Fluorescence Imaging Spectroscopy Microscope

• Subject Area: Plant Sciences
• Term: Funded in 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 553896058

Confocal laser scanning microscopy (CLSM) is widely used in molecular cell biology to identify and localize fluorescently tagged proteins in live cells with high spatial and temporal resolution. Despite its utility, a deeper understanding of dynamic protein behaviour, molecular interactions, and precise cellular localization is needed. Therefore, advanced techniques such as Fluorescence Lifetime Imaging Microscopy (FLIM), Fluorescence Anisotropy (FA) detection, and Fluorescence Correlation Spectroscopy (FCS) are required to exceed conventional CLSM capabilities by adding time-resolved detectors that are sensitive to single photons. FLIM, a technique measuring the duration a fluorophore remains excited before returning to its ground state, precisely detects protein-protein interactions (PPI) in vivo by measuring Förster Resonance Energy Transfer (FRET). This allows for accurate measurements of protein complexes in vivo, essential for understanding intricate and dynamic cellular processes at the molecular level. The simultaneous acquisition of FA complements FLIM as it delivers information about the orientation and rotational mobility of proteins with unique accuracy and precision which is vital for studying PPIs and complex behaviour in live cells over time and in response to intrinsic or extrinsic cues (e.g., heat or chemical stresses). FCS, which detects the mobility and number of single molecules in the observation volume, provides even more high-quality quantitative data. Pulsed Interleaved Excitation (PIE) enhances the FIS-M setup by enabling precise and simultaneous multi-colour FLIM, FA, and FCS measurements. An additional area detector enables 2-fold higher resolution and FCS-measurements at higher concentrations. The integration of FLIM, FA, and FCS in the requested FIS-M, provides a comprehensive and state-of-the-art toolset for studying the dynamic molecular behaviour of protein complexes in live cells as well as their underlying function. This advanced capability is indispensable for addressing intricate biological questions and advancing our understanding of fundamental cellular processes. This unique FIS-M setup allows to measure in vivo how proteins interact with each other and with other cellular components, how these interactions are regulated, and how they contribute to cellular function and dysfunction with the highest spatial and temporal resolution. The proposed FIS-M would provide Goethe University (GU) researchers access to cutting-edge imaging techniques that were previously unavailable. This advanced microscopy setup would enable them to explore molecular dynamics in live cells with unprecedented detail. Such advancement in research capabilities would benefit the research groups intending to use the described techniques and greatly support current and future collaborative efforts, making it a highly valuable resource for the entire scientific community at GU.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Fluoreszenzbildgebungsspektroskopie-Mikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Goethe-Universität Frankfurt am Main
• Leader: Professorin Dr. Yvonne Stahl