Correlative light and scanning electron microscopy (CLSEM) for analysis of bacterial infection of polarized...

Infection of mammalian host cells by bacterial pathogens is a highly dynamic process and microscopy is instrumental to reveal the cellular and molecular details of host-pathogen interactions. Correlative light and electron microscopy (CLEM) combines the advantages of three-dimensional live cell imaging with ultrastructural analysis. The analyses of adhesion to, and invasion of polarized epithelial cells by pathogens often deploys scanning electron microscopy (SEM), since surface structures of the apical brush border can be analyzed in detail. Most available CLEM approaches focus on relocalization of separated single cells in different imaging modalities, but are not readily applicable to polarized epithelial cell monolayers, since orientation marks on substrate are overgrown during differentiation. To address this problem, we developed a simple and convenient workflow for correlative light and scanning electron microscopy (CLSEM), using gold mesh grids as carrier for growth of epithelial cell monolayers, and for imaging infection. The approach allows fast live cell imaging of bacterial infection of polarized cells with subsequent analyses by SEM. As examples for CLSEM applications, we investigated trigger invasion by Salmonella enterica, zipper invasion by Listeria monocytogenes, and the enterocyte attachment and effacement phenotype of enteropathogenic Escherichia coli. Our study demonstrates the versatile use of gold mesh grids for CLSEM of the interaction of bacterial pathogens with the apical side of polarized epithelial cells.