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

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