Simultaneous Surface-Near and Solution Fluorescence Correlation Spectroscopy.

Area of Science:

• Analytical Chemistry
• Spectroscopy
• Surface Science

Background:

• Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying molecular dynamics.
• Existing FCS methods typically measure bulk solution properties, lacking surface-specific information.
• Simultaneous measurement of surface and bulk concentrations is crucial for understanding interfacial phenomena.

Purpose of the Study:

• To develop and validate a novel optical configuration for simultaneous surface-confined and solution fluorescence correlation spectroscopy (FCS).
• To characterize the distinct detection volumes generated by undercritical angle fluorescence (UAF) and supercritical angle fluorescence (SAF).
• To enable simultaneous determination of bulk solution and surface-near concentrations.

Main Methods:

• Utilized a unique optical setup for tightly focused excitation and separated detection of UAF and SAF.
• Employed FCS to characterize two distinct detection volumes: a standard confocal volume (UAF) and a surface-confined volume (~200 nm axial extent, SAF).
• Validated the method through characterization of free diffusion and comparison with analytical models and simulations.

Main Results:

• Achieved the first simultaneous measurement of surface-confined and solution FCS.
• Demonstrated that SAF creates a highly surface-confined detection volume, distinct from the UAF confocal volume.
• Successfully characterized the axial extents of both detection volumes using FCS.

Conclusions:

• The developed FCS technique allows for simultaneous quantification of molecular concentrations in bulk solutions and near surfaces.
• This method provides a significant advancement for studying interfacial processes and surface-bound molecules.
• Opens new avenues for research in areas requiring precise surface and bulk concentration analysis.