2-photon microscope (Imaging / Electrophysiology)

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Two-Photon microscopy has become an indispensable method to visualize cellular processes within living tissue. Relying on infrared light that can penetrate tissue with less scattering than visual light, this modern technique permits excitation of fluorophores in significantly deeper tissue layers than conventional fluorescence microscopy. This allows for imaging of cells in a depth of approximately 150 µm in acute slice preparations, which are routinely used as model systems for neuroscience or immunological research in many work groups at the Otto-von-Guericke-University Magdeburg. Although there is an ongoing strong interest in work with acute slice preparations, the only available 2-photon microscopes at the Medical Faculty Magdeburg campus have been specifically customized for in vivo experiments with animals. A temporary retooling of these setups for slice work would be too laborious and too time consuming to be feasible. The requested 2-photon microscope is therefore intended as a dedicated setup for work with tissue slices, offering users in particular the possibility to combine 2-photon imaging with electrophysiological recordings. The new microscope will replace an inoperable outdated 2-photon prototype (Till Photonics, originally purchased in 1999) in the Institute of Physiology, whose image quality can no longer meet current standards. To reduce costs, existing instrumentation for electrophysiological experiments (micromanipulators, motorized stage etc.) and additional components from the original setup will be re-used with the modern 2-photon microscope. Apart from the use of the microscope in the applicants’ projects (see form sheets "Research"), it will also become a part of the core facility "Multidimensional microscopy and cellular diagnostics", making it available for all interested researchers at the OVGU Magdeburg. The chosen configuration of the microscope will allow for sophisticated live-cell imaging experiments, in which red and green fluorophors can be simultaneously visualized with high spatial and temporal resolution. Therefore, the microscope needs to contain two separate laser lines (one tuneable femtosecond laser and one fixed wavelength laser) that permit independent excitation of two fluorophores near their optimal absorption cross sections. Moreover, the configuration of the microscope scan head needs to support fast and precise scanning of lines, areas, and volume elements within the slice preparation. Lastly, the detectors should possess a rapid gating mechanism ("gated photomultipliers") to exclude the risk that massive light exposure during optogenetic stimulation with an external laser can cause any detector damage. Taken together, the procurement of a state–of-the-art 2-photon microscope with such properties should significantly facilitate the progress in several DFG-funded research projects at the campus Magdeburg.