Deal 2018 Identifying Molecular Contributors of Autofluorescence of Neoplastic and Normal Colon Sections Using Excitation Scanning Hyperspectral Imaging.pdf

Autofluorescence, the endogenous fluorescence present in cells and tissues, has historically beenconsidered a nuisance in biomedical imaging. Many endogenous fluorophores, specifically, collagen, elastin,nicotinamide adenine dinucleotide, and flavin adenine dinucleotide (FAD), are found throughout the humanbody. In fluorescence imaging scenarios, these signals can be prohibitive as they can outcompete signals introducedfor diagnostic purposes. However, autofluorescence also contains information that has diagnostic value.Recent advances in hyperspectral imaging have allowed the acquisition of significantly more data in a shortertime period by scanning the excitation spectra of fluorophores. The reduced acquisition time and increased signal-to-noise ratio allow for separation of significantly more fluorophores than previously possible. We propose toutilize excitation-scanning hyperspectral imaging of autofluorescence to differentiate neoplastic lesions fromsurrounding non-neoplastic “normal” tissue. The spectra of isolated autofluorescent molecules are obtainedusing a custom inverted microscope (TE-2000, Nikon Instruments) with an Xe arc lamp and thin-film tunablefilter array (VersaChrome, Semrock, Inc.). Scans utilize excitation wavelengths from 360 to 550 nm in 5-nmincrements. The resultant molecule-specific spectra are used to analyze hyperspectral image stacks fromnormal and neoplastic colorectal tissues. Due to a limited number of samples, neoplastic tissues examined hereare a pool of both colorectal adenocarcinoma and adenomatous polyps. The hyperspectral images are analyzedwith ENVI software and custom MATLAB scripts, including linear spectral unmixing. Initial results indicate theability to separate signals of endogenous fluorophores and measure the relative concentrations of fluorophoresamong healthy and diseased states, in this case, normal colon versus neoplastic colon. These results suggestpathology-specific changes to endogenous fluorophores can be detected using excitation-scanning hyperspectralimaging. Future work will focus on expanding the library of pure molecules, exploring histogram distancemetrics as a means for identifying deviations in spectral signatures, and examining more defined disease states.