Skip to main content
. Author manuscript; available in PMC: 2020 Mar 27.
Published in final edited form as: Methods. 2019 Jul 22;174:27–41. doi: 10.1016/j.ymeth.2019.07.019

Figure 4.

Figure 4

RESCue STED attenuates photobleaching compared to conventional STED. Two regions of the same nucleus of a fixed U-2 OS cell immunostained for the nuclear pore protein Nucleoporin 153 with Abberior Star Red were each imaged 30 times using conventional STED (upper sequence) or RESCue STED (lower sequence). In an initial confocal probing step of 5 μs duration, a decision level of 30 photon counts was used to determine whether STED mode should be used at the respective pixel. For RESCue STED, four decision times with corresponding lower thresholds (lTh) were used for early laser shutdown: 1 count after 14% of the pixel dwell time, 3 counts after 24%, 6 counts after 37% and 16 counts after 79%. Upper threshold (uTh) that triggered premature laser shutoff and signal extrapolation was 50 counts. Pixel size was set to 15 nm with a dwell time of 40 μs, laser power at the sample was 1.7 μW for 640 nm excitation and 103 mW for 775 nm STED in both conditions. Scale bars: 500 nm. Note that the colour lookup table is different for frame #1 vs. the later frames.