A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation.

Area of Science:

• Biophysics
• Physical Chemistry
• Materials Science

Background:

• Fluorescence Correlation Spectroscopy (FCS) is a sensitive technique for analyzing dynamic systems.
• Confocal microscopy is standard for FCS, but surface-specific analysis requires specialized setups like Total Internal Reflection (TIR)-FCS.
• Existing TIR-FCS systems often require complex configurations.

Purpose of the Study:

• To present a combined confocal and TIR-FCS setup.
• To enable simultaneous surface-sensitive and bulk measurements.
• To demonstrate the setup's versatility with various fluorescent samples.

Main Methods:

• Utilized economic low-power pulsed diode lasers for excitation.
• Integrated time-correlated photon counting for simultaneous fluorescence lifetime and FCS measurements.
• Enabled easy switching between TIR and epi-illumination for comparative analysis.

Main Results:

• Successfully performed simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode.
• Demonstrated the capability for fluorescence lifetime correlation spectroscopy at surfaces.
• Measured diffusion coefficients of dye molecules, labeled polymers, and nanoparticles using both confocal and TIR-FCS.

Conclusions:

• The combined setup offers a versatile platform for surface-sensitive and bulk dynamic analysis.
• The system allows for direct comparison of surface and bulk properties.
• This approach enhances the study of molecular diffusion in various environments.