Confocal-Laser-Scanning-Microscope

• Subject Area: Neurosciences
• Term: Funded in 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 491176901

An inverse confocal laser-scanning microscope of the latest generation is requested as a replacement for an existing Laserscanning Microscope. Here, a higher sensitivity should be given in comparison to conventional confocal microscopes in order to keep the light exposure of the tissue to be examined as low as possible. In conventional confocal microscopes, such as the LSM to be replaced, much of the emitted light is discarded by a pinhole, thus reducing sensitivity. Since extensive analyses were not possible due to the slow scanning speeds of the previous system and due to the limited measuring times, a much faster scanning speed of the system is necessary. To increase the sampling rate, scanning multiple image lines at the same time would be preferable to a faster scanning speed of a single line, as this is the only way to keep the exposure time per pixel constant leading to no signal-to-noise ratio deterioration.As a functional extension compared to the existing LSM, the requested microscope is supposed to be equipped with an incubator for the registration of cell cultures. For the analysis of subcellular compartments, the lateral spatial resolution of the requested device, in contrast to conventional laser scanning microscopes, must be around 120nm and therefore beyond the diffraction limit. Here, an optical method for increasing the lateral resolution rather than a software solution such as a pure deconvolution calculation is preferable. Five excitation wavelengths (405nm, 488nm, 561nm, 594nm, 639nm) are required for the most common dyes and a possibility to extent these by further laser lines is necessary. In addition, the microscope should also contain a spectral detector, which enables a simultaneous detection of several emission wavelength ranges, especially for multiple labeled fixed or vital preparations. By linear demixing, the emission signals of spectrally overlapping dyes could be separated and thus their proportions could be quantified for each image pixel. This would significantly expand the variety of dyes usable for our scientific projects, as spectral overlap becomes a less critical factor. In addition, the spectral decomposition would significantly increase the sensitivity and thus the signal-to-noise ratio.The requested laser-scanning microscope will be installed centrally in the Lobeda Research Centre at the University Hospital Jena and will be accessible for working groups from different clinics of the Faculty of Medicine. The operation of the microscope is secured by experienced personnel and a long-proven training concept ensuring the integration of new users.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Konfokales Laser-Scanning-Mikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Friedrich-Schiller-Universität Jena
• Leader: Professor Dr. Knut Holthoff