Figure 2.

(A) Jablonski diagram: once an organic dye absorbs a photon it is excited from the ground state S₀ to the first excited singlet state S₁ (k_ex) and can return to S₀ by emission of a photon (k_fl, fluorescence). The dye can undergo numerous excitation-emission cycles before it enters a non-fluorescent triplet state T₁ via intersystem crossing (k_ISC). Once in the triplet state, the dye can either return directly to S₀ (k_t) via different, competing pathways or can be destroyed by singlet oxygen (photobleaching). The reduction of the dye in its triplet state to a non-fluorescent radical anion A⁻ (k_red, red), which can subsequently be oxidized (k_ox, blue) promotes the depopulation of T₁ to S₀. The depopulation of the triplet state can also start with the oxidation reaction followed by the reduction step; (B–D) Exemplary photophysical behavior of organic dyes. Fluorescence transients of Cy5-labeled dsDNA in aqueous PBS buffer (B). The removal of oxygen leads to reduced triplet quenching and increased blinking. (C) Addition of 2 mM Trolox(TX)/Troloxquinone (TXQ) enables stable and prolonged fluorescence over minutes (D). Photostabilization by the “geminate recombination” mechanism [48] (E). A time series of frames showing the photostability of Alexa568 dyes in different buffers (2 mM TX/TXQ, left column; 1% beta-mercaptoethanol (ME; center), and a combination of both (right column)), oxygen was removed in all cases (see text for details). Scale bar: 10 μm. The last row shows transients from a confocal microscope that illustrate the differences in blinking and photobleaching behavior of the dye. (F) Total number of detected photons before photobleaching or long-lived dark states for the dyes Alexa568 and ATTO647N in different buffers (figures in panel A, E and F adapted by permission from Holzmeister et al., Geminate recombination as a photoprotection mechanism for fluorescent dyes, Angewandte Chemie, 2014).