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

Abstract

Electrostatic interactions between dielectric surfaces and different fluorophores used in ultrasensitive fluorescence microscopy are investigated using objective-based Total Internal Reflection Fluorescence Correlation Spectroscopy (TIR-FCS). The interfacial dynamics of cationic rhodamine 123 and rhodamine 6G, anionic/dianionic fluorescein, zwitterionic rhodamine 110 and neutral ATTO 488 are monitored at various ionic strengths at physiological pH. As analyzed by means of the amplitude and time-evolution of the autocorrelation function, the fluorescent molecules experience electrostatic attraction or repulsion at the glass surface depending on their charges. Influences of the electrostatic interactions are also monitored through the triplet-state population and triplet relaxation time, including the amount of detected fluorescence or the count-rate-per-molecule parameter. These TIR-FCS results provide an increased understanding of how fluorophores are influenced by the microenvironment of a glass surface, and show a promising approach for characterizing electrostatic interactions at interfaces.

Measuring Surface Binding Thermodynamics and Kinetics by Using Total Internal Reflection with Fluorescence Correlation Spectroscopy: Practical Considerations

“…27,28 This correction is necessary only if the excitation intensity is high enough so that the measured G(τ) are dependent on it. In addition, the G(0) values must be extrapolated from G(τ) at low τ because when τ = 0 the autocorrelation functions contain contributions from shot noise.…”

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.…”

Coupling of fluorescence correlation spectroscopy with capillary and microchannel analytical systems and its applications

“…With similar method, other FCS‐based techniques were also combined into the microchannel including two‐focus FCCS , scanning FCS , total internal reflection FCS (TIR‐FCS or TIR‐FCCS) , zero‐mode waveguides FCS , and twin‐focus photothermal correlation spectroscopy (Twin‐PhoCS) . These FCS techniques demonstrate the versatility of characterizing on flow properties in the channel.…”

“…In the proximity of an interface, the normal resolution can be significantly increased using TIR microscopy. In order to overcome the disadvantage of one‐beam FCS, two‐beam FCCS, or line‐scan FCS on flow profiling in the close proximity of an interface caused by the limited resolution of these techniques, new TIR‐FCS or TIR‐FCCS configurations were developed to measure velocity profiles in the interface . For example, Yordanov et al tuned the evanescent wave penetration depth of TIR and the velocity at different distances less than 200 nm from the interface was measured .…”