Multimodal, Confocal Super-Resolution STED Microscope with integrated Fast Lifetime Imaging Modality

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 543363886

We apply for a microscope system with superresolution capacity provided by stimulated emission depletion (STED) combined with a fast fluorescence lifetime imaging (FLIM) modality. This system will allow us to study our key research objectives both in live cells and organisms as well as in fixed samples. We aim at characterizing the process and control of bacterial ribosome biogenesis and maturation at unprecedented spatial resolution with the STED system. This will be done by analyzing the localization of fluorescently tagged components such as e.g. the GTPase ObgE or the DEAD-box helicase DeaD involved in different phases of ribosome biogenesis in the bacterial nucleoid and upon pharmacological manipulation in live cells. We will disentangle the fine structure of nucleoids in their native and also in a genetically perturbed configuration, e.g. upon knockdown of involved proteins, and investigate structural und functional consequences for ribosome biogenesis. In mammalian cells in culture and multicellular model organisms we will study 1) the mechanism and control of maturation of nascent polypeptides during their synthesis by the ribosome where we seek to elucidate how access of enzymes modifiying the N-terminus of a nascent polypeptide is coordinated and which factors are involved, 2) the folding mechanism of hetero-oligomeric protein complexes and 3) the processes at play under protein folding stress, i.e. when proteins aggregate. To this end we will perform fluorescence cross correlation spectroscopy (FCCS) and we will use FLIM for fluorescence lifetime correlation spectroscopy (FLCS) that allow to study molecular dynamics in living cells. When done with a STED microscope, it is possible to measure diffusion processes at the nanoscale due to the very small observation volume. To collect a maximum of photons emanating from the protein complexes present in this small volume during the FCCS and FLCS experiments, the system requires very sensitive detectors with the lowest possible dead time. The size of protein aggregates investigated in our laboratory enables not only FLIM/FCCS analyses but also studies by STED microscopy. In order to cover a broad range of fluorescence labels the STED microscope will be equipped with two lasers for emission depletion. This will not only allow to employ cell permeable label systems (SNAP- and Halo-tags) that are suited for use with bacteria and mammalian cells in culture but will also enable us to analyze model organisms not amenable to the use of such cell permeable labels but rather depend on the classic fluorescent protein labels. A pulsed white light excitation laser will cover the full spectrum of excitation wavelengths ranging from 440 to 790 nm allowing for optimal adaptation to the excitation maximum of the label and optimal signal yield and make the novel near IR labels and fluorescent proteins accessible for experiments.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Multimodales, konfokales Super-Resolution STED-Mikroskop mit Fast Lifetime Imaging Modalität
• Instrumentation Group: 5000 Labormikroskope
• Applicant Institution: Universität Konstanz
• Leader: Professorin Dr. Elke Deuerling