Chlamydia psittaci infected cell studies by 4Pi Raman and atomic force microscopy.

Chlamydia psittaci is an avian bacterial pathogen that can cause atypical pneumonia in humans via zoonotic transmission. It is a Gram-negative intracellular bacterium that proliferates inside membrane bound inclusions in the cytoplasm of living eukaryotic cells. The study of such cells with C. psittaci inside without destroying them poses a significant challenge. We demonstrated in this work the utility of a combined multitool approach to analyze such complex samples. Atomic force microscopy was applied to obtain high-resolution images of the surface of infected cells upon entrance of bacteria. Atomic force microscopy scans revealed the morphological changes of the cell membrane of Chlamydia infected cells such as changes in roughness of cell membrane and the presence of micro vesicles. 4Pi Raman microscopy was used to image and probe the molecular composition of intracellular bacteria inside intact cells. Information about the structure of the inclusion produced by C. psittaci was obtained and it was found to have a similar molecular fingerprint as that of an intracellular lipid droplet but with less proteins and unsaturated lipids. The presented approach demonstrates complementarity of various microscopy-based approaches and might be useful for characterization of intracellular bacteria.