The study of pathogens and their interactions with host cells has advanced hand-in-hand with developments in optical microscopy. Whereas microbiology benefits enormously from modern imaging technologies, for example, digital imaging and confocal microscopy, it also presents unique challenges. To overcome these, microbiologists are adept at customising imaging methods, and recently there have been studies using state-of-the-art quantitative imaging methods to probe host-pathogen interactions at the single-cell level. Of particular interest are the studies using combined light and electron microscopy methods, bi-arsenical tetra-cysteine tag labelling and automated image-acquisition and analysis for high-throughput/high-content experimentation. These applications demonstrate how imaging methodologies, adapted for microbiology, continue to open avenues for studies that previously have proven inaccessible.