Nomenclature

In previous studies, the terms reticular pseudodrusen , pseudo-reticular drusen and pseudodrusen were used for different en face modalities, and the term subretinal drusenoid deposits was used for cross-sectional histology and spectral-domain OCT. In the present article, we use the term pseudodrusen for standard en face ophthalmoscopy (color fundus photography, infrared reflectance [IR], red-free [RF], and autofluorescence [AF]) and the term subretinal drusenoid deposits for the collections of material seen in the subretinal space in histologic specimens, AOSLO and OCT.

Patients and Controls

Study patients with AMD and age-similar subjects with normal retinas were recruited from the clinical research registry of the Department of Ophthalmology of the University of Alabama at Birmingham and through the Retina Service. The patients had been diagnosed with AMD previously. To assess the disease severity, stereoscopic color digital 30 degree fundus photographs were taken with an FF450 Plus fundus camera (Carl Zeiss Meditec, Dublin, California) after pupil dilation, and photographs were graded by a masked, experienced grader using the Age-Related Eye Disease Study 2 (AREDS2) severity scale for AMD. Disease severity ranged from early to advanced (AREDS grade 2–11). Participants in normal macular health met the criteria for AREDS grade 1 in both eyes. Exclusion criteria included diabetes, history of retinal vascular occlusions, and any signs or history of hereditary retinal dystrophy. Subjects were also excluded for reasons that might potentially prevent successful imaging, such as poor fixation, significant media opacity, irregular pupil shape, poor dilation, or refractive errors beyond ±6 diopters (D) spherical and ±3 D cylinder. The inclusion criteria for normal comparison subjects were the same, with the additional criteria of age greater than 50 years, no clinically significant cataract, and best-corrected visual acuity of 20/25 or better.

High-Resolution AOSLO Imaging

Imaging was conducted with a new-generation AOSLO that was developed in our laboratory, as described. This instrument was based on an earlier prototype that used a microelectric-mechanical system–based deformable mirror. The new instrument addressed several major obstacles that impede imaging of older patients with AMD. First, the pupil size decreases and the wavefront aberration increases with aging. Second, many older patients have cataract, which significantly affects wavefront detection and impairs AO wavefront correction. Third, in patients with intraocular lenses, it is very common that the proliferation and transformation of lens epithelial cell remnants lead to posterior capsular opacification or fibrosis over the intraocular lens. Although capsulotomy can make an opening on the opacified posterior capsule, the clear pupil often has an irregular shape for imaging. This may not only reduce the useful pupil size but may also cause complicated light scattering that impedes AO operation.

To address these challenges, we developed an advanced wavefront detection and correction strategy. We designed a high-speed Shack-Hartmann wavefront sensor based on a complementary metal-oxide-semiconductor (CMOS) camera (MicroVista-NIR; Intevac, Santa Clara, California). The camera’s spectral response is optimized for the imaging light used by AOSLO, enabling the AO system to be operated at a loop frequency up to 100 Hz. Most previously reported AO systems for retinal imaging run under 30 Hz. High-speed wavefront sensing significantly facilitates advanced control strategy based upon Zernike mode correction, thereby allowing for AO correction of wavefronts in eyes with the optical problems described above. Furthermore, we adopted a high-speed deformable mirror (Hi-Speed DM97-15; ALPAO SAS, Montbonnot St. Martin, France) with 97 actuators with stroke up to 30 μm, which provides improved ability to compensate for increased high orders and amplitudes of wavefront aberration due to aging. The AOSLO pupil size was set at 5.6 mm in diameter. After AO correction, the root-mean-square wave aberration was reduced to less than 0.05 μm in most eyes, reaching the criterion for diffraction-limited resolution for the light used in imaging. A low-coherence light source, a superluminescent diode (Broadlighter S840-HP; Superlum, Carrigtwohill Co, Cork, Ireland) was employed for producing high-fidelity retinal images. The imaging light power measured at the cornea was 500 μW, which is about 1/26 of the maximum permitted exposure limits set by the American National Standards Institute. The AOSLO records continuous videos from the eye with a frame rate of 15 Hz.

All participants underwent best-corrected visual acuity measurement by the Electronic Visual Acuity protocol. Pupils were dilated with 1.0% tropicamide and 2.5% phenylephrine hydrochloride. The subject’s head was aligned and stabilized using a head mount with a chin rest. A fixation target consisting of a moving bright green dot formed by the light from a laser diode (DJ532-10- 532; Thorlabs, Newton, New Jersey) on a calibrated grid. The grid, on white paper, was placed in front of the eye via a pellicle beam splitter (BP208, Thorlabs) to help the subject’s fixation. The wavelength of the diode laser was 532 nm, and the light was coupled into a single-mode fiber and collimated to form a light dot of 1.5 mm diameter on the grid paper. The light power was adjusted to 2 mW at the output of the fiber. The subject saw the light dot on the back of the grid paper through a pellicle beam splitter. During imaging, the dot was moved on the grid paper to direct the subject’s view angle. At each grid point, the light dot stopped for 3–5 seconds so that 45–75 frames were acquired. Videos were recorded continuously across an area of 15 × 15 degrees. An AOSLO imaging session lasted approximately 1 hour. Data presented were collected from 1 imaging session for each subject. Before images were recorded, the gain of the AOSLO photodetector was titrated to obtain proper image brightness and contrast according to a real-time histogram of a retinal video, and it remained constant through the whole imaging session.

AOSLO Image Processing and Analysis

Image distortions caused by nonlinearities in the resonant scanner and by eye movements were eliminated by customized software. Registered images were averaged to enhance signal-to-noise ratio. Images of various retinal locations were manually aligned on a cell-to-cell basis to create a continuous montage (Photoshop; Adobe Systems, Mountain View, California).

AOSLO image pixel size was computed from an image of a precisely calibrated dot grid placed at the retinal plane of a model eye. The extent of retina affected by the underlying subretinal drusenoid deposits at different progression stages was measured at multiple locations, using the Ruler Tool of Photoshop, and then averaged.

Multimodal Imaging

In addition to stereoscopic color digital 30 degree fundus photographs, enface IR (λ = 830 nm); RF (λ = 560 nm); and AF (excitation, 488 nm; emission >600 nm) images were acquired with the confocal SLO of the Spectralis (Heidelberg Engineering, Carlsbad, California). Fields of view of 30 × 30 degrees were digitized at 768 × 768 pixels. Retinal cross-sections were imaged using the Spectralis spectral-domain OCT (λ = 870 nm; acquisition speed, 40,000 A-scans per second; scan depth, 1.9 mm; digital depth resolution, 3.5 μm per pixel in tissue; lateral resolution in tissue 14 μm). In each study eye, 97 B-scans were acquired across a 15 × 10 degree area of the central macula to create a volume.

Pseudodrusen Identification

Multiple imaging modalities can disclose pseudodrusen with differing specificity and sensitivity. It is recommended that detection should be confirmed using more than 1 modality so as to improve accuracy. The identification of pseudodrusen in our study was based on their presence in at least 2 en face imaging modalities and in spectral-domain OCT. In en face imaging, pseudodrusen appear as an interlacing collection of ribbons or a dotlike pattern of yellow-white lesions (in color fundus photography); as a pattern of hyporeflective or hyper-reflective spots (in IR reflectance); or as a pattern of small hypoautofluorescent areas against a background of mild hyperautofluorescence (in AF). Although a reticular pattern was often seen in color photography, it was not a criterion for diagnosis. In spectral-domain OCT, subretinal drusenoid deposits were hyper-reflective mounds internal to the RPE. Axial microstructure and location of subretinal drusenoid deposits were evaluated by spectral-domain OCT, using the nomenclature of Spaide and Curcio for the 4 outer retinal hyper-reflective bands.

Multimodal Image Registration

Color fundus photographs and IR, RF and AF images taken with the Spectralis SLO were registered manually by use of retinal vessels and capillaries as invariant landmarks. Color fundus photographs and the SLO IR image were then magnified and registered with the AOSLO montage by using retinal vessels and capillaries as landmarks. Then pseudodrusen apparent on standard funduscopy were localized in AOSLO images and examined with high resolution.

Subretinal Drusenoid Deposits Classification

Lesions were scored by the 3-stage grading system introduced by Zweifel and coworkers. Specific lesions at each stage and surrounding photoreceptors were examined by AOSLO and by en face OCT.