Autofluorescence imaging with near-infrared excitation:normalization by reflectance to reduce signal from choroidal fluorophores

Abstract

Purpose: We previously developed reduced-illuminance autofluorescence imaging (RAFI) methods involving near-infrared (NIR) excitation to image melanin-based fluorophores and short-wavelength (SW) excitation to image lipofuscin-based flurophores. Here, we propose to normalize NIR-RAFI in order to increase the relative contribution of retinal pigment epithelium (RPE) fluorophores.

Methods: Retinal imaging was performed with a standard protocol holding system parameters invariant in healthy subjects and in patients. Normalized NIR-RAFI was derived by dividing NIR-RAFI signal by NIR reflectance point-by-point after image registration.

Results: Regions of RPE atrophy in Stargardt disease, AMD, retinitis pigmentosa, choroideremia, and Leber congenital amaurosis as defined by low signal on SW-RAFI could correspond to a wide range of signal on NIR-RAFI depending on the contribution from the choroidal component. Retinal pigment epithelium atrophy tended to always correspond to high signal on NIR reflectance. Normalizing NIR-RAFI reduced the choroidal component of the signal in regions of atrophy. Quantitative evaluation of RPE atrophy area showed no significant differences between SW-RAFI and normalized NIR-RAFI.

Conclusions: Imaging of RPE atrophy using lipofuscin-based AF imaging has become the gold standard. However, this technique involves bright SW lights that are uncomfortable and may accelerate the rate of disease progression in vulnerable retinas. The NIR-RAFI method developed here is a melanin-based alternative that is not absorbed by opsins and bisretinoid moieties, and is comfortable to view. Further development of this method may result in a nonmydriatic and comfortable imaging method to quantify RPE atrophy extent and its expansion rate.

Figure 1

Reduced-illuminance autofluorescence imaging (RAFI) results comparing SW- and NIR-excitation in healthy eyes and in retinal disease. (A, B) Reduced-illuminance autofluorescence in a representative young normal (N1) with dark irides and in an older normal (N16) with light irides. Calibration (lower right) applies to all images. (C, D) Stargardt disease with large central RPE atrophy in patients with light (P6) or dark (P5) irides. Arrows, superonasal boundary between atrophy and neighboring healthier region. Arrowheads, heterogenous-appearing region infero-nasal to fovea. (E, F) Nonneovascular AMD with geographic atrophy in two patients (P15, P14) with dark irides. Arrows, regions of geographic atrophy. Arrowhead, foveal preservation in P15. (G, H) Retinitis pigmentosa with parafoveal and perimacular degeneration in two patients (P16, P17) with light irides. Arrows, inferonasal perimacular region with RPE disease. Arrowheads, parafoveal region with RPE atrophy. Image contrasts are individually adjusted for visibility of features.

Figure 2

Range of reflectance (REF) images with NIR illumination showing different appearances of RPE and choroid likely associated with differences in absorption and backscatter at different depths. (A) Healthy subjects showing no obvious choroidal features (N4, dark irides), barely visible choroidal features (N6, light irides), and clearly visible choroidal features (N7, light irides, arrow). (B) Stargardt patients (STGD1) with central lesions showing a parafoveal ring of low NIR-REF signal (P4, arrowheads), and those high to low NIR-REF transition at the border of RPE atrophy (P7, P12, arrowheads). All three patients had light irides. Optical coherence tomography (OCT) and SW-RAFI images are also shown. Horizontal dashed lines depict the location and extent of the OCT. (C) A patient (P25, dark irides) with choroideremia (CHM) demonstrating the relationship between the relatively retained central retinal pigmentation corresponding to relatively low NIR-REF signal. Another CHM patient (P27, light irides) demonstrating NIR-REF in the case of chorioretinal degeneration. The brighter NIR-REF images were recorded with the standard sensitivity setting, whereas the dimmer images recorded with a reduced sensitivity. White arrows, retinal regions with no obvious photoreceptors or choroid remaining. Black arrows, retinal regions with retained choroidal structure but without detectable photoreceptors. Arrowheads, correspondence between the boundaries of the low NIR-REF signal and the associated OCT scans.

Figure 3

Quantitation of NIR-REF, NIR-RAFI, and NIR-RAFIn signals in a group of healthy subjects. (A, B) Representative results in younger (N3) and older (N17) healthy subjects. Both had lighter irides. (C) Mean horizontal and vertical profiles of signal intensity in younger (gray) and older (black) subjects. (D) NIR-RAFIn signal at five central locations (Inset, C, central; S, superior; I, inferior; T, temporal; N, nasal) across all healthy subjects as a function of age and eye color. Dashed lines depict linear regression.

Figure 4

Comparison of RPE atrophy and neighboring healthier retina obtained with NIR modalities versus SW-RAFI in different retinal diseases. Representative examples of STGD1 patients with light irides (A, B), RP patient with dark irides (C), and CHM patient with light irides (D) are shown. NIR-REF image of the CHM patient is obtained with nonstandard detector sensitivity in order to avoid saturation. Calibration shown on lower right applies to all panels.

Figure 5

Imaging in RPE65-LCA and RHO-ADRP patients in whom SW-RAFI is not usable either because of the lack of lipofuscin resulting from underlying pathophysiology or because of concerns about the light load. (A) NIR-RAFIn results in RPE65-LCA patients P23 and P24. (B) NIR-RAFIn results in RHO-ADRP patients P21 and P22. Optic nerve head and major blood vessels are traced (white) to better interpret the context of the images shown. (C) Bone-spicule–like pigment in two patients with RHO-ADRP.