Trans-retinal cellular imaging with multimodal adaptive optics

Zhuolin Liu, Johnny Tam, Osamah Saeedi, Daniel X Hammer, Zhuolin Liu, Johnny Tam, Osamah Saeedi, Daniel X Hammer

Abstract

Adaptive optics (AO), when coupled to different imaging modalities, has enabled resolution of various cell types across the entire retinal depth in the living human eye. Extraction of information from retinal cells is optimal when their optical properties, structure, and physiology are matched to the unique capabilities of each imaging modality. Despite the earlier success of multimodal AO (mAO) approaches, the full capabilities of the individual imaging modalities were often diminished rather than enhanced when integrated into multimodal platforms. Furthermore, many mAO designs added unnecessary complexity, making clinical translation difficult. In this study, we present a novel mAO system that combines two complementary approaches, scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT), in one instrument using a simplified optical design, flexible alternation of scanning modes, and independent focus control. The mAO system imaging performance was demonstrated by visualization of cells in their mosaic arrangement across the full depth of the retina in three human subjects, including microglia, nerve fiber bundles, retinal ganglion cells and axons, and capillaries in the inner retina and foveal cones, peripheral rods, and retinal pigment epithelial cells in the outer retina. Multimodal AO is a powerful tool to capture the most complete picture of retinal health.

Keywords: (010.1080) Active or adaptive optics; (110.4500) Optical coherence tomography; (170.3880) Medical and biological imaging; (170.4460) Ophthalmic optics and devices.

Conflict of interest statement

The authors declare that there are no conflicts of interest related to this article.

Figures

Fig. 1
Fig. 1
Schematic of the FDA mAO retinal imaging system (flattened for clarity). AL: adaptive lens, APD: avalanche photodiode, D1-3: dichroic beamsplitters, DG: diffraction grating, DM: deformable mirror, Gh, Gv: galvanometer scanners, I: iris, P: pinhole, PBS: pellicle beamsplitter, RSh: resonant scanner, SHWS: Shack-Hartmann Wavefront Sensor, SM1-8: spherical mirrors, TS: translation stage.
Fig. 2
Fig. 2
Slow scan mode for simultaneous SLO/OCT imaging. The SLO raster and OCT B-Scan are acquired simultaneously with Gv scanner.
Fig. 3
Fig. 3
Predicted system optical performance. PSF spot diagrams at the retina (left) with flattened DM. The solid circles denote diffraction-limited blur size. Beam displacement at DM and eye pupil planes (right) for ± 1.8° vertical (V) and horizontal (H) scans. Dashed line represents lenslet pitch at DM and eye pupil planes.
Fig. 4
Fig. 4
Cellular structures of the center macula using AOSLO. (A) Red square in subject S2 denotes location imaged with AOSLO. (B) Montage of the macula vessels by 3x3 overlapping 2° FOV AOSLO videos reveals both the big blood vessels and smallest capillaries. (*) denotes the center of FAZ. Scale bar in (B) also applies to (C). The photoreceptor mosaic in (C) was generated by shifting the system focus to the photoreceptor layer, and 1 airy disc pinhole was used for confocal imaging. Cones at foveal center labeled as yellow box in (C) is showed in zoomed in view in (D). Simultaneous collected AOOCT single B-Scan shows distinct retinal layers in (E). The OCT image is displayed in logarithmic scale. Keys: ILM: inner limiting membrane; NFL: nerve fiber layer; GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; ELM: external limiting membrane; IS/OS: inner segment/ outer segment junction; COST: cone outer segment tip; and RPE: retinal pigment epithelium.
Fig. 5
Fig. 5
Foveal cone mosaics from three subjects imaged with AOSLO. Cones are resolved across the fovea in all three subjects (top row). Cones are identified with semi-automatic software and the resultant cone locations (dots) and Voronoi maps calculated (second row).
Fig. 6
Fig. 6
Photoreceptor mosaics in peripheral retina using AOSLO. (A) Red square at 7.5°-10° temporal to the fovea in subject S2 denotes location imaged with AOSLO. (B) Montage of the cone and rods by 4x4 overlapping 0.75° FOV AOSLO videos. (C) Simultaneous collected AOOCT B-Scan at the same patch of retina indicated as green dashed line in (B) shows paired hyper-reflections that originate from the segments of the photoreceptors in the outer retina. Photoreceptor mosaics at single location (12° temporal retina) (D) in S1, and (E) in S3. Scale bar in (D) also applies for (E). AOSLO images are displayed with logarithmic intensity, and the AOOCT B-scan is displayed with linear scale.
Fig. 7
Fig. 7
Simultaneous imaging with independent focus control of the FDA mAO system (Visualization 2and Visualization 3) on subject S2 at 4° inferior and temporal to the fovea shows cellular details across the thickness of retina. (A) Simultaneous AOSLO and AOOCT focus controlled by DM, and (B) independent AOSLO focus control by AL. White arrows indicate the estimated focus plane in depth.
Fig. 8
Fig. 8
AOOCT cross-sectional and en face images extracted from the photoreceptor-RPE complex in S1 at 7° temporal retina. Total 40 volumes are averaged. (A) Averaged B-scan and corresponding A-scan profile reveal distinct reflectance bands corresponding to IS/OS, COST, ROST, and RPE layers. En face projection shows mosaic of (B) cones, and (C) RPEs. The cell locations were identified in (D) where yellow dots denote cone centers and cyan denotes the RPE Voronio map. Nonlinear scanner artifact is not corrected in (B)-(D). (E) 2-D power spectra of (B) and (C) are superimposed and color coded (cones: yellow; RPE cells: cyan).
Fig. 9
Fig. 9
Inner retina cells and structures imaged with averaged of 154 AOOCT volumes (Visualization 4). (A) Three-dimensional perspective of a registered and averaged AOOCT volume at 12° temporal to the fovea in subject S1, where green dashed line denotes corresponding cross-section shown in (B). Red arrow indicates same GCL soma in B and E. Scale bar in C also applies to D–E. (C) Star-like microglial cells sparsely cover the surface of the ILM. (D) A complex web of nerve fiber bundles of varying size project across the NFL. Some have a diameter as large as 30 μm (blue arrow). Others are as small as 3 μm (green arrow), which matches the known caliber of a single large GC axon. GCL somas appear between the overlying bundles (black arrow). (E) A mosaic of GCL somas of varying size tile the layer. Red arrow points to a large soma, thought to be a parasol RGC. Additional projection views of capillary networks are showed in (F and G) at IPL-INL and INL-OPL layer respectively. (H) Composition of (C, E-G) with segmented features of interest shows the spatial arrangement of retinal structure at different depths.

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