Multifunctional adaptive optics optical coherence tomography allows cellular scale reflectometry, polarimetry, and angiography in the living human eye

Abstract

Clinicians are unable to detect glaucoma until substantial loss or dysfunction of retinal ganglion cells occurs. To this end, novel measures are needed. We have developed an optical imaging solution based on adaptive optics optical coherence tomography (AO-OCT) to discern key clinical features of glaucoma and other neurodegenerative diseases at the cellular scale in the living eye. Here, we test the feasibility of measuring AO-OCT-based reflectance, retardance, optic axis orientation, and angiogram at specifically targeted locations in the living human retina and optic nerve head. Multifunctional imaging, combined with focus stacking and global image registration algorithms, allows us to visualize cellular details of retinal nerve fiber bundles, ganglion cell layer somas, glial septa, superior vascular complex capillaries, and connective tissues. These are key histologic features of neurodegenerative diseases, including glaucoma, that are now measurable in vivo with excellent repeatability and reproducibility. Incorporating this noninvasive cellular-scale imaging with objective measurements will significantly enhance existing clinical assessments, which is pivotal in facilitating the early detection of eye disease and understanding the mechanisms of neurodegeneration.

Fig. 1.

Cellular-scale multifunctional imaging of the retinal nerve fiber layer using AO-OCT. Measuring AO-OCT-based reflectance, angiogram, retardance, and optic axis orientation reveals cellular and subcellular features (e.g., individual retinal nerve fiber bundles and striated glial septa), polarization properties (e.g., well-being of nerve fiber parallel structure, density, and orientation), and their dynamics (e.g., capillary blood flow), all of which are key clinical features in glaucoma and other neurodegenerative diseases.

Fig. 2.

Schematic diagram of multifunctional AO-OCT system. (left) OCT engine and (right) AO sample arm with a stimulation channel. Of note, the same system design is used to build another duplicated machine for on-going studies in non-human primates, which includes a fluorescence channel (more details are described in Subsection S1.3 in Supplement 1 for interested readers).

Fig. 3.

Characterizing the imaging resolution (a), (b), and the reflectance, retardance, and optic axis orientation measurements (c) of the multifunctional AO-OCT. (a) En-face images of a USAF 1951 test target with a lens focal length of 75 mm (left) and 30 mm (right), respectively. The red lines, labeled with “X” and “Y,” denote the positions of the amplitude profiles shown in (b). We found a 1/e² beam waist spot radius of 2.8 µm using Gaussian fitting. (c) Cross-sectional images of Scotch tape corresponding to AO-OCT reflectance (left), retardance (middle), and optic axis orientation (right). (d) shows the normalized amplitude reflectance up to 2.0 mm (refractive index of 1.5) along the axial scan position denoted by the red arrow in c (left) as a function of scan depth. (e) shows the single-pass cumulative phase retardation and optic axis orientation up to 2.0 mm along the axial scan position denoted by the red arrow in c (middle and right). The white bar denotes 200 µm.

Fig. 4.

Collage of en-face image pairs, each showing multifunctional AO-OCT reflectance (left) and angiogram (right), captured from the right eye of a 37 y/o subject with high myopia (Sph: -7D; H001) at circled numbered positions in the Spectralis SLO image. (0) Central ONH (see Subsection 3.2.2). (1-5) Radial striation patterns of Müller septa are visible with a width as narrow as a couple of micrometers as denoted by the yellow arrowheads, well aligned with radial peripapillary capillaries in the corresponding angiogram (see Subsection 3.2.1). (6-8) The RNFBs continue to extend toward the temporal retina along the arcuate as predicted by Jansonius et al.’s model [98] (see Subsection 3.2.3). At location (8), the individual GCL somas are visible by adjusting the image brightness (9X brighten). The white bars denote 100 µm. The angiograms were bandpass filtered to improve the contrast.

Fig. 5.

Representative AO-OCT imaging results of peripapillary RNFL reflectance, angiogram, retardance, and optic axis orientation in the right eye of 37 y/o subject with high myopia (Sph: -7D; H001) at location 1 (see Fig. 4). (a) En face image pair of reflectance (left) and angiogram (right). (b) Cross-sectional images of reflectance, angiogram, retardance, and optic axis orientation along the red line as denoted in (a). Twenty cross-sectional images (25 µm) were averaged along the direction perpendicular to the RNF orientation to reduce speckle noise while maintaining its parallel structure. The yellow and green arrowheads denote the positions of individual Müller septa and RPCs, respectively. The blue dashed curved lines denote the automatically segmented anterior and posterior surface of the pRNFL. The white bars denote 100 µm. The angiograms were bandpass filtered to improve the contrast.

Fig. 6.

Measurements of peripapillary RNFL reflectance, thickness, birefringence, optic axis, Müller cell process spacing, RP capillary density/spacing in two healthy subjects (H001 and H002). (a) Spectralis 12° circle scan. The green boxes denote the AO-OCT imaging locations. (b) AO-OCT cross-sectional image for each location. The yellow arrowheads denote the manually identified Müller septa. The star indicates the septum aligned with the capillary. (c) pRNFL reflectance, thickness, birefringence, optic axis, and Müller cell process spacing are plotted against the manually determined pRNFBs orientation from the AO-OCT reflectance volume.

Fig. 7.

Repeatability test results of peripapillary RNFL measurements at location (1) in the right eye of a well-trained subject (H001). (a) The impact of involuntary eye motions on the pRNFL imaging for five consecutive weeks. The image on the left merges the five AO-OCT en face images, each color-coded by the week, i.e., baseline (red), week 1 (green), week 2 (blue); week 3 (cyan), and week 4 (magenta), respectively. The corresponding spatial distributions of the eye positions tracked by our software are shown on the right. The color-coded stars denote the mean positions of the eye motion trace. The color-coded isoline contours encompass 3SDs of the eye motion trace. (b) The repeatability test results of the reflectance, retardance, and optic axis orientation measurements on the anterior surface of the pRNFL within the overlap area. The means and SDs of each measure are shown on the left. Their volume correlation coefficients are shown on the right.

Fig. 8.

Multifunctional AO-OCT reflectometry and polarimetry reveal the detailed view of prelaminar and laminar tissue in a 37 y/o subject with high myopia. (a) Spectralis OCT image with 1:1 aspect ratio. The red square denotes the corresponding cross-sectional area imaged by the multifunctional AO-OCT. The red dashed-line square denotes the corresponding area image (see Section S4 and Fig. S5 in Supplement 1). (b) Cross sections of AO-OCT reflectance, retardance, and optic axis with varying focus. The color change in the retardance and optic axis images represents their value change, which follows the color maps shown on the bottom right. The red arrows denote the width of dark trunk-like structures in the laminar cribrosa (aka LC pore); however, the width varies significantly depending on which cross-section is used. The striation pattern is visible in the laminar region and, to a lesser extent in the prelaminar region (see also Visualization 1). The yellow arrowheads denote one of the dark partitions that continues across the entire FOV. (c) En-face images of AO-OCT reflectance, retardance, and optic axis orientation at different depths (Z0, Z1, …, Z5). The green and red asterisks denote the axon bundle’s center positions, illustrating a tortuous path through the prelaminar and laminar regions. We mask out unreliable pixels of optic axis orientation with a retardance smaller than 5°. The white bars denote 100 µm unless otherwise specified.

Fig. 9.

Multifunctional AO-OCT reflectometry and polarimetry reveal the detailed view of prelaminar and laminar tissue in a 57 y/o healthy subject with a relatively deep cup (H003). (a) Spectralis OCT image with 1:1 aspect ratio. The red square denotes the corresponding cross-sectional area imaged by the multifunctional AO-OCT. (b) Cross sections of AO-OCT reflectance, retardance, and optic axis orientation after the reconstruction. The color change in the retardance and optic axis images represents their value change, which follows the color maps shown on the bottom row. The yellow arrowheads denote one of the dark partitions that continues across the entire FOV. The periodic appearance of these dark partitions forming the striation pattern is more visible in the laminar region. (c) En-face images of AO-OCT reflectance at the depths near Z3, visualizing an intricated collagenous fiber network. The red, green, and blue arrowheads indicate isolated collagenous fiber bundles, respectively; some of them bridge the axon bundle and appear to be under-stressed/stretched (see also Visualization 3). (d) En-face images of AO-OCT reflectance, retardance, and optic axis orientation at different depths (Z1, Z2, …, Z4). We mask out unreliable pixels with a retardance smaller than 5°. The white bars denote 100 µm unless otherwise specified.