confocal laser scanning microscope for live imaging

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 446967193

Live imaging represents a central research method of the laboratory of Prof. Großhans. Building on classic immunohistology of fixed samples, developmental processes are examined in their dynamics on length scales between subcellular structures up to the tissue level. The time scale ranges from 100 ms to hours. Dynamic data document the history and future of a selected object in time and are therefore much more information-rich than images of fixed embryos. Dynamic image data allow an analysis of temporal changes in the measured parameters, such as cell cross-sectional area, junction length, myosin activity, for example. Dynamic multimodal data can be used to investigate whether a correlation of e.g. high amount of myosin with the course of the cross-sectional area several seconds later (cross correlation). In most cases, fluorescence labeling is carried out using genetically encoded dyes (GFP and variants) that are expressed as fusion proteins or reporter proteins. In many cases - and in the future more and more - genetic labeling takes place at the endogenous locus, so that under certain circumstances a low signal can be expected.Three qualities are central to live imaging: signal quality (signal-to-noise ratio), recording speed, image resolution. State-of-the-art microscopes for live imaging use strategies to increase signal detection or reduce phototoxicity. A current strategy for increasing sensitivity, resolution and speed is the use of an area detector which is placed in the light path instead of the pinhole. This method solves two problems: detection (almost) of all photons and increased spatial resolution beyond the Abbe criterion. Furthermore, with suitable illumination, the area detector can be used to simultaneously detect several lines (up to 8 in the case of slit excitation), which leads to a much higher scanning speed. For an acceptable signal quality in live imaging, hybrid detectors are required, which are characterized by high signal dynamics and a quantum yield of up to 50%.A significant extension of the functionality of confocal systems is the use of UV lasers in the range between 355 nm (limited by the optical transmission of glass) and 400 nm. Pulsed UV lasers generate an energy density that unspecifically destroy all structures in the focus volume. Continuous wave UV lasers are used for the specific opening of chemical bonds (uncaging).

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokales Laserscanning Mikroskop für Lebendbildgebung
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Philipps-Universität Marburg
• Leader: Professor Dr. Jörg Großhans