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. 2013 Aug 23;4(9):1710-23.
doi: 10.1364/BOE.4.001710. eCollection 2013.

In vivo dark-field imaging of the retinal pigment epithelium cell mosaic

Drew Scoles  1 , Yusufu N Sulai, Alfredo Dubra
Affiliations

In vivo dark-field imaging of the retinal pigment epithelium cell mosaic

Drew Scoles et al. Biomed Opt Express. .

Abstract

Non-invasive reflectance imaging of the human RPE cell mosaic is demonstrated using a modified confocal adaptive optics scanning light ophthalmoscope (AOSLO). The confocal circular aperture in front of the imaging detector was replaced with a combination of a circular aperture 4 to 16 Airy disks in diameter and an opaque filament, 1 or 3 Airy disks thick. This arrangement reveals the RPE cell mosaic by dramatically attenuating the light backscattered by the photoreceptors. The RPE cell mosaic was visualized in all 7 recruited subjects at multiple retinal locations with varying degrees of contrast and cross-talk from the photoreceptors. Various experimental settings were explored for improving the visualization of the RPE cell boundaries including: pinhole diameter, filament thickness, illumination and imaging pupil apodization, unmatched imaging and illumination focus, wavelength and polarization. None of these offered an obvious path for enhancing image contrast. The demonstrated implementation of dark-field AOSLO imaging using 790 nm light requires low light exposures relative to light safety standards and it is more comfortable for the subject than the traditional autofluorescence RPE imaging with visible light. Both these factors make RPE dark-field imaging appealing for studying mechanisms of eye disease, as well as a clinical tool for screening and monitoring disease progression.

Keywords: (110.1080) Active or adaptive optics; (170.4460) Ophthalmic optics and devices; (170.4470) Ophthalmology; (290.4210) Multiple scattering.

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Figures

Fig. 1
Fig. 1
AOSLO image plane apertures in front of the detector: a) traditional confocal pinhole, approximately one Airy disk diameter (ADD), and b) large pinhole with centered filament.
Fig. 2
Fig. 2
AOSLO confocal (left) and dark-field (right) retinal images in four different subjects, all collected at the foveal center (center of fixation). The confocal images show the cone photoreceptor mosaic, while the dark-field images show the characteristic hexagonal RPE cell mosaic. The scale bar is 100 μm across.
Fig. 3
Fig. 3
AOSLO confocal (left) and dark-field (right) retinal images in four different subjects, all collected at 10° temporal to fixation. The confocal images show the cone and rod photoreceptor mosaic, while the dark-field images show the hexagonal RPE cell mosaic with significant cross-talk from the photoreceptor mosaic. The scale bar is 100 μm across.
Fig. 4
Fig. 4
Comparison of SD-OCT data and an AOSLO dark-field image (subject AD_1025). The en face view shown in B was created by coarsely segmenting the SD-OCT signal from the choroid over the area highlighted in panel A over the depth range indicated by the blue band. Panel D, shows the same retinal area as B as seen using AOSLO dark-field imaging. Scale bars are: A) 500 μm; C) 500 μm horizontal and 100 μm vertical; B) & D) 100 μm.
Fig. 5
Fig. 5
Confocal (photoreceptor) and dark-field (RPE) images collected simultaneously (subject JC_0616) at approximately 0.8° from fixation. Panels A-B and C-F show cones recorded with 1 ADD pinhole and dark-field recorded with a 16 ADD pinhole and 1 ADD filament, respectively. Panels B and E show the cone and RPE cell centers marked with crosses and circles superimposed to the images in A and D, respectively. Panel C shows the dark-field image with cone centers and RPE centers superimposed, while panel F adds the cell borders, determined as Voronoi cells derived from the estimated cell centers. The scale bar is 10 μm across.
Fig. 6
Fig. 6
Dark-field AOSLO images of the RPE mosaic at the center of fixation in volunteer JC_0616 collected using a 1 ADD thick filament and different pinhole diameters: A) 16, B) 12, C) 8 and D) 4 ADDs. The scale bar is 100 μm across.
Fig. 7
Fig. 7
Dark-field AOSLO images of the RPE mosaic at the center of fixation of volunteer JC_0616 collected using a 16 ADD diameter pinhole and either 1 (A) or 3 ADD thick filament (B). The scale bar is 100 μm across.
Fig. 8
Fig. 8
Time-averaged retinal point-spread function (PSF) recorded from research volunteer JC_0616 (left), focused on the photoreceptor layer (logarithmic color scale). The central and right panels show the radial average and integral, respectively, compared to that of a single retinal layer theoretical PSF (red solid lines).
Fig. 9
Fig. 9
Dark-field AOSLO images of the RPE mosaic at the center of fixation of volunteer JC_0616, using a 16 ADD diameter pinhole, 1 ADD thick filament and 790 nm (A) and 680nm (B) light. The scale bar is 100 μm across.
Fig. 10
Fig. 10
Effect of pupil apodization on image quality and contrast at 10° temporal to fixation in volunteer JC_0616 using 790 nm illumination, 16 ADD pinhole, 1 ADD filament (A) and with a centered 3 mm diameter circular block in the imaging (B) or the illumination paths (C). The scale bar is 100 μm across.
Fig. 11
Fig. 11
Comparison of autofluorescence to dark-field RPE imaging in AD_1025 at 3° superior and 9° temporal from fixation. A) dark-field image, B) autofluorescence images collected using 565 nm excitation and 625±45 nm emission. The scale bar is 100 μm across.
Fig. 12
Fig. 12
RPE images collected in a patient DW_1188 with central serous retinopathy. The SD-OCT image in panel A shows the 187 µm thick fluid collection that separates the retina from the RPE (below). En face AOSLO images of the area between the white arrows in A show RPE morphology in confocal mode (B), as well as in dark-field mode (C) at this location approximately 6° superior to fixation. The scale bar is 100 μm across.
Fig. 13
Fig. 13
AOSLO dark-field view from the fovea of volunteer JC_0616, with ‘*’ denoting the point of maximum cone density. The scale bar is 100 μm across.
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