Tailoring Cyanine Dark States for Improved Optically Modulated Fluorescence Recovery

Abstract

Cyanine dyes are well known for their bright fluorescence and utility in biological imaging. Yet, cyanines also readily photoisomerize to produce non-emissive 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 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.

Simple imaging protocol for autofluorescence elimination and optical sectioning in fluorescence endomicroscopy

“…The original OPIOM protocol exploiting resonant modulated monochromatic light can be used when the backward isomerization after forward photoswitching is thermally driven [31]. Nevertheless, the Speed-OPIOM protocol [32] used in this study should be preferentially adopted when the forward and backward photoswitching processes of the reversibly photoswitchable fluorescent labels can be photochemically governed at two distinct wavelengths (e.g., in RSFPs [34], cyanines [63], and spirobenzopyrans [64]). Indeed, the Speed-OPIOM response is 2 times higher than the OPIOM one and accordingly provides a better signal-to-noise ratio.…”

Macroscale fluorescence imaging against autofluorescence under ambient light

“…When the backward isomerization that follows forward photoswitching is thermally driven, one can use the original OPIOM protocol, which exploits the resonant modulation of monochromatic light, to eliminate spectral interferences 23 . Alternatively, when the forward and backward photoswitching processes are photochemically governed at two distinct wavelengths, as in many reversibly photoswitchable labels (e.g., RSFPs 17 , 18 , azobenzenes 43 – 45 , cyanines 14 , 46 , 47 , diarylethenes 15 , 48 , and spirobenzopyrans 49 ), one should preferentially rely on the Speed OPIOM protocol that is used in this study since it gives rise to an amplitude that is twice that of the collected signal and, accordingly, a higher signal-to-noise ratio. Our approach can also be applied to nonphotoswitching fluorophores if their photophysical scheme can be dynamically reduced to a two-state model on a sufficiently long time scale.…”

Anti-Stokes fluorescence microscopy using direct and indirect dark state formation

“…Additionally we would like to point out that the presented OADF and UCF modalities are not necessarily limited to DNA-AgNCs specific dark states, but that triplet and other dark states, present in organic fluorophores or fluorescent proteins could potentially also be applied. [33][34][35][36] We gratefully acknowledge financial support from the ''Center for Synthetic Biology'' at Copenhagen University funded by the UNIK research initiative of the Danish Ministry of Science, Technology and Innovation (Grant 09-065274), bioSYNergy, University of Copenhagen's Excellence Programme for Interdisciplinary Research, the Villum Foundation (Project number VKR023115), the Carlsberg Foundation (CF14-0388), the Danish Council of Independent Research (Project number DFF-7014-00027).…”