Confocal Laser-Scanning-Microscop

• Subject Area: Microbiology, Virology and Immunology
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 538190998

Confocal laser scanning microscopy (CLSM) is a central analytical method to study cellular processes in infectious diseases research. This includes the investigation of molecular mechanisms of gene regulation in pathogens as well as the underlying processes of host-pathogen interactions. The research groups at the Institute of Molecular Infection Biology (IMIB) at the University of Würzburg perform many experiments that specifically require a technically highly equipped state-of-the-art CLSM. In addition, planned and future research includes scientific questions that cannot be addressed with the limited analysis tools and technical equipment of the currently used CLSM. Thus, we are here applying for a high-performance confocal microscopy platform to replace the previous CLSM, an almost 12-year-old device. The planned work includes, e.g., the establishment of novel organoid-based infection models for pathogens and commensals, the analysis of RNA-based therapeutics, the subcellular localisation of RNAs and proteins in bacterial cells, as well as the discovery and characterisation of high-molecular complexes in phage-infected bacteria. These projects will be carried out both with fixed samples as well as under live-cell imaging conditions. Especially for the latter, it is essential that the CLSM is set-up in a biosafety level 2 (S2) laboratory, as the projects will be carried out with infectious agents of the risk group 2. For their projects, the groups of the IMIB require an excitation spectrum extended into the near-infrared range, complete spectral freedom, gentle and sample-preserving conditions for live-cell analysis in an S2 laboratory, as well as increased resolution into the nanometre range. The equipment of the here requested replacement device combines the novel white light laser technology, with a continuous excitation spectrum of 440-790 nm, an optimised beam path, and four highly sensitive detectors. In addition, an adaptive deconvolution process increases the resolution range to 200 nm. Built-in imaging tools that allow for measurements based on fluorescence lifetimes, will help to answer questions about the effects of changes in the cellular milieu on bacteria themselves or even on the amount and localization of bacterial RNAs or proteins. Therefore, the CLSM that we apply for in this proposal, will be an essential, state-of-the-art, and powerful replacement instrument that will allow us to investigate the molecular mechanisms of bacterial pathogens and infection processes. This CLSM will significantly expand the range of resolution required to visualise subcellular RNA- and protein-based regulatory mechanisms in bacteria. The resulting data will substantially expand our knowledge of gene regulation and virulence mechanisms of widespread pathogens and thus pave the way for new diagnostic and therapeutic approaches.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokales Laser-Scanning-Mikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Julius-Maximilians-Universität Würzburg
• Leader: Professorin Dr. Cynthia Mira Sharma