Targeted ocular spectroscopy sheds new light on retinal health

In a recent study published in Journal of Biomedical Optics, Researchers demonstrate multimodal functionality of targeted ocular fluorescence spectroscopy in vitro and in vivo.

Study: Targeted fundus spectroscopy. Image credit: PeopleImages.com – Yuri A / Shutterstock.com

Record

Some typical structural and functional changes occur in the eyes, especially in the fundus, due to eye diseases such as diabetic retinopathy (DR), age-related macular degeneration (AMD), and glaucoma. Neurological diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) can also lead to changes in the retina, such as thinning of the retinal nerve fiber layer (RNFL) and changes in hemodynamics.

Given the highly heterogeneous characteristics and composition of the fundus, biomarkers are either widely dispersed throughout this tissue or localized to specific regions. For example, β-amyloid plaques spread throughout the retina of AD patients, while DR patients have localized hemorrhages.

Standard imaging techniques do not provide enough data on the retinal changes caused by these diseases compared to targeted ocular diffuse reflectance spectroscopy (DRS). Ocular DRS methods allow spectral analysis of specific parts of the fundus, including the optic disc, peripheral retina, and fovea between 500 and 800 nanometers (nm).

Diffuse reflectance and fluorescence spectroscopy can also elucidate the effect of factors such as lipofuscin accumulation, RNFL structural changes, blood absorption spectrum, and melanin spectral profile, all of which affect the optical properties of retinal tissues.

About the study

In the current study, the researchers identify key features of the targeted ocular spectroscopy technology in vitro using a reference target and a model eye. The reference target was an ultra-high-definition screen with a grid of eight different colors, with the bottom camera placed in front of it and collecting only the light emitted by the screen. The OEMI-7 eye model, a seven-millimeter pupil that accurately simulates the human eye, helped validate these DRS acquisitions.

Subsequently, in vivo Imaging and DRS were used to assess blood oxygen saturation (StO₂) in the optic nerve head and paraphobia of eight healthy study participants who provided informed consent prior to the study. These subjects were between 27 and 35 years of age, had no systemic disease or medication, and had normal eye exam results.

The pointing light emitting diode (LED) illuminated the exact position of the actual spectral acquisition area (ROSA), which allowed the camera to register its position. A two-step acquisition sequence was used, followed by combined imaging and targeted spectroscopy.

The location of the DRS acquisition region was determined based on ROSA image segmentation. Spectra were acquired by moving the ROSA to six different positions within the field of view of the reference target for spectral analysis.

Bandpass filters isolate the excitation illumination for green fluorescence imaging. In comparison, long-pass filters allowed exclusive imaging and spectral capture of the light emitted by the fluorescence.

The spectral analysis involved three processing steps where ambient light spectral contributions were removed from the spectrum and then the effect of the illumination source spectrum was determined. The light spectrum was then normalized to correct for differences in signal intensity.

Study findings

The model eye acquired reflectance spectra from the blood vessels, the retina near the optic nerve head, the optic nerve head, and the retina away from the optic nerve head (D). Blood vessels and optic nerve showed distinctly different reflectance spectra. Similarly, the eye model helped perform fluorescence analysis for four regions, with only blood vessels and the optic nerve head emitting fluorescence signals.

Five-second DRS acquisitions corresponded to 13 acquired spectra and were performed on the optic nerve head and parafobia for all eight participants. The mean absorption spectra for both sites showed inter-individual variability.

All previous methods for assessing blood oxygen saturation in the eye had limited sensitivity and, as a result, allowed relative assessment of StO₂ only for large blood vessels of the fundus of the eye. In the current study, blood oxygen saturation measurements taken at different sites resulted in different values ​​of StO₂.

Lower oxygen saturation and greater interindividual variability in StO₂ were observed in the parafobia rather than the optic nerve head, ranging from 30.4–58.4% and 62.1–69.7%, respectively.

An ocular oximetry algorithm was applied to the acquired spectra in vivo and demonstrated the possibility of evaluating the presence of different fluorophores/chromophores that can be used to diagnose different retinal pathologies. More specifically, this approach targeted specific regions of interest identified via wide-field fluorescence and acquired an entire emission spectral profile of these molecules.

conclusions

The multimodal system presented in this study enabled simultaneous and continuous imaging and targeted spectroscopy at the fundus of the eyes. In addition, it showed high sensitivity, spectral resolution, and short acquisition speed for the detection of retinal biomarkers. It is noteworthy because other systems, such as hyperspectral imaging, trade off between spectral resolution and acquisition speed.

In addition, this technology obtained distinct spectral profiles in the various regions tested during in vitro and during in vivo test. In conclusion, targeted ocular spectroscopy could open new ways to diagnose and treat eye diseases over time.