Live Cell Fluorescence Microscopy – From Sample Preparation to Numbers and Plots

Abstract

AbstractFluorescence 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 cover glass. This allows for live cell imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. We use Cellpose software for cell segmentation and custom-written Fiji (ImageJ) macros for automated analysis of various cell parameters. 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 overviewFrom fluorescence microscopy to numbers and plots – a generalized workflow