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

Abstract

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.

Measurement of expansion factor and distortion for expansion microscopy using isolated renal glomeruli as landmarks

“…The concept of using landmarks or fiduciary markers for relocation has also played important roles in other fields of microscopy. In correlated light and electron microscopy, notches in the mesh grid have been used as landmarks for relocating the area of interest when switching from live cell imaging to electron microscopy modality [25]. In stochastic optical reconstruction microscopy (STORM), fluorescent microspheres have been used as landmarks to track the stage movement and reconstruct the STORM images [26].…”

High definition DIC imaging uncovers transient stages of pathogen infection cycles on the surface of human adult stem cell-derived intestinal epithelium

“…We could for this category not unequivocally distinguish between sudden detachment or IEC invasion in the absence of overt surface perturbation. In other cases, we observed unambiguous Salmonella invasion through a small digitated IEC surface rim transiently formed around the bacterium (Fig 3B - middle row panels, movie will be provided upon publication), or through somewhat more pronounced donut-like ruffles which begun resembling those elicited in Salmonella -infected polarized MDCK cells (Fig 3B, bottom row panels) (46–48).…”

Current Methods, Common Practices, and Perspectives in Tracking and Monitoring Bioinoculants in Soil

“… Wrede et al (2008) used a scanning electron microscope in conjunction with an optical fluorescence system to study microbial mats from Cold Seep communities, simultaneously visualizing and distinguishing bacterial and archaeal microorganisms and their associated biogenic minerals. Instruments in which FM is correlated with scanning EM are often complemented by additional techniques that exploit the possibility of performing different measurements at the same point of interest in a sample, switching between instruments, or integrating multiple analysis methods in the same instrument, with software that manages coordinates and recognizes image patterns ( Kommnick et al, 2019 ). For example, Liu et al (2020) used confocal FM, time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, and scanning electron microscopy (SEM) in an integrated system to document, through imaging and both organic and inorganic chemical analysis, the interaction of Brachypodium roots with a strain of Pseudomonas spp., a typical plant growth-promoting soil bacterium.…”