Illuminating native fluorescence signatures for cancer detection

Autofluorescence describes light emitted from a naturally occurring substance when exposed to light of a shorter wavelength. It has been shown that cancer-related changes in tissue composition can be responsible for shifts in autofluorescence intensity of biological samples. Several prior studies have worked to characterize the spectral properties of autofluorescent molecules using spectrofluorometers and microscope systems. Here, we quantitatively characterized endogenous fluorophores using spectrofluorometry and fluorescence microscopy with the objective of establishing a reference spectral library for subsequent spectral unmixing of hyperspectral images of mouse tissues during colorectal cancer (CRC) progression. Endogenous fluorophores of interest include collagen, elastin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), protoporphyrin IX (PPIX), tryptophan, and tyrosine. In the tumor microenvironment (TME), collagen is overproduced and remodeled affecting immune cell infiltration and treatment resistance; elastin degradation generates fragments to promote or inhibit tumor development; NADH and FAD play essential roles in reduction-oxidation reactions; PPIX precedes heme in the heme biosynthesis pathway; and tryptophan and tyrosine are autofluorescent amino acids. Concentration fluctuations of these endogenous fluorophores are directly related to autofluorescent properties which contribute to shifts in bulk autofluorescence spectra concurrent with tissue restructuring caused by the TME. Current hyperspectral fluorescence microscopy techniques utilize emission-scanning hyperspectral imaging (Em-HSI). However, this method requires long acquisition times that would be incompatible with real-time endoscopic screening. Here, we utilize a novel excitation-scanning hyperspectral imaging (Ex-HSI) approach to establish a comprehensive spectral library of several biologically-relevant autofluorescent molecules for downstream spectral unmixing of mouse CRC tissue spectral image stacks.