置的舞台上，并聚焦在细胞上。在显微镜的软件中，设置 Z 堆栈设置，以便它们跨越整个细胞体积。总垂直距离取决于细胞大小，光学切片的数量取决于目标的数值孔径。接下来，将焦点设置为相对于中央焦点平面移动。要对脂质液滴进行成像，请实验性地设置绿色通道中的光强度和曝光时间。成像时要小心，因为BODIPY是一种非常明亮的荧光色素，可能会迅速光漂白。此外，尽量减少曝光时间，避免过度饱和。在切换到蓝色通道之前，先捕获完整的绿色通道 Z 堆栈，以防止移动脂质液滴模糊。要对细胞边界进行成像，请实验性地设置蓝色通道中的光强度和曝光时间。如果可能，在显微镜控制软件中设置和使用自动化实验工作流程，以便于在标准化条件下对多个样品进行成像。重要的是，必须使用相同的设置获取所有图像，以便对样本进行适当的比较。一旦成像条件得到优化，请关注细胞，并在绿色和蓝色通道中成像它们。每个样本的多个视场进行图像，以获得健壮、具有代表性的数据。将蓝色和绿色通道 Z 堆栈图像保存为 16 位多层 TIFF 文件。请确保在相应的文件名中包含绿色或蓝色单词。打开 ImageJ 中的显微图像，并删除包含大量在采集过程中移动的单元格的任何图像堆栈。这些显示在各个 Z 部分的不同位置。接下来，删除蓝色通道中包含高荧光非细胞颗粒的任何图像堆栈。这通常是由成像表面的污垢或培养介质中的杂质引起的，并可能干扰其附近细胞的检测。此外，删除包含大量死细胞的任何图像堆栈。这些图像将显示与活细胞相比，蓝色荧光增加的细胞。虽然样本中存在几个死细胞通常不是问题，在分析过程中这些细胞会自动丢弃，但一些死细胞或死亡细胞有时可能通过分段算法被识别为活细胞，从而扭曲报告的结果。若要在 MATLAB 中开始分析，请先创建一个主文件夹，然后将所有 MATLAB 脚本复制到此位置。接下来，创建包含各种酵母物种名称的子文件夹，并复制相应的 TIFF 图像到这些位置。现在，启动 MATLAB，打开脚本 MAIN。m，然后运行脚本。在菜单中，选择要分析的酵母品种并开始图像处理。使用电子表格编辑器或统计包，按要求检查和处理输出文件。工作流生成分号分离的 CSV 文件和带检测到的细胞对象和脂滴的 TIFF 文件。此处显示的是野生型 S.pombe 细胞，这些细胞生长在复杂的 YES 介质或定义的 EMM 介质中。与在 YES 培养的细胞相比，EMM 介质中检测到的脂质液滴更少，每单位细胞体积的染色强度更高。此外，EMM介质中形成的单个脂质液滴较大，总染色强度增加。这与先前在EMM中生长的细胞中储存脂质含量增加的发现是一致。接下来，这些图像显示了S.japonicus细胞从指数和早期固定培养生长在 YES 培养。进入固定阶段的细胞显示，每单位细胞体积的脂滴数量明显减少，而体积正常化脂滴荧光强度在两种条件下略有下降。早期的固定相脂质液滴通常大小中等大，与指数级生长细胞的液滴相比，总荧光强度要稍高。在S.cerevisiae细胞中，与呈指数生长的细胞相比，固定细胞每单位体积的脂质液滴要少一些。然而，它们的体积正常化液滴荧光强度几乎翻了一番。整体脂液滴含量的急剧上升是由于在固定相中单个脂质液滴的荧光强度和体积要高得多。按照此过程，可以在统计包（如 R）中聚合输出数据，以创建汇总结果的绘图，并针对脂滴含量的任何观察到的差异运行统计测试。原则上，图像处理管道可用于分析其他微生物中的脂液滴含量或分析其他点状亚细胞结构。

Research

JoVE Journal

Immunology and Infection

Using Luciferase to Image Bacterial Infections in Mice

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

促进药物发现：斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Isolation, Processing and Analysis of Murine Gingival Cells

小鼠牙龈细胞的分离，处理和分析

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

半自动化的方法准备抗体鸡尾酒人外周血中免疫表型分析

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

全自动微流控装置在白色念珠菌生物膜形成中的可视化

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

利用广域跟踪平台对大型线虫种群进行自动行为分析

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

用荧光显微镜分析膜受体扩散和簇簇的图像处理协议

Particle tracking on a video sequence and the posterior analysis of their trajectories is nowadays a common operation in many biological studies. Using ...