High-speed TIRF-SIM/SIM super-resolution microscope for live-cell imaging

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 496847469

Our understanding of complex dynamic cellular processes has advanced dramatically in recent years due to the development of cutting-edge super-resolution microscopy techniques that overcome the resolution limit of conventional light microscopy. Structured illumination microscopy (SIM) has proved to be one of the most competitive approaches given its relatively simple setup and broad applicability due to the use of a wide range of classical fluorophores and standard sample preparation protocols. Furthermore, the significant increase in acquisition speed due to spatial light modulator (SLM) devices for pattern generation now facilitates the routine use of SIM for live-cell imaging and the precise spatio-temporal investigation of dynamic subcellular processes on the molecular level. The participating groups in this proposal have so far gained critical insight into molecular mechanisms that control key cellular processes with physiological and pathological relevance. In particular, we have successfully studied processes ranging from DNA damage repair, golgi stress and endo-lysosomal damage response to cell migration and adhesion. The requested SIM microscope system will facilitate dissecting these molecular mechanisms with significantly higher resolution in space and time. Furthermore, many dynamic structures that are critical for cellular behaviour, such as the cortical actin cytoskeleton and adhesion sites are localized near the plasma membrane. Thus, we will combine super resolution live-cell imaging with total internal reflection fluorescence microscopy (TIRF-SIM) to take advantage of the dramatically improved signal-to-noise ratio for studying the molecular processes in these cell regions. Multi-colour read-outs and high-speed live-cell imaging combined with flexible ultra-fast switching between different imaging modes (TIRF-SIM/SIM/TIRF) will help deciphering the spatio-temporal coordination of key molecules identified in our previous studies with potential regulatory factors.Beyond steady state measurements, we will also combine the different imaging modalities with acute drug treatments and light-based manipulation of related signal networks components with an integrated photostimulation device. Using the wide range of fluorescence read-out capabilities, we will be able to perform multiple cell response measurements upon perturbations including organelle morphodynamics, protein localization and cytoskeletal and adhesion reorganization. Such perturbation-response studies will greatly advance our understanding of causalities in the signal networks and help dissecting complex regulatory mechanisms. Together, the unique combination of system components in the requested microscope will facilitate the dissection of a multitude of cellular processes with very high spatio-temporal precision and thus decisively expand our understanding of underlying molecular mechanisms.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: TIRF-SIM/SIM-Mikroskop für Lebendzelluntersuchungen
• Instrumentation Group: 5040 Spezielle Mikroskope (außer 500-503)
• Applicant Institution: Universität Duisburg-Essen
• Leader: Professorin Perihan Nalbant, Ph.D.