Non-invasive in vivo imaging of human corneal microstructures with optical coherence microscopy

Abstract

Non-invasive imaging systems with cellular-level resolution offer the opportunity to identify biomarkers of the early stage of corneal diseases, enabling early intervention, monitoring of disease progression, and evaluating treatment efficacy. In this study, a non-contact polarization-dependent optical coherence microscope (POCM) was developed to enable non-invasive in vivo imaging of human corneal microstructures. The system integrated quarter-wave plates into the sample and reference arms of the interferometer to enable deeper penetration of light in tissues as well as mitigate the strong specular reflection from the corneal surface. A common-path approach was adopted to enable control over the polarization in a free space configuration, thus alleviating the need for a broadband polarization-maintained fiber. The POCM achieved volumetric imaging of corneal microstructures, including endothelial cells over a field of view 0.5 × 0.5 mm² with an almost isotropic resolution of ∼2.2 µm and a volume (500 × 500 × 2048 voxels) rate of 1 Hz. A self-interference approach between the corneal surface and underlying layers was also developed to lessen the corneal curvature and axial motion artifacts, thus enabling high-resolution imaging of microstructures in the anterior cornea, including squamous epithelial cells, wing epithelial cells, basal epithelial cells, sub-basal nerve plexus, and stromal keratocytes.

Fig. 1.

Schematic of POCM for in vivo human corneal imaging. PC, polarization controller; FC, fiber collimator; QWP, quarter-wave plate; DCE, dispersion compensation elements; DG, diffractive grating; M, mirror; ACD, achromatic doublet; 10X Obj, 10X-microscopic objective; BS, beam splitter; FT, fixation target; IL, imaging lens.

Fig. 2.

Interference signals in three different arrangements of the reference arm quarter-wave plate (QWP) with the fast axis of the QWP in the sample arm set at 45° from horizontal. (a) Interference signal with the relative angle of 0° between QWPs (blue trace). (b) Interference signal with the relative angle of 22.5° between QWPs (red trace). (c) Interference signal with the relative angle of 45° between QWPs (green trace). (d) Corresponding depth profiles of the three configurations. The inserts highlight depth profiles in the three configurations corresponding to the point spread function (PSF) from the self-interference between the front and back surface of the glass (d1) and the PSF from the interference with the reference arm (d2).

Fig. 3.

POCM Enface images of the resolution chart over a field of view of 0.5 × 0.5 mm² with the corresponding intensity profiles over the 6th elements (horizontal and vertical orientations) of the 7th group in four configurations. (a1) 1000 × 1000 A-scans with the relative angle of QWPs set at 0^. (b1) Corresponding intensity profiles. (a2) 500 × 500 A-scans with the relative angle of QWPs set at 0^. (b2) Corresponding intensity profiles. (a3) 1000 × 1000 A-scans with the relative angle of QWPs set at 45^. (b3) Corresponding intensity profiles. (a4) 500 × 500 A-scans with the relative angle of QWPs set at 45^. (b4) Corresponding intensity profiles.

Fig. 4.

POCM images of the cornea in two healthy subjects. (a1) and (a2) Cross-sectional images along the slow-scanning axis of Subjects 1 and 2, respectively, showing the axial motion as well as a blink (gap in (a2)) during the acquisition. (a1) exhibits a false double-layer of the surface induced by the configuration with QWPs at 45^ whereas (a2) shows a strong specular reflection from the surface in the configuration with QWPs at 0^ (black arrows). The top part of the image corresponds to the interference of the cornea with the reference arm whereas the botom part corresponds to the self-interference, which is not affected by the axial motion. (b1) and (b2) Enface images of the corneal surface of Subjects 1 and 2, respectively. Hyperreflective features (blue arrows) are visible on the surface, presumably immune cells or infiltrates from the tear film. Low-contrast squamous epithelial cells and their hyporeflective nuclei (black arrowhead) are also visible in (b2). (c1) and (c2) Enface images of the mid-stroma showing keratocytes (white arrowheads) as well as a nerve branching (green arrow). (d1) and (d2) Enface images of the endothelium showing endothelial cells.

Fig. 5.

POCM images of the anterior cornea of the two subjects from the self-interference region in Fig. 4. (a1) and (a2) Enface images of squamous epithelial cells in both subjects showing some hyporeflective nuclei and cell boundaries. (b1) and (b2) Enface images of wing epithelial cells in both subjects. (c1) and (c2) Enface images of basal epithelial cells in the two subjects. (d1) and (d2) Enface images of sub-basal nerve plexus. (e1) and (e2) Enface images of the anterior stroma exhibiting keratocytes and a nerve branching in (e1).