April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Mathematical modeling of retinal pigment epithelium (RPE) autofluorescence (AF) with Gaussian mixture models and non-negative matrix factorization (NMF)
Author Affiliations & Notes
  • Ansh Johri
    Ophthalmology, New York University School of Medicine, New York, NY
  • Robert Post
    Ophthalmology, New York University School of Medicine, New York, NY
  • Bhaskar Ganti
    Ophthalmology, New York University School of Medicine, New York, NY
  • Amani A Fawzi
    Ophthal-Feinberg School of Med, Northwestern University, Chicago, IL
  • Paul Sajda
    Biomedical Engineering, Columbia University, New York, NY
  • Thomas Ach
    Ophthalmology, University of Alabama at Birmingham, Birmingham, AL
  • Christine A Curcio
    Ophthalmology, University of Alabama at Birmingham, Birmingham, AL
  • Theodore Smith
    Ophthalmology, New York University School of Medicine, New York, NY
  • Footnotes
    Commercial Relationships Ansh Johri, None; Robert Post, None; Bhaskar Ganti, None; Amani Fawzi, None; Paul Sajda, None; Thomas Ach, None; Christine Curcio, None; Theodore Smith, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3827. doi:
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      Ansh Johri, Robert Post, Bhaskar Ganti, Amani A Fawzi, Paul Sajda, Thomas Ach, Christine A Curcio, Theodore Smith; Mathematical modeling of retinal pigment epithelium (RPE) autofluorescence (AF) with Gaussian mixture models and non-negative matrix factorization (NMF). Invest. Ophthalmol. Vis. Sci. 2014;55(13):3827.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

To devise a mathematical algorithm that can extract individual spectral fluorophor components and their spatial localizations from hyperspectral autofluorescence (AF) emission data taken from RPE and Bruch’s membrane (BrM) human donor flat mounts (ex vivo).

 
Methods
 

Step 1: Hyperspectral cube acquisition: The AF of eleven pure human RPE/BrM flatmounts was studied at 3 locations (fovea, parafovea and periphery) via excitation at wavelengths 436-460 nm and 480-510 nm at 40X magnification. The corresponding hyperspectral emission data (hypercubes of two spatial and one spectral dimension) were captured using the Nuance FX camera (Caliper Life Sciences, US). (Further details in K. Agarwal abstract); Step 2: Gaussian modeling: We fit the original RPE spectra with mixtures of four Gaussian curves (Fig. 1), which provided single peak, smooth candidates for individual fluorophor components; Step 3: NMF modeling: We used these candidate spectra to initialize an NMF technique that factors the entire hypercube to recover constituent source spectra and their spatial localizations minimizing error. We also initialized the NMF with the emission signal from a patch of bare BrM because BrM, underlying the RPE, contributes its signal throughout.

 
Results
 

NMF models with Gaussian/BrM initialization consistently decomposed RPE AF hypercubes into smooth individual candidate spectra with histologically plausible localizations within the flat-mount images (Fig. 2). For example, the shorter wavelength spectral component C3 localized to BrM (Fig. 2, Spatial Abundance C3), while the other four, emitting from 575 nm to 700 nm, localized to the lipofuscin compartment.

 
Conclusions
 

The Gaussian/NMF mixture model enabled consistent recovery of candidate spectra for individual RPE fluorophor emission signals with histologically plausible localizations. These spectra should now be matched to their corresponding biochemical components with techniques like imaging mass spectroscopy.

 
 
Figure 1: Gaussians components fitted to the RPE emission spectrum of a 50-year-old female donor. The sum of the Gaussians (solid pink) closely follows the contours of the RPE signal.
 
Figure 1: Gaussians components fitted to the RPE emission spectrum of a 50-year-old female donor. The sum of the Gaussians (solid pink) closely follows the contours of the RPE signal.
 
 
Figure 2: NMF result. Five abundant components are recovered, four of which localize adjacent to nuclei, where lipofuscin is expected. C3 localizes mostly to bare BrM, with some transmission through RPE nuclei.
 
Figure 2: NMF result. Five abundant components are recovered, four of which localize adjacent to nuclei, where lipofuscin is expected. C3 localizes mostly to bare BrM, with some transmission through RPE nuclei.
 
Keywords: 701 retinal pigment epithelium • 551 imaging/image analysis: non-clinical • 582 ipofuscin  
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