Zahumensky Jakub, Malinsky Jan
Department of Functional Organization of Biomembranes, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague, Czech Republic.
Biol Methods Protoc. 2024 Oct 11;9(1):bpae075. doi: 10.1093/biomethods/bpae075. eCollection 2024.
Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: (2022), DOI: 10.1128/spectrum.01961-22; (2022), DOI: 10.1128/spectrum.02489-22; (2023), DOI: 10.1242/jcs.260554.
生物样品的荧光显微镜图像包含有价值的信息，但需要进行严格分析，才能准确可靠地确定所研究对象的蛋白质定位、荧光强度和形态变化。传统上，用于显微镜观察的细胞是用化学物质固定的，这可能会引入应激。分析通常只关注共定位，涉及手动分割和测量，既耗时又可能引入偏差。我们的新工作流程通过在显微镜盖玻片上用一个小琼脂糖块轻轻固定细胞来解决这些问题。这种方法适用于有细胞壁的细胞（酵母、真菌、植物、细菌），便于在接近其自然环境的条件下对其进行活细胞成像，并能在延时实验中添加化学物质。该方案的主要重点是所展示的分析工作流程，它几乎适用于任何细胞类型——我们描述了使用Cellpose软件进行细胞分割，然后使用自定义编写的Fiji（ImageJ）宏对多个参数进行自动分析。使用提供的R markdown脚本或可用的绘图软件可以轻松处理结果。我们的方法有助于对大型数据集进行无偏差的批量分析，提高荧光显微镜研究的效率和准确性。我们最近的出版物中使用了所报道的样品制备方案和Fiji宏：（2022年），DOI：10.1128/spectrum.01961 - 22；（2022年），DOI：10.1128/spectrum.02489 - 22；（2023年），DOI：10.1242/jcs.260554。
