使用自动图像处理分析裂变和萌芽酵母中的脂滴含量
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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)。
Materials
| 12-bit monochromatic CCD camera Hamamatsu ORCA C4742-80-12AG | Hamamatsu | or equivalent | |
| Adenine hemisulfate salt, ≥99% | Merck | A9126-25G | |
| BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene) | Thermo Fisher Scientific | D3922 | for neutral lipid staining |
| D-(+) – Glucose, ≥99.5% | Merck | G7021 | |
| Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable | Thermo Fisher Scientific | D1976 | for negative staining of cells |
| Dimethyl sulfoxide, ≥99.5% | Merck | D4540 | or higher purity, keep anhydrous on molecular sieves |
| EMM broth without dextrose | Formedium | PMD0405 | medium may also be prepared from individual components |
| Fiji/ImageJ software | NIH | or equivalent; for visual inspection of microscopic data | |
| High precision cover glasses, 22×22 mm, No 1.5 | VWR | 630-2186 | use any # 1.5 cover glass |
| Image Processing Toolbox for MATLAB, version 10.0 | Mathworks | ||
| Lectin from Glycine max (soybean) | Merck | L1395 | for cell immobilization on slides |
| MATLAB software, version 9.2 | Mathworks | ||
| Microscope slide, 26 x 76 mm, 1 mm thickness | Knittel Glass | L762601.2 | use any microscope slide fitting your microscope stage, clean thoroughly before loading cells |
| Olympus CellR microscope with automatic z-axis objective movement | Olympus | or equivalent | |
| pentaband filter set | Semrock | F66-985 | brightfield, green and blue channels are sufficient |
| Signal Processing Toolbox for MATLAB, version 7.4 | Mathworks | ||
| SP supplements | Formedium | PSU0101 | |
| standard office computer capable of running MATLAB | |||
| Statistics and Machine Learning Toolbox for MATLAB, version 11.1 | Mathworks | ||
| Universal peptone M66 for microbiology | Merck | 1070431000 | |
| UPLSAPO 60XO objective | Olympus | or equivalent | |
| Yeast extract | Formedium | YEA03 | |
| Yeast nitrogen base without amino acids | Formedium | CYN0405 |
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