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Proceedings Paper

Demystifying autofluorescence with excitation scanning hyperspectral imaging
Author(s): Joshua Deal; Bradley Harris; Will Martin; Malvika Lall; Carmen Lopez; Paul Rider; Carole Boudreaux; Thomas Rich; Silas J. Leavesley
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Paper Abstract

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.

Paper Details

Date Published: 20 February 2018
PDF: 8 pages
Proc. SPIE 10497, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI, 1049715 (20 February 2018); doi: 10.1117/12.2290818
Show Author Affiliations
Joshua Deal, Univ. of South Alabama (United States)
Bradley Harris, Univ. of South Alabama (United States)
Will Martin, Univ. of South Alabama (United States)
Malvika Lall, Univ. of South Alabama (United States)
Carmen Lopez, Univ. of South Alabama (United States)
Paul Rider, Univ. of South Alabama (United States)
Carole Boudreaux, Univ. of South Alabama (United States)
Thomas Rich, Univ. of South Alabama (United States)
Silas J. Leavesley, Univ. of South Alabama (United States)


Published in SPIE Proceedings Vol. 10497:
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVI
Daniel L. Farkas; Dan V. Nicolau; Robert C. Leif, Editor(s)

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