Two-photon fluorescence correlation spectroscopy as a tool for measuring molecular diffusion within human skin.

Area of Science:

• Biophotonics
• Materials Science
• Pharmacology

Background:

• Quantitative imaging of biological tissues is crucial for pharmaceutical applications.
• Existing methods lack the precision for detailed molecular analysis in complex matrices like skin.

Purpose of the Study:

• To demonstrate the proof-of-principle for a combined two-photon fluorescence microscopy (TPM) and fluorescence correlation spectroscopy (FCS) technique.
• To quantitatively measure fluorophore concentration and diffusion dynamics within human skin.
• To assess molecular interactions and binding within the skin matrix.

Main Methods:

• Excised human skin samples were used.
• Samples were exposed to rhodamine B (RB) and rhodamine B isothiocyanate (RBITC).
• TPM-FCS measurements were conducted at tissue depths of 0-20 μm.

Main Results:

• Significant differences in diffusion coefficients were observed at skin depths of 5 and 10 μm (P<0.05).
• Median diffusion coefficients were 4.0×10⁻¹³ m²/s for RB and 2.0×10⁻¹³ m²/s for RBITC.
• RBITC diffusion suggests binding to skin biomolecules, indicating a larger effective molecular volume.

Conclusions:

• The TPM-FCS method provides quantitative data on fluorophore concentration and diffusion in human skin.
• The technique can differentiate between free and bound fluorophores, revealing molecular interactions.
• TPM-FCS shows potential for tracking molecular interactions within complex biological environments like skin.