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Major signal increase in fluorescence microscopy through dark-state relaxation


We report a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 μs, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5–25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 μs. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes. Please visit methagora to view and post comments on this article

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Figure 1: Photobleaching of GFP and Atto532 decreases with inter-pulse break Δt = 1/f for one- and two-photon excitation.
Figure 2: Total fluorescence signal generated by one-photon excitation (G1p) for GFP and Atto532 for a given number of excitation pulses (1.4 × 106).
Figure 3: Energy diagram of a typical organic fluorophore, indicating the major molecular pathways for excitation (Exc), fluorescence (Fl), intersystem crossing (ISC), relaxation (dashed lines) and photobleaching (bleach).
Figure 4: Total fluorescence signal generated by two-photon excitation (G2p) for GFP and Atto532 for a given number of excitation pulses (1.4 × 106).
Figure 5: The pulse duration does not affect the total fluorescence signal G2p of two-photon excitation.
Figure 6: Two-photon fluorescence images of Escherichia coli cells expressing the fluorescent protein Venus are brighter when recorded in the D-Rex mode.


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We thank A. Miyawaki (Laboratory for Cell Function and Dynamics, Riken) for supplying the fluorescent protein Venus. We also thank S. Jakobs, S. Verrier and D. Ouw for preparation of the samples, A. Giske, R. Kellner and K. Willig for help with the experimental setup, and V. Westphal and A. Egner for fruitful discussions. Finally, we thank A. Schönle for support with the IMSPECTOR software.

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Correspondence to Stefan W Hell.

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Donnert, G., Eggeling, C. & Hell, S. Major signal increase in fluorescence microscopy through dark-state relaxation. Nat Methods 4, 81–86 (2007).

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