250 kHz, 1.5 µm resolution SD-OCT for in-vivo cellular imaging of the human cornea

Abstract

We present the first spectral domain optical coherence tomography (SD-OCT) system that combines an isotropic imaging resolution of ~1.5 µm in biological tissue with a 250 kHz image acquisition rate, for in vivo non-contact, volumetric imaging of the cellular structure of the human cornea. OCT images of the healthy human cornea acquired with this system reveal the cellular structure of the corneal epithelium, cellular debris and mucin clusters in the tear film, the shape, size and spatial distribution of the sub-basal corneal nerves and keratocytes in the corneal stroma, as well as reflections from endothelial nuclei. The corneal images presented here demonstrate the potential clinical value of the new high speed, high resolution OCT system for non-invasive diagnostics and monitoring the treatment of corneal diseases.

Fig. 1

Schematic of the 250 kHz SD-OCT system. CL – collimator; DCP – dispersion compensation prisms; FC – broadband fiber coupler; FFT – fast Fourier transform; L1 to L4 – broadband achromat doublets; M – mirror; MEFO - Multi-element Focusing Objective; MO – microscope objective; NDF – neutral density filter; PC – polarization controllers; X,Y – a pair of galvanometric scanners; VPHG – volume phase holographic grating.

Fig. 2

Sample and reference arm spectra measured at the detection end of the SD-OCT system (A). Axial PSF measured in free space (B). Sensitivity roll-off (C). Image of an USAF target acquired with a 10x microscope objective (D). Magnified view of the USAF target marked with the blue square (E). Red and green lines show the intensity profiles of group 7, elements 6 and 7 of the USAF target image (F).

Fig. 3

Typical cross-sectional H&E histology image of a healthy human cornea with red arrows marking keratocytes in the stroma (A). EPI – epithelium, BM – Bowman’s membrane, STR – stroma. Representative B-scan acquired in-vivo from a healthy human cornea with the blue and red arrows marking the tear film and keratocytes respectively (B). A volumetric OCT image of the anterior cornea with yellow arrows marking hyper-reflective features in the tear film (C). Enface image of the tear film showing hyper-reflective structures (yellow arrows) that may correspond to cellular debris and mucin clusters (D). Enface images of the corneal epithelium acquired at different depths and showing the cellular structure of the epithelium (E-G). White dots inside the cells correspond to reflections from the cellular nuclei. Enface UHR-OCT image of a larger area of the corneal epithelium (H) and a corresponding IVCM image (J).

Fig. 4

Typical volumetric image of a healthy human anterior cornea (A). The same image showing the sub-basal corneal nerves, located in the basal cell layer of the epithelium (B). A typical cross-sectional histological image with GFAP marked corneal nerves (C, brown color). An UHR-OCT enface image of sub-basal corneal nerves, acquired in-vivo from a healthy subject (D). A typical IVCM image of sub-basal corneal nerves (E).

Fig. 5

UHR-OCT volumetric images of the corneal stroma showing keratocyte cells in the anterior and middle stroma respectively (A and D). Corresponding enface UHR-OCT images with red arrows marking thin stromal nerves (B and E). IVCM images of the anterior and middle stroma respectively (C and F) showing keratocytes and thin stromal nerves (red arrows).

Fig. 6

Cross-sectional UHR-OCT image of the healthy human cornea, acquired in-vivo with a 10x microscope objective with the imaging beam focused in the posterior cornea (A). Magnified view of the area in Fig. 6(A) marked with the red dashed line rectangle (B). 5X magnification was applied in axial direction to allow viewing of the corneal endothelium (END), Descemet’s membrane (DM) and the pre-Descemet’s layer (PDL). Keratocytes located at the boundary between the posterior stroma and the PDL are marked with red arrows. Typical H&E histological image of the healthy posterior human cornea (C). Enface IVCM image of the cellular structure of the corneal endothelium (D). Enface UHR-OCT image acquired in-vivo from a healthy corneal endothelium (E) from a single plane inside the endothelial layer at a depth location marked with the blue arrow in Fig. 6(B). The white hyper-reflective dots that appear arranged in a hexagonal pattern, most likely correspond to reflections from endothelial cell nuclei. FFT map of the UHR-OCT image shown in Fig. 6(E) and (F). The radius of the ring structure in the FFT map corresponds to the mean distance between the reflective white dots in the UHR-OCT image (Fig. 6(E)). The length of the radius was measured to be ~20 µm, which correlated very well with the average size of the healthy endothelial cells that was determined from the IVCM image shown in Fig. 6(D). Figure 6(G) shows a maximum intensity projection OCT image of the corneal endothelium that was acquired ex-vivo. The image clearly shows the hexagonal structure of the corneal endothelial cells.

Fig. 7

Enface, maximum intensity projection image of the corneal endothelium that was acquired in-vivo from a healthy subject at the maximum camera speed of 250 kHz (A). A small region in the image (red line rectangle) shows hexagonally shaped endothelial cells. A magnified view of this area is shown in Fig. 7(B). Cross-sectional B-scans from the same 3D data set, corresponding to locations marked with the blue and orange arrows in the enface image (C and D), show loss of image contrast due to fast axial eye motion.