Quantitative fluorescence spectroscopy in turbid media using fluorescence differential path length spectroscopy

Area of Science:

• Biomedical Optics
• Spectroscopy
• Photonics

Background:

• Quantitative analysis of fluorophores in turbid media is challenging.
• Existing methods may not fully account for complex optical properties.
• Accurate measurements are crucial for applications in biological imaging and diagnostics.

Purpose of the Study:

• To develop and validate a novel technique, fluorescence differential path length spectroscopy (FDPS), for quantitative fluorophore investigation.
• To assess the impact of varying optical properties and probe geometry on FDPS measurements.
• To establish the relationship between differential fluorescence signal, absorption, and path length.

Main Methods:

• Developed FDPS, utilizing similar probe geometry to differential path length spectroscopy (DPS).
• Conducted phantom studies across a range of optical properties (reduced scattering coefficient mu(s)' and absorption coefficient mu(a)).
• Investigated the influence of two fiber diameters (400 µm and 800 µm) on the differential fluorescence signal.

Main Results:

• The differential fluorescence signal exhibited variations of 1.4x (400 µm) and 2.2x (800 µm) across the biologically relevant scattering range (0.5–5 mm⁻¹).
• Signal attenuation due to absorption at the excitation wavelength followed Lambert-Beer's law.
• The effective path length was found to be equal to the differential path length.

Conclusions:

• FDPS provides a quantitative method for investigating fluorophores in turbid media.
• The technique's performance is influenced by optical properties and fiber geometry.
• FDPS offers a promising approach for accurate in-situ fluorescence measurements in biological tissues.