Inverse STED Superresolution Microscope

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 541518226

In this proposal, the CECAD Imaging Facility is applying for a STED microscope as a replacement for their old Leica STED system that was purchased in 2013. STED is one of our highly demanded techniques in the imaging facility over many years now. With the new STED, we want to increase the sensitivity of the measurements even further to allow better STED imaging in several aspects. Firstly, less photobleaching enables the possibility to use more STED laser power e.g in fixed cells to further increase the resolution, since the STED beam power directly impacts on the effective PSF size and by that, the resolution that can be reached. Secondly, more sensitive imaging leads to less phototoxicity, a factor that is crucial in live cell imaging and highly needed for the requested research project in this proposal. STED itself works with high light doses so any kind of adaptive illumination or extracting information in a smart way to achieve a better signal to noise ratio is very important. Thirdly, several groups are moving from 2D cell culture approaches into tissue which is limited within our system so far due to scattering issues especially of the STED donut. Adaptive optics could compensate for these issues by shaping the beam front to compensate for the scattering. We will evaluate the benefits of this technique with our own samples in the near future. In the fourth point, even though not strictly for the STED purpose but for the confocal part of the microscope, we have the need to extend the imaging spectrum to the near infrared to give our users the possibility to image 6-7 fluorophores at the same time, a feature which is not covered by any other microscope in the platform but demanded more and more. This can be achieved by a new version of white light laser ranging deeper in the NIR or dedicated laser diodes covering this spectrum up on request. The new STED system should have an intuitive user interface due to the multiuser environment within the facility. We ask for an inverse microscope since especially for live-cell imaging, glass-bottom dishes are our preferred solution. The system needs to be equipped with an incubation chamber and 1Ph excitation lasers to cover the full visible spectrum (405 nm - 640 nm + NIR if possible). Taken together, we were very happy with the performance of our now depreciated old STED system but looking very much forward to offer the next generation of possibilities with a new STED microscope to all of our users on campus.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Inverses STED Superresolutionsmikroskop
• Instrumentation Group: 5090 Spezialmikroskope
• Applicant Institution: Universität zu Köln
• Leader: Dr. Astrid Schauss