Leica Microsystems CMS GmbH Workshop

The new TauSTED technology (1) sets the new standard for straightforward gentle STED using lifetime-based information. Stimulated emission depletion (STED) induces changes in the spatial fluorescence lifetime distribution, therefore the combination of STED and fluorescence lifetime can benefit applications requiring nanoscale resolution in fixed and living specimens. Pioneering developments using lifetime-derived information gave rise to gated STED (2) and recent implementations based on FLIM phasor analysis (3, 4). Inspired by this approach, we developed TauSTED, that combines the optical signals from STED and the physical information from the fluorescence lifetime at confocal speeds. This approach uses phasor analysis in a novel way, delivering outstanding STED resolution and image quality, while removing background noise, even at low excitation and STED light dose. 

This workshop will focus on explaining the underlying principles of TauSTED, how it is implemented on the STELLARIS confocal microscope as a TauSense tool (5), and in combination with the FALCON approach (6). We will use fixed samples typically imaged with STED: single labelled nuclear pores and multicolour labelled cytoskeleton (microtubules, actin) and mitochondria. We will in addition show the extended live cell imaging capabilities afforded by TauSTED, thanks to greater access gentle STED imaging conditions.

1. Alvarez LAJ, Schwarz U, Friedrich L, Foelling J, Hecht F and Roberti M J.  Application Note: TauSTED: pushing STED beyond its limits with lifetime Nat Methods (2021) 

2. Vicidomini G, Moneron G, Han KY, Westphal V, Ta H, Reuss M, Engelhardt J, Eggeling C, Hell SW. Sharper low-power STED nanoscopy by time gating. Nat Methods (2011), Jun 5; 8(7):571-3. 

3. Lanzano L et al. Encoding and decoding spatio-temporal information for super-resolution microscopy. Nat Communications (2015), 6, 6701. 

4. Tortarolo et al. Photon-separation to enhance the spatial resolution of pulsed STED microscopy. Nanoscale, (2019), 11, 1754-1761. 

5. Roberti M J, Ortiz Lopez L, Ossato G, Steinmetz I, Haas P, Hecht F and Alvarez LAJ. Application Note: TauSense: a fluorescence lifetime-based tool set for everyday imaging. Nat Methods (2020) https://www.nature.com/articles/d42473-020-00364-w  

6. 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/d42473-019-00261-x