April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Quantitation of Hyperspectral Autofluorescence (AF) from Human Retinal Pigment Epithelium (RPE) Ex Vivo
Author Affiliations & Notes
  • Camellia Nabati
    Ophthalmology, NYU, New York, NY
  • Ansh Johri
    Ophthalmology, NYU, New York, NY
  • Robert Post
    Ophthalmology, NYU, New York, NY
  • Paul Sajda
    Biomedical Engineering, Columbia University, New York, NY
  • Thomas Ach
    Ophthalmology, The University of Alabama at Birmingham, BIRMINGHAM, AL
  • Christine A Curcio
    Ophthalmology, The University of Alabama at Birmingham, BIRMINGHAM, AL
  • Theodore Smith
    Ophthalmology, NYU, New York, NY
  • Footnotes
    Commercial Relationships Camellia Nabati, None; Ansh Johri, None; Robert Post, None; Paul Sajda, None; Thomas Ach, None; Christine Curcio, None; Theodore Smith, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 3844. doi:https://doi.org/
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    • Get Citation

      Camellia Nabati, Ansh Johri, Robert Post, Paul Sajda, Thomas Ach, Christine A Curcio, Theodore Smith; Quantitation of Hyperspectral Autofluorescence (AF) from Human Retinal Pigment Epithelium (RPE) Ex Vivo. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3844. doi: https://doi.org/.

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

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Abstract
 
Purpose
 

Quantify the hyperspectral AF signal from RPE/Bruch’s membrane (BrM) flat mounts.

 
Methods
 

Hyperspectral AF images (hypercubes) were captured from 66, 40X fields in 11 RPE/BrM flat mounts from human donor eyes using techniques described in detail in the abstract submitted by K. Agarwal. Briefly, for each 40X field, the hypercube has the two spatial dimensions of the field, and at each spatial point the photon counts recorded at each wavelength, hence the third or spectral dimension. For reproducible quantification of these data, exposure times were calibrated so that photon counts per spectral channel fell within the 12-bit linear range of the detector and then were offset by the dark current. Scaled counts-per-second were determined by exposure time (Eqn. 1) and calibrated to a standard fluorescent reference (courtesy of F Delori) to correct for any variation in power of the excitation light, yielding quantified hypercubes with units of photon counts per second at each point and wavelength.

 
Results
 

The Root Mean Square(RMS ) difference of quantified hypercubes from repeat imaging of the same location was within the noise level (dark current) of the Nuance detector, establishing reproducibility. Separation of RPE signal from BrM (Fig. 2) and further mathematical analyses of the hypercubes (see abstract of A. Johri) therefore extracted reliable quantitative RPE lipofuscin spectra for individual constituents and their corresponding spatial co-localizations.

 
Conclusions
 

Hyperspectral AF images of human RPE flatmounts may be reliably quantified for use as a surrogates for measurement of abundant of lipofuscin components. Such quantitative information can help guide the analysis of RPE physiology and biochemistry.

 
 
Equation 1: Counts = total photon counts from camera exp=exposure time (seconds) bin=binning (default 1) gain=camera gain (default 3) Full Scale= 4096 for 12-bit acquisitions
 
Equation 1: Counts = total photon counts from camera exp=exposure time (seconds) bin=binning (default 1) gain=camera gain (default 3) Full Scale= 4096 for 12-bit acquisitions
 
 
Figure 2: 2a: RGB image from quantified hypercube (49 y/o M donor, exc. 436 nm.) Green line: Pure BrM. Red Line: RPE that overlies BrM 2b: Quantified emission curves. Green, pure BrM. Red, RPE and BrM combined. Magenta, pure RPE signal (subtraction of the two)
 
Figure 2: 2a: RGB image from quantified hypercube (49 y/o M donor, exc. 436 nm.) Green line: Pure BrM. Red Line: RPE that overlies BrM 2b: Quantified emission curves. Green, pure BrM. Red, RPE and BrM combined. Magenta, pure RPE signal (subtraction of the two)
 
Keywords: 701 retinal pigment epithelium  
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