Klinisch-Spektroskopische Diagnostik

Intracellular infections are difficult to study as the host cell protects the pathogen from direct observation from the outside. Transmission electron microscopy (TEM) is the most commonly used method for subcellular analysis. However, samples preparation is based on fixation which prevents continuous observation. Here, we focus on the obligate intracellular bacterium Chlamydia abortus. It causes infections primarily in small ruminant livestock, and can also be transmitted to humans, where it can cause disease. Diagnosis is difficult, requiring PCR or cell culture. At the moment, non-invasive methods for the direct study of intracellular infections are rare and not yet established in routine analysis. In this study, we present 3D confocal Raman imaging as a non-invasive tool to investigate and characterize the infection directly inside intact host cells without the need for any purification step and compare the results to established conventional TEM. A 2D cell culture infection model with Buffalo Green Monkey kidney cells was employed and infection with C. abortus S26/3 was characterized at different time points post infection. Using multivariate statistical data analysis, high-quality false colour image stacks were generated from the Raman data. Two chlamydia morphoforms, elementary body (EB) and reticulate body (RB), could be distinguished based on their Raman spectral features: RBs are characterized by prominent lipid and nucleic acid signals while EBs revealed higher carbohydrate and protein signals. This provides complementary information to TEM analysis where morphoforms are differentiated based on size and contrast. The complementary nature of both imaging methods is discussed in the manuscript.