Three-Dimensional Simultaneous Imaging of Nucleic Acids and Proteins During Influenza Virus Infection in Single Cells Using Confocal Microscopy

Three-dimensional imaging is a powerful tool for examining the spatial distribution of intracellular molecules like nucleic acids, proteins, and organelles in cells and tissues. Multicolor fluorescence imaging coupled with three-dimensional spatial information provide a platform to explore the relationship between different cellular features and molecules. We have previously developed a pipeline to study the intracellular localization of influenza virus genomic segments within an infected cell. Here, we describe the staining of multiple viral RNA segments in cells infected with influenza virus by combined fluorescence in situ hybridization (FISH) and immunofluorescence and quantification of colocalization between viral segments. This chapter will cover the acquisition and analysis of 3D images by the widely used laser scanning confocal microscope. These strategies can be applied to a wide range of biological processes and modified to examine colocalization of other cellular features.