Live Cell Fluorescence Microscopy - An End-to-End Workflow for High-Throughput Image and Data Analysis

Abstract

Fluorescence microscopy images of biological samples contain valuable information but require rigor-ous 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 in-volves 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 cover glass. 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 addi-tion 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: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554. Graphical overview O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=38 SRC="FIGDIR/small/587214v3_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1ffb66borg.highwire.dtl.DTLVardef@1214a99org.highwire.dtl.DTLVardef@a8b39aorg.highwire.dtl.DTLVardef@1a744d3_HPS_FORMAT_FIGEXP M_FIG C_FIG From fluorescence microscopy to numbers and plots - a generalized workflow