Triplet-State Investigations of Fluorescent Dyes at Dielectric Interfaces Using Total Internal Reflection Fluorescence Correlation Spectroscopy

Abstract

The triplet-state kinetics of several fluorescent dyes used in ultrasensitive fluorescence microscopy are investigated using total internal reflection fluorescence correlation spectroscopy (TIR-FCS). A theoretical outline of the correlation analysis and the physical aspects of evanescent excitation and fluorescence emission at dielectric interfaces are given. From this analysis, the rates of intersystem crossing and triplet decay are deduced for fluorescein, ATTO 488, rhodamine 110, rhodamine 123, and rhodamine 6G in aqueous buffer solutions. All investigated dyes show slightly higher triplet rates at the dielectric interface compared to bulk solution measurements. We attribute this enhancement to possible modifications of the dyes' photophysical properties near a dielectric interface. In the case of rhodamine 6G, the impact of changes in the dye concentration, ionic strength of the solvent, and potassium iodide concentration are also investigated. This leads to a better understanding of the influences of dye-dye, dye-solvent, and dye-surface interactions on the increased triplet intersystem crossing and triplet decay rates. The study shows that analysis of triplet-state kinetics by TIR-FCS not only results in a better understanding of how the photophysical properties of the dyes are affected by the presence of an interface, but also provides a means for probing the microenvironment near dielectric interfaces.

Permeability of anti-fouling PEGylated surfaces probed by fluorescence correlation spectroscopy

“…1(b–e) were used to test the hypothesis of size-dependent, sieve-like behavior for PEG brushes. Because R6G exhibits surface dependent diffusion and photophysics [38,58,59], Alexa was chosen as the primary probe for these studies. Four generations of Janus-type linear-dendrons were analysed.…”

Electrostatic Interactions of Fluorescent Molecules with Dielectric Interfaces Studied by Total Internal Reflection Fluorescence Correlation Spectroscopy

“…Triplet-state kinetics of organic dye molecules in the vicinity of glass surfaces has been investigated previously at high ionic strengths ( i.e. , fully screened surfaces) [36]. Extracting the triplet-state kinetic rates from the data on unscreened surfaces should in principle be possible, but is beyond the scope of this work.…”

“…The level of irradiance experienced by a fluorescent molecule at the surface may be greater than the irradiance from the incident laser beam by up to a factor of five [7]. Under our experimental conditions the angle of incidence (AOI) is fixed to a value slightly below 64° (estimated as 63.7°), which generates an enhancement factor of η ~4 (see reference [36] for a discussion of AOI settings). The discontinuity in the refractive index also produces a significant modification of the angular dependence of the emitted fluorescence, which has a maximum of emission in the direction of the critical angle ( i.e.…”

“…The structure factor was in this work fixed to 0.18 [32]. The parameter, T̄ is the steady state probability of the molecules to be in the triplet state, and τ̄ T denotes the steady state triplet relaxation time [36]. To fit the TIR-FCS data, a multidimensional least-squares minimization algorithm written in Matlab (Mathworks Inc., Natick, MA, USA) was used to estimate the unknown parameters of the Equation above, i.e.…”

“…With the former method we have investigated diffusion of dianionic and cationic organic dyes molecules in pure aqueous solution at glass interfaces [31,32]. In addition, we have further applied it for investigation of DNA-hybridization, single enzymes kinetics, triplet-state dynamics, and protein and surfactant interactions at bare and coated glass surfaces [33–36]. …”