Mapping absolute tissue endogenous fluorophore concentrations with chemometric wide-field fluorescence microscopy

Area of Science:

• Biophysics
• Chemical Imaging
• Molecular Imaging

Background:

• Endogenous fluorophores are crucial biomarkers in biological tissues.
• Accurate quantification of these fluorophores is essential for disease diagnosis.
• Existing imaging methods often lack precision in determining absolute concentrations.

Purpose of the Study:

• To develop and validate a chemometric wide-field fluorescence microscopy technique.
• To enable precise imaging of spatial distribution and absolute concentration of endogenous fluorophores.
• To assess the utility of this method for differentiating tissue types, specifically lung cancer.

Main Methods:

• Utilized nonnegative factorization with spatial diversity for spectral signature and spatial distribution learning.
• Employed broadband excitation and detection for fluorescence imaging.
• Derived absolute concentration maps by comparing with pure fluorophore standards.

Main Results:

• Successfully mapped concentrations of tryptophan, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide in lung tissue.
• Observed a significant decrease in all measured fluorophore concentrations from normal to cancerous tissues (squamous cell carcinoma).
• Demonstrated superior accuracy in tissue discrimination using absolute fluorophore concentrations compared to relative fluorescence intensity.

Conclusions:

• The developed chemometric microscopy technique provides accurate absolute quantification of endogenous fluorophores.
• Absolute fluorophore concentration mapping offers a more reliable approach for tissue characterization and disease diagnosis.
• This method enhances biological relevance and simplifies comparative analysis of imaging techniques.