Photoisomerization of Heptamethine Cyanine Dyes Results in Red-Emissive Species: Implications for Near-IR, Single-Molecule, and Super-Resolution Fluorescence Spectroscopy and Imaging

[Image: see text] Photoisomerization kinetics of the near-infrared (NIR) fluorophore Sulfo-Cyanine7 (SCy7) was studied by a combination of fluorescence correlation spectroscopy (FCS) and transient state (TRAST) excitation modulation spectroscopy. A photoisomerized state with redshifted emission was identified, with kinetics consistent with a three-state photoisomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced redshift in the emission spectrum of SCy7. We show how this red-emissive photoisomerized state contributes to the blinking kinetics in different emission bands of NIR cyanine dyes, and how it can influence single-molecule, super-resolution, as well as Förster resonance energy transfer (FRET) and multicolor readouts. Since this state can also be populated at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also readouts not relying on high excitation conditions. However, this additional red-emissive state and its photodynamics, as identified and characterized in this work, can also be used as a strategy to push the emission of NIR cyanine dyes further into the NIR and to enhance photosensitization of nanoparticles with absorption spectra further into the NIR. Finally, we show that the photoisomerization kinetics of SCy7 and the formation of its redshifted photoisomer depend strongly on local environmental conditions, such as viscosity, polarity, and steric constraints, which suggests the use of SCy7 and other NIR cyanine dyes as environmental sensors. Such environmental information can be monitored by TRAST, in the NIR, with low autofluorescence and scattering conditions and on a broad range of samples and experimental conditions.