Development of Feasible Methods to Image the Eyelid Margin Using In Vivo Confocal Microscopy.

Purpose: To develop a feasible method to image eyelid margin structures using in vivo confocal microscopy (IVCM) for use in clinical research. Second, to assess the association between IVCM and meibography images.

Methods: IVCM was performed on the central upper eyelid margin of 13 healthy participants (31 ± 5 years). Overall morphology montages (1600 × 1600 μm) were created of 3 participants. Single frames (400 × 400 μm) of 10 participants were imaged to determine the feasibility of measuring eyelid features. Meibography was performed with EASYTEARview+ in the same 10 participants. ImageJ software was used to quantify image structures.

Results: In the montages, structures of rete ridges, meibomian gland openings, and the lid wiper region were observed. The maximum possible montage size, using multiple single frames, was approximately 5200 × 1500 × 150 μm in the X, Y, and Z directions, respectively. The mean number, density, area, perimeter, and shortest and longest diameters of rete ridges of the 9 nonoverlapped frames were 12 ± 2/frame, 73 ± 5/mm, 2504 ± 403 μm, 250 ± 33 μm, 40 ± 6 μm, and 84 ± 13 μm, respectively. Sampling analysis determined at least 5 nonoverlapped frames were necessary to accurately represent the parameters of the ridges. The mean areas of 3 meibomian openings were 785 ± 784 μm, 1036 ± 963 μm, 950 ± 1071 μm, 848 ± 954 μm, 737 ± 831 μm, 735 ± 743 μm, and from 30 μm to 130 μm at 20-μm depth intervals, respectively. No significant association between IVCM and meibography parameters (P = 0.53) was found.

Conclusions: Imaging rete ridges with IVCM should include at least 5 nonoverlapping single frames in the upper eyelid margin. At least 3 openings imaged between 30 and 130 μm at 20-μm depth intervals are recommended to determine the opening area.