Several bacteria are adapted to colonize and live together with multicellular organisms. There are some pathogenic bacteria that are even able to invade eukaryotic cells for different purposes, usually to escape the immune response or antibiotic treatment, but also to take advantage of growth in the nutrient rich host cell environment. Intracellular bacteria commonly are associated with chronic infections and difficult to treat. Studying intracellular bacteria in order to gain more knowledge about their pathogenesis requires sophisticated methods that deliver specific information from the bacteria while residing in the host cell environment. In this regard imaging methods are superior to follow both, the bacteria and host cell, at the same time without the necessity of extracting the bacteria.

Confocal Raman micro spectroscopy is a powerful tool that allows to make hyperspectral false-color images after scanning of a biological sample in a non-destructive and label-free way. We demonstrate that it is possible to obtain such images of endothelial cells containing Staphylococcus aureus (1), a prominent hospital-related pathogen that is assumed to use intracellular lifestyle to persist in the body. Raman spectroscopic scans using a 532 nm laser and a low step size of 0.25 µm for scanning were generated and bacteria could be specifically detected using the spectral unmixing algorithm N-FINDR (2). Additionally, it was possible to visualize host cell organelles and particles such as lipid droplets, the nucleus of the host cell or peri-nuclear region. Host cell organelles that appear similar to bacteria through their size and shape could be well discriminated from the bacteria through the specific Raman spectrum. False-color images revealed the morphology and different location of the bacteria in the host cell even in three dimensions by confocal scanning in Z. The detection was verified through immunofluorescence labelling and fluorescence microscopy afterwards.

The Raman spectra extracted from the images further could be used to analyze growth changes based on the chemical information delivered by the spectra such as a relative change in the nucleic acid and protein amount. This information helps to follow the intracellular bacteria in their growth behavior and detect changes due to changing host cell conditions.

Additionally, it is shown that Raman-based imaging can be extended from single host cells in vitro to in situ tissue samples as we demonstrate for symbiotic bacteria detected in special pockets near to the hindgut of larvae of the forest cockchafer. Through this approach production of poly-3-hydroxybutyrate, an important bacterial storage compound, could be detected and located to different areas and bacterial populations in the Raman image.

We conclude that confocal Raman spectroscopy has the potential to become a valuable imaging tool for studying pathogenic bacteria in host cells and tissues because of the label-free and specific detection capacity that allows to analyse even living samples. Further, both bacteria and host cells can be studied at the same time. A combination with other imaging methods such as fluorescence microscopy allows to study the bacteria in more detail, i.g. the role of specific proteins in overall changes of the bacteria growth.

Acknowledgements go to the financial support by the BMBF (FKZ 01EO1002 and FKZ 01EO1502) and the EU within the Framework Program 7 (P4L, Grant agreement no.: 224014), and C. Beleites for help with “R”.

The authors declare no conflict of interests.

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