20 February 2018 Demystifying autofluorescence with excitation scanning hyperspectral imaging
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Autofluorescence has historically been considered a nuisance in medical imaging. Many endogenous fluorophores, specifically, collagen, elastin, NADH, and FAD, are found throughout the human body. Diagnostically, these signals can be prohibitive since they can outcompete signals introduced for diagnostic purposes. Recent advances in hyperspectral imaging have allowed the acquisition of significantly more data in a shorter time 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. Here, we propose to utilize excitation-scanning of autofluorescence to examine tissues and diagnose pathologies.

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.
Conference Presentation
© (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Joshua Deal, Joshua Deal, Bradley Harris, Bradley Harris, Will Martin, Will Martin, Malvika Lall, Malvika Lall, Carmen Lopez, Carmen Lopez, Paul Rider, Paul Rider, Carole Boudreaux, Carole Boudreaux, Thomas Rich, Thomas Rich, Silas J. Leavesley, 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); doi: 10.1117/12.2290818; https://doi.org/10.1117/12.2290818

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