Demystifying autofluorescence with excitation-scanning hyperspectral imaging

Area of Science:

• Biomedical Optics
• Medical Imaging
• Spectroscopy

Background:

• Autofluorescence from endogenous molecules (collagen, elastin, NADH, FAD) historically hinders medical imaging.
• These autofluorescent signals can obscure diagnostically relevant signals.
• Advances in hyperspectral imaging enable faster data acquisition and improved signal-to-noise ratios.

Purpose of the Study:

• To investigate the utility of excitation-scanning hyperspectral imaging for analyzing tissue autofluorescence.
• To differentiate and quantify endogenous fluorophores for pathological diagnosis.
• To explore pathology-specific alterations in endogenous fluorophore concentrations.

Main Methods:

• Utilized excitation-scanning hyperspectral imaging with a custom microscope and tunable filter array.
• Acquired spectra across excitation wavelengths from 360 nm to 550 nm in 5 nm increments.
• Analyzed hyperspectral image stacks using linear spectral unmixing (LSU) and principal component analysis (PCA).

Main Results:

• Successfully separated signals from endogenous fluorophores in tissue samples.
• Quantified relative concentrations of fluorophores in healthy versus diseased tissues.
• Demonstrated the potential to detect pathology-specific changes in autofluorescence.

Conclusions:

• Excitation-scanning hyperspectral imaging can effectively utilize autofluorescence for tissue examination and disease diagnosis.
• Pathology-specific changes in endogenous fluorophores are detectable using this advanced imaging technique.
• Future work will expand the molecular library and investigate more defined disease states.