Quantitation of Hyperspectral Autofluorescence (AF) from Human Retinal Pigment Epithelium (RPE) Ex Vivo

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

 

 

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