Tailoring cyanine dark states for improved optically modulated fluorescence recovery

Cyanine dyes are well-known for their bright fluorescence and utility in biological imaging. However, cyanines also readily photoisomerize to produce nonemissive dark states. Co-illumination with a secondary, red-shifted light source on-resonance with the longer wavelength absorbing dark state reverses the photoisomerization and returns the cyanine dye to the fluorescent manifold, increasing steady-state fluorescence intensity. Modulation of this secondary light source dynamically alters emission intensity, drastically improving detection sensitivity and facilitating fluorescence signals to be recovered from an otherwise overwhelming background. Red and near-IR emitting cyanine derivatives have been synthesized with varying alkyl chain lengths and halogen substituents to alter dual-laser fluorescence enhancement. Photophysical properties and enhancement with dual laser modulation were coupled with density functional calculations to characterize substituent effects on dark state photophysics, potentially improving detection in high background biological environments.