Workshop 5: Tau-STED - application of lifetime measurement in super-resolution microscopy

Fluorescence lifetime provides a new perspective for STED nanoscopy. This workshop will focus on Tau-STED, a new concept for STED Nanoscopy that exploits the fluorescence lifetime gradient induced by the STED beam in a novel way to identify and remove uncorrelated background, improve image quality and increase image resolution in an automated way. This is only possible thanks to the unique combination of STED, ultra-fast photon-counting detectors, and FLIM as provided by the FALCON approach (Alvarez et al, Nat Methods 2019). Tau-STED identifies the signal from the STED process to differentiate it from background using fluorescence lifetime. It identifies the fluorescence lifetime gradient generated by the STED process with the help of FLIM Phasor analysis to deliver the desired resolution at dramatically lower light dose, and to push the resolution beyond what conventional STED provides. The experiment raw data is always available for validation and quantification of results. We will work with a variety of single and multicolor fixed samples, focusing on structures typically observed with STED (nuclear pores, microtubules, mitochondria). The lower excitation and STED light dose protect the sample during imaging, and enable acquiring more frames for large volume imaging or time-lapse experiments, which we will demonstrate with living cells. Alvarez L, Widzgowski B, Ossato G, van den Broek B, Jalink K, Kuschel L, Roberti MJ, and Hecht F: “SP8 FALCON: a novel concept in fluorescence lifetime imaging enabling video-rate confocal FLIM”, Nat Methods (2019). https://www.nature.com/articles/d4247... ================== Presenter: Steffen Dietzel, Head of the Core Facility Bioimaging, Ludwig-Maximilians-Universität München (Germany) Mariano Gonzalez Pisfil, Staff Scientist at the Core Facility Bioimaging, Ludwig-Maximilians-Universität München (Germany) Daniel Smeets, Application Specialist Confocal Microscopy EMEA, Leica Microsystems GmbH (Germany) ================== This video is recorded on 26.11.2021 during the online FLIM symposium - Beginners Guide to Fluorescence Lifetime Imaging (FLIM) in Life Sciences takes place on November 23. to 27. 2020 via Zoom. ================== Website: https://webapp.biotec.tu-dresden.de/e...