Confocal laser scanning microscope with fast fluorescence lifetime imaging module

• Subject Area: Plant Sciences
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 540803833

Imaging of fluorescently labelled cellular structures using confocal laser scanning microscopy (CLSM) is one of the key methodologies to study cellular processes with high spatial and temporal resolution in various biological contexts. In our own research, we employ a broad portfolio of methods that rely heavily on confocal microscopy, such as localisation and expression studies of fluorescently labelled proteins, live imaging of fluorescent indicators of Ca2+, pH or membrane microviscosity, formation and dynamics of fluorescently labelled endomembrane and biocondensate compartments, and Förster resonance energy transfer (FRET)-based techniques to study the kinetics of complex dissociation and protein-protein interactions. Overall, research in the applicant labs would not be possible without the use of confocal laser scanning microscopy. With this grant application we aim to replace an existing CLSM purchased in 2010 which is now outdated, prone to repair and uneconomical to maintain, which is a limiting factor for our ongoing imaging-based research. Future research objectives will require the simultaneous use of multiple fluorescent probes. Therefore, the future system must be able to operate up to four different fluorescence channels in parallel, with specifications for fluorescence excitation and detection ranging from the ultraviolet to the near-infrared region of the spectrum. In addition, the required instrument should be able to simultaneously acquire intensity and lifetime information. The acquisition of fluorescence lifetime information is required for applications such as subtraction of autofluorescence that spectrally overlaps with fluorescent reporters, imaging of cellular pH and membrane microviscosity, and dynamic measurements of enzyme complex formation and protein-protein interactions. The system therefore requires a pulsed laser source and highly sensitive detectors capable of operating in photon-counting mode. For dynamic imaging of cellular structures smaller than 500 µm (subdomains of endosomes, membrane domains and bio-condensates), it will be necessary to image 3D volumes with high spatial and temporal resolution. Therefore, the instrument must be equipped with an ultra-fast scanning option combined with adaptive image processing software that can compute images on the fly. Our demand for a CLSM equipped with a fast fluorescence lifetime imaging module and ultra-fast scan optics cannot be served by other microscope systems in house or in the central core imaging facility on campus (NIC) due the amount of user time that will be required by the applicant groups and due to technical limitations, such as the specification of the pulsed laser sources and the demand for ultra-fast fluorescence lifetime acquisition. Overall, the realisation of the current and future research goals of the applicant groups will depend critically on the acquisition of the requested confocal laser scanning microscope

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokales Laserscanning-Mikroskop mit Modul zur schnellen Fluoreszenz-Lebenszeit-Bildgebung
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Ruprecht-Karls-Universität Heidelberg
• Leader: Professorin Dr. Karin Schumacher