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

This folder contains all raw data underlying the results presented in a manuscript, submitted to J. Phys. Chem. B, and entitled:

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

Authored by:

Elin Sandberg^a, Joachim Piguet^a, Uliana Kostiv^a, Glib Baryshnikov^b, Haichun Liu^a, Jerker Widengren^a,*

^a Royal Institute of Technology (KTH), Experimental Biomolecular Physics, Dept. Applied Physics, Albanova Univ Center 106 91 Stockholm, Sweden

^b Linköping University, Dept. Science and Technology, Campus Norrköping, 601 74 Norrköping, Sweden

* Corresponding author: Email:  jwideng@kth.se, Phone: +46-8-7907813

Data files containing the raw data and the results of the analysis are grouped according to the order of the figures in the manuscript.

ABSTRACT

The 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 photo-isomerized state with red-shifted emission was identified, with kinetics consistent with a three-state photo-isomerization model. Combining TRAST excitation modulation with spectrofluorimetry (spectral-TRAST) further confirmed an excitation-induced red-shift in the emission spectrum of SCy7. We show how this red emissive photo-isomerized 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 be populated also at moderate excitation intensities, it can also more broadly influence fluorescence readouts, also when 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 red-shifted photo-isomer 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.