Fig. 3a–b.

Photophysics and photochemistry of fluorophores. a Left: Jabłoński energy diagram representing energy states and transitions of a fluorophore. S ₀ ground singlet state, S ₁ excited singlet state, T ₁ triplet state, F ^●− radical state. Different compounds can affect brightness and photostability or shift the fluorophore into a radical state. (i) absorption spectra of H₂O and D₂O, and correlated enhancements of the fluorescence emissions of different fluorophores in D₂O versus H₂O for the visible range of light. Adapted from [72] with permission. (ii) Cyclooctatetraene (COT) quenches the triplet state by quickly transferring fluorophores back into the ground state and thus stabilizes the fluorescence. Adapted with permission from [73]. (iii) A reducing and oxidizing system (ROXS) accelerates the transition of a fluorophore from its triplet state back to the electronic ground state by performing fast sequential reducing and oxidizing steps. Adapted with permission from [74]. (iv) The radical states of some dyes (e.g., the Alexa Fluor 488 fluorophore, as shown in black here; red indicates the radical) possess an absorption peak in the UV range. By exciting the radicals with UV light to higher intermediate states, they can be quickly brought back down to their electronic ground state. Adapted with permission from [75]. b Different fluorophore structures: (i) Barrel structure of the photoactivatable green fluorescent protein (paGFP) and a close-up of its chromophore. (ii) Overview of organic dye classes. c Different photochemical and conformational changes that affect fluorescence: (i) photoactivation of paGFP [76], (ii) green-to-red photoconversion of mEos2 [77], (iii) reversible cis/trans-photoswitching of Dronpa [78], (iv) cleavage of a photocage from a rhodamine [79], (v) reversible fluorescence quenching of Cy5 by covalent binding of a thiol [80], and (vi) reversible cyclization of rhodamine HMSiR [81]