Spectra of autofluorescent molecules were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Scans utilized excitation wavelengths from 360 nm to 550 nm in 5 nm increments. The resultant spectra were used to examine hyperspectral image stacks from various collaborative studies, including an atherosclerotic rat model and a colon cancer study. Hyperspectral images were analyzed with ENVI and custom Matlab scripts including linear spectral unmixing (LSU) and principal component analysis (PCA). Initial results suggest the ability to separate the signals of endogenous fluorophores and measure the relative concentrations of fluorophores among healthy and diseased states of similar tissues. These results suggest pathology-specific changes to endogenous fluorophores can be detected using excitationscanning hyperspectral imaging. Future work will expand the library of pure molecules and will examine more defined disease states.
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Joshua Deal, Bradley Harris, Will Martin, Malvika Lall, Carmen Lopez, Paul Rider, Carole Boudreaux, Thomas Rich, Silas J. Leavesley, "Demystifying autofluorescence with excitation scanning hyperspectral imaging," Proc. SPIE 10497, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI, 1049715 (20 February 2018);