Cellular structural and functional imaging of donor and pathological corneas with label-free dual-mode full-field optical coherence tomography

Abstract

In this study, a dual-mode full-field optical coherence tomography (FFOCT) was customized for label-free static and dynamic imaging of corneal tissues, including donor grafts and pathological specimens. Static images effectively depict relatively stable structures such as stroma, scar, and nerve fibers, while dynamic images highlight cells with active intracellular metabolism, specifically for corneal epithelial cells. The dual-mode images complementarily demonstrate the 3D microstructural features of the cornea and limbus. Dual-modal imaging reveals morphological and functional changes in corneal epithelial cells without labeling, indicating cellular apoptosis, swelling, deformation, dynamic signal alterations, and distinctive features of inflammatory cells in keratoconus and corneal leukoplakia. These findings propose dual-mode FFOCT as a promising technique for cellular-level cornea and limbus imaging.

Fig. 1.

Schematic of the dual-mode FFOCT system and imaging processing workflow. A: schematic of the dual-mode FFOCT setup based on Linnik interferometer. B: in static mode, four-phase modulation scheme is applied with reference arm PZT to extract single static image, and multiple static FFOCT images are acquired and averaged to improve SNR. C: in dynamic mode, a time-series of interferograms are acquired without reference arm modulation, then undergo frame normalization, singular value decomposition for motion artifacts removal, cumulative sum operation for SNR improvement and finally power spectral density analysis to obtain the dynamic FFOCT images. BS: Beam splitter; PZT: Piezoelectric transducer; LED: light emitting diode.

Fig. 2.

Dual-mode FFOCT imaging of donor cornea. A, B, C: squamous epithelial cell layer. D, E, F: wing epithelial cell layer. G, H, I: basal epithelial cell layer. J, K, L: subbasal nerve layer. M, N, O: stromal layer. P: static image of subbasal nerve plexus (SNP). Q: static image of Bowman’s layer. R: corneal nerve traversing through the stroma. S: tomographic image of a donor cornea approximately 550 µm thick. T: 3D stack of en face static images of a cornea approximately 550 µm thick. U: 3D stack of en face dynamic images of corneal epithelium approximately 40um thick. (Scale-bar: 50 µm)

Fig. 3.

FFOCT static images of keratoconus samples. A: tomographic image with irregular epithelial thickness (9-30 µm). B: basal nerve distribution. C: stroma layer, arrow refers to stromal nerve. D: a gap between the epithelium and stroma. E: en face image of stromal cells breaking into the epithelial layer. The arrow points to the keratocyte. F: bowman’s layer loss. (Scale-bar: 50 µm)

Fig. 4.

Dual-mode FFOCT imaging of keratoconus epithelial cells. A, B: superficial epithelial cells. C, D: zoomed images of the ROIs marked in A and B. E, F: basal epithelial cells. G, H: zoomed images of the ROIs marked in A and B. I, J: manifestations of keratoconus epithelial cells. K-N: zoomed images of the ROIs marked in I and J. (Scale-bar: A, B, E, F, I, J 50 µm; C, D, G, H, K, L, M, N 10 µm)

Fig. 5.

FFOCT static images of corneal leukoplakia samples. A: tomographic image. B: corneal neovascularization or lymphatic vessels. C: edematous corneal basal cells. D: Langerhans cells below the basal layer. (Scale-bar: 50 µm)

Fig. 6.

Dual-mode FFOCT imaging of corneal leukoplakia epithelial cells. A, B: superficial epithelial cells. C, D: basal epithelial cells. E-T: different manifestations of epithelial cells in corneal leukoplakia samples due to different inflammatory responses. G-L: zoomed images of the ROIs marked in E and F. O-T: zoomed images of the ROIs marked in M and N. U: static image of scar fibrous tissue. V: dynamic image of inflammatory cells. W, X: zoomed images of the ROIs marked in V. (Scale-bar: A-F, M, N, U, V 50 µm; G-L, O-T, W, X 10 µm)

Fig. 7.

Dual-mode FFOCT imaging of limbus. A: limbal crypt structure. B-D: epithelial cells with different manifestations in the limbal crypt. E: POV structure. The arrow points to cristae. F: 3D stack of en face static images of limbus. G, H: superficial epithelial cells in limbus. I-P: different manifestations of limbal basal cells. K-P: zoomed images of the ROIs marked in I and J. Q: static image of blood vessels or lymphatic vessels in limbal stroma. R: dynamic image of intraluminal cells. S: superimposed image of dual-mode images. (Scale-bar: A, E, G-J, Q-S 50 µm; B-D, K-P 10 µm)