Combined Fluorescence Fluctuation and Spectrofluorometric Measurements Reveal a Red-Shifted, Near-IR Emissive Photo-Isomerized Form of Cyanine 5

Abstract

Cyanine fluorophores are extensively used in fluorescence spectroscopy and imaging. Upon continuous excitation, especially at excitation conditions used in single-molecule and super-resolution experiments, photo-isomerized states of cyanines easily reach population probabilities of around 50%. Still, effects of photo-isomerization are largely ignored in such experiments. Here, we studied the photo-isomerization of the pentamethine cyanine 5 (Cy5) by two similar, yet complementary means to follow fluorophore blinking dynamics: fluorescence correlation spectroscopy (FCS) and transient-state (TRAST) excitation–modulation spectroscopy. Additionally, we combined TRAST and spectrofluorimetry (spectral-TRAST), whereby the emission spectra of Cy5 were recorded upon different rectangular pulse-train excitations. We also developed a framework for analyzing transitions between multiple emissive states in FCS and TRAST experiments, how the brightness of the different states is weighted, and what initial conditions that apply. Our FCS, TRAST, and spectral-TRAST experiments showed significant differences in dark-state relaxation amplitudes for different spectral detection ranges, which we attribute to an additional red-shifted, emissive photo-isomerized state of Cy5, not previously considered in FCS and single-molecule experiments. The photo-isomerization kinetics of this state indicate that it is formed under moderate excitation conditions, and its population and emission may thus deserve also more general consideration in fluorescence imaging and spectroscopy experiments.

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

“…For SMLM with cyanine dyes, it has recently been reported that photoisomerized states of Cy5 with redshifted excitation spectra may lead to unwanted FRET-mediated excitation transfer between closely (<10 nm) located non-photoisomerized and photoisomerized fluorophores. 36 Thus, in the context of SMLM, the fact that such photoisomerized states also can show redshifted emission, as very recently shown for Cy5, 53 and shown for NIR cyanines in this work, makes it necessary to also account for this emission source in the analyses of the resulting blinking. Beyond SMD and SRM experiments, since the redshifted emissive state is generated already at relatively low excitation intensities ( Figure 3 D), excitation and emission spectra of NIR cyanine dyes can be expected to be altered in a range of experiments.…”