Photoswitching mechanism of cyanine dyes

Abstract

Cyanine dyes have been shown to undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state upon illumination at different wavelengths. The photochemical mechanism by which switching occurs has yet to be elucidated. In this study, we have determined the mechanism of photoswitching by characterizing the kinetics of dark state formation and the spectral and structural properties of the dark state. The rate of switching to the dark state depends on the concentration of the primary thiol in the solution and the solution pH in a manner quantitatively consistent with the formation of an encounter complex between the cyanine dye and ionized thiol prior to their conjugation. Mass spectrometry suggests that the photoconversion product is a thiol-cyanine adduct in which covalent attachment of the thiol to the polymethine bridge disrupts the original conjugated pi-electron system of the dye.

Figure 1

Spectral and kinetic analyses of the photoconversion of Cy5 in the presence of βME. (A) Absorption spectra of Cy5-COOH. The inset shows fluorescence of single Cy5 molecules anchored to a surface. (B) After excitation by a red laser, the Cy5 fluorescence disappears (inset), and the absorption at 650 nm is diminished, while a new peak at 310 nm appears. (C) Following UV illumination, the 650 nm absorption of the Cy5 solution is only partially recovered because of photobleaching of some Cy5 molecules. In the single-molecule-imaging assay with antiphotobleaching buffer, nearly all of the Cy5 molecules recover to the fluorescent state (inset). (D) The rate constant (k_off) for switching the dye off, normalized by the red (633 nm) laser intensity (I), is plotted as a function of [βME] at pH 9.85 (black dots); the fit to eq 4 in the SI (red line) is also shown. (E) The fraction of deprotonated thiol normalized by the dissociation constant of the encounter complex (F_RS⁻/K_d) is shown as a semilogarithmic plot against pH. The red curve shows the fit of the data to eq 5 in the SI.

Scheme 1. Kinetic Pathway for Dark State Formation

Figure 2

ESI-LC/MS measurements of the dark state of Cy5-COOH in the presence of βME. The panels show the cyanine dye−thiol mixture (A) before and (B) after red laser illumination as well as (C) after reactivation by UV excitation. The peak that appears for [M − 2H]⁻ at |m/z| 655.21 corresponds to [Cy5-COO]⁻. After red illumination, a new peak appears for the singly charged species [M − H]⁻ at |m/z| 733.22. This new peak disappears after recovery by UV illumination. Because of substantial spontaneous recovery to the fluorescent state during sample preparation and measurement, the original dye peak at m/z 655.21 is not completely diminished in panel B.

Scheme 2. Proposed Structural Model of the Dark State