Princová Jarmila, Schätz Martin, Ťupa Ondřej, Převorovský Martin
Faculty of Science, Charles University.
Faculty of Science, Charles University;
J Vis Exp. 2019 Jul 17(149). doi: 10.3791/59889.
Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.
脂质代谢及其调控在基础生命科学、应用生命科学和生物技术领域均备受关注。就此而言，多种酵母物种被用作脂质代谢研究的模型或用于工业脂质生产。脂滴是高度动态的储存体，其细胞内成分是脂质代谢状态的便捷指标。荧光显微镜是定量分析细胞脂滴的首选方法，因为它依赖广泛可用的设备，且能对单个脂滴进行分析。此外，显微图像分析可实现自动化，大幅提高整体分析通量。在此，我们描述了一种实验和分析工作流程，用于自动检测和定量描述三种不同模式酵母物种中的单个脂滴：裂殖酵母粟酒裂殖酵母和日本裂殖酵母，以及芽殖酵母酿酒酵母。使用BODIPY 493/503对脂滴进行可视化，将细胞不可渗透的荧光葡聚糖添加到培养基中以帮助识别细胞边界。对细胞进行绿色和蓝色通道的三维落射荧光显微镜观察，所得的z-stack图像由MATLAB管道自动处理。该程序以表格形式输出关于细胞脂滴含量和单个脂滴特征的丰富定量数据，适合在主要电子表格或统计软件包中进行下游分析。我们提供了在影响细胞脂质代谢的各种条件下脂滴含量的示例分析。
