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Published on: June 12, 2013
Looking Inside Non-Destructively: Label-Free, Raman-Based Visualization of Intracellular Coxiella burnetii
Nancy Unger1,2, Simone Eiserloh1,2, Frauke Nowak3
1Center for Sepsis Control and Care, Jena University Hospital, 07747 Jena, Germany.
Abstract:
The life cycle of intracellular pathogens is often complex and can include different morphoforms. Treatment of intracellular infections and unperturbed studying of the pathogen inside the host cell are frequently challenging. Here, we present a Raman-based, label-free, non-invasive, and non-destructive method to localize, visualize, and even quantify intracellular bacteria in 3D within intact host cells in a Coxiella burnetii infection model. C. burnetii is a zoonotic obligate intracellular pathogen that causes infections in ruminant livestock and humans with an acute disease known as Q fever. Using statistical data analysis, no isolation is necessary to gain detailed information on the intracellular pathogen's metabolic state. High-quality false color image stacks with diffraction-limited spatial resolution enable a 3D spatially resolved single host cell analysis that shows excellent agreement with results from transmission electron microscopy. Quantitative analysis at different time points post infection allows to follow the infection cycle with the transition from the large cell variant (LCV) to the small cell variant (SCV) at around day 6 and a gradual change in the lipid composition during vacuole maturation. Spectral characteristics of intracellular LCV and SCV reveal a higher lipid content of the metabolically active LCV.
Insights
This study introduces a novel Raman spectroscopy method to visualize and quantify intracellular bacteria like Coxiella burnetii within host cells without labels. This technique offers detailed insights into pathogen metabolism and life cycle stages, aiding infection research.
Area of Science:
- Microbiology
- Cell Biology
- Spectroscopy
Background:
- Intracellular pathogens present complex life cycles and challenges for study and treatment.
- Visualizing and quantifying pathogens within host cells non-invasively is crucial for understanding infection dynamics.
Purpose of the Study:
- To develop and validate a label-free, non-invasive Raman spectroscopy method for 3D imaging and quantification of intracellular bacteria.
- To analyze the metabolic state and life cycle progression of Coxiella burnetii within host cells.
Main Methods:
- Raman spectroscopy for label-free, non-destructive imaging of intact host cells.
- 3D image reconstruction and statistical data analysis.
- Comparative analysis with transmission electron microscopy.
Main Results:
- Successful localization, visualization, and quantification of intracellular Coxiella burnetii in 3D.
- Demonstrated agreement between Raman imaging and transmission electron microscopy.
- Observed transition from large cell variant (LCV) to small cell variant (SCV) and changes in lipid composition during infection progression.
- Identified spectral differences indicating higher lipid content in metabolically active LCV.
Conclusions:
- Raman spectroscopy provides a powerful tool for studying intracellular pathogens in their native environment.
- The method allows for detailed analysis of pathogen morpho-transitions and metabolic states without cell isolation.
- This technique advances the study of obligate intracellular bacteria and infection models like Q fever.
