STED Microscope


Figure 4: Unlocking uncomplicated multi-color experiments with time-resolved detection. Four-color imaging showing nuclear pores (NUP968, STAR RED), Golgi membrane (GM130, STAR 635), Golgi medial rims (Giantin, STAR ORANGE), and actin (STAR 580 phalloidin) in fixed cells. (A) Confocal image without time-resolved detection shows only two colors due to limited separability illustrated in (C). (B) Detail of the orange (upper image) and red (lower image) detection channels with separation by lifetime illustrated in (D). (E) Final, fully separated image. Scale bars: (A) 5 μm, (B and E) 2 μm.


STAR 580, and STAR RED and STED 635, respectively, are too similar to be discriminated (Figure 4A). However, they can be separated using another characteristic measure of fluorescence: the lifetime of the excited state, measured


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by the time that passes between absorption and emission of a photon. Time-resolved detection is needed to record this information, and an easy and versatile implementa- tion was recently released with TIMEBOW. Analyzing the


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