Abstract
Autofluorescence, the endogenous fluorescence present in cells and tissues, has historically been considered a nuisance in biomedical imaging. Many endogenous fluorophores, specifically, collagen, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide (FAD), are found throughout the human body. In fluorescence imaging scenarios, these signals can be prohibitive as they can outcompete signals introduced for 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 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. We propose to utilize excitation-scanning hyperspectral imaging of autofluorescence to differentiate neoplastic lesions from surrounding non-neoplastic "normal" tissue. The spectra of isolated autofluorescent molecules are obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with an Xe arc lamp and thin-film tunable filter array (VersaChrome, Semrock, Inc.). Scans utilize excitation wavelengths from 360 to 550nm in 5-nm increments. The resultant molecule-specific spectra are used to analyze hyperspectral image stacks from normal and neoplastic colorectal tissues. Due to a limited number of samples, neoplastic tissues examined here are a pool of both colorectal adenocarcinoma and adenomatous polyps. The hyperspectral images are analyzed with ENVI software and custom MATLAB scripts, including linear spectral unmixing. Initial results indicate the ability to separate signals of endogenous fluorophores and measure the relative concentrations of fluorophores among healthy and diseased states, in this case, normal colon versus neoplastic colon. These results suggest pathology-specific changes to endogenous fluorophores can be detected using excitation-scanning hyperspectral imaging. Future work will focus on expanding the library of pure molecules, exploring histogram distance metrics as a means for identifying deviations in spectral signatures, and examining more defined disease states.
Highlights
Exogenous fluorescence labels have been used for biological imaging since the 1940s.1 Coons et al used fluorescein chemically bound to an antibody to visualize that antibody in tissue sections, creating the field of immunofluorescence microscopy
Hyperspectral imaging was initially performed via spectral reflectance, advancements in both imaging technology and processing allow measurement of absorbance and fluorescence
Hyperspectral imaging is especially relevant in fields, such as fluorescence microscopy, where the entire method is predicated on identifying a molecule based on its interaction with light
Summary
Exogenous fluorescence labels have been used for biological imaging since the 1940s.1 Coons et al used fluorescein chemically bound to an antibody to visualize that antibody in tissue sections, creating the field of immunofluorescence microscopy. Hyperspectral imaging began with remote sensing and geologic applications by NASA.[9,10] Subsequent applications of hyperspectral imaging have reached virtually every field of science, including agriculture,[11] archaeology,[12] astronomy,[13] biomedicine,[14] crime scene investigation,[15] environmental science,[16] eye care,[17] food processing,[18] forensics,[19] and surveillance.[20] hyperspectral imaging was initially performed via spectral reflectance, advancements in both imaging technology and processing allow measurement of absorbance and fluorescence The basis of this technique is that every object interacts with light in a unique and wavelengthdependent manner, and the resultant measurements are termed as spectral signatures. Hyperspectral imaging is especially relevant in fields, such as fluorescence microscopy, where the entire method is predicated on identifying a molecule based on its interaction with light
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