Lipofuscin is a fluorescent lipid/protein complex in the retinal pigment epithelium (RPE) that accumulates with age and contributes to the pathogenesis of retinal dystrophies. Therefore, quantification of lipofuscin, through fundus autofluorescence (FAF) imaging, is important in diagnosis and monitoring the progression of the diseases. However, the measured AF signal is affected by the excitation light intensity, the detector sensitivity, and the optical properties of the ocular media anterior to the RPE.
We previously developed a simultaneous visible-light OCT and FAF imaging technology which is able to eliminate the pre-RPE attenuations. Further, we implemented two reference targets with known fluorescence efficiency and reflectivity in the retinal intermediate imaging plane to eliminate the effects of system fluctuations. However, we used an ND filter to reduce the fluorescence signal of the commercially available reference target to FAF level which brought the necessity of a separate reflection target for OCT.
In this study, we introduce a customized reference standard target to serve as a common target for both AF and OCT. This reference target is composed of PMMA and synthesized A2E, the major fluorophore of lipofuscin. Homogenous mixtures of PMMA resist and A2E were prepared with an optimum concentration of A2E to avoid the necessity of the ND filter. A2E-PMMA solution was spin-coated on a silicon wafer. The fluorescent coating with 400nm thickness serves as the common FAF and OCT reference target. Using A2E in reference fabrication provides a similar excitation and emission spectrums to RPE lipofuscin, and thus a better reference for lipofuscin/A2E quantification.
Rhodopsin, the light-sensing molecule in the outer segments of rod photoreceptors, is responsible for converting light into neuronal signals in a process known as phototransduction. Rhodopsin is thus a functional biomarker for rod photoreceptors. We developed a novel technology based on visible-light optical coherence tomography (VIS-OCT) for in vivo molecular imaging of rhodopsin. The depth resolution of OCT allows the visualization of the location where the change of optical absorption occurs and provides a potentially accurate assessment of rhodopsin content by segmentation of the image at the location. A broadband supercontinuum laser, whose filtered output was centered at 520 nm, was used as the illuminating light source. To test the capabilities of the system on rhodopsin mapping we imaged the retina of albino rats. The rats were dark adapted before imaging. An integrated near infrared OCT was used to guide the alignment in dark. VIS-OCT three-dimensional images were then acquired under dark- and light- adapted states sequentially. Rhodopsin distribution was calculated from the differential image. The rhodopsin distributions can be displayed in both en face view and depth-resolved cross-sectional image. Rhodopsin OCT can be used to quantitatively image rhodopsin distribution and thus assess the distribution of functional rod photoreceptors in the retina. Rhodopsin OCT can bring significant impact into ophthalmic clinics by providing a tool for the diagnosis and severity assessment of a variety of retinal conditions.
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