Quantitative characterization of biological liquids for third-harmonic generation microscopy

Area of Science:

• Biophotonics
• Nonlinear Optics
• Microscopy

Background:

• Third-harmonic generation (THG) microscopy visualizes unstained biological tissues.
• THG signal intensity depends on nonlinear susceptibility, refractive index, and dispersion.
• Understanding these properties is crucial for interpreting THG images.

Purpose of the Study:

• To quantify third-order nonlinear susceptibilities of biological solutions.
• To assess THG imaging sensitivity and specificity for physiological changes.
• To evaluate the impact of index-matching liquids on THG images.

Main Methods:

• Quantitative measurement of third-order nonlinear susceptibilities for various solvents and solutions.
• THG imaging experiments using a 1.05-1.25 micrometer excitation range.
• Analysis of THG signal contributions from different biological components.

Main Results:

• Established quantitative nonlinear susceptibility values for water, ethanol, glycerol, and physiological solutions.
• Demonstrated that THG imaging at 1.2 micrometers has limited sensitivity for detecting ion concentration variations.
• Identified non-aqueous structures, like lipid bodies, as significant sources of THG signal.
• Illustrated the influence of index-matching liquids on THG image contrast and signal intensity.

Conclusions:

• THG imaging at ~1.2 micrometers shows low specificity and sensitivity for physiological ion concentration changes.
• Lipid bodies are more robust signal generators in THG microscopy compared to aqueous physiological components.
• Quantitative susceptibility data aids in interpreting biological THG images and developing new applications.