Development of feasible in vivo confocal microscopy methods to image eyelid margin in clinical research

Abstract

Purpose : Arbitrary protocols have been used for imaging eyelid structures using in vivo confocal microscopy (IVCM). The aim of this study was to develop a feasible method to image eyelid margin structures that could be used in clinical research.

Methods : IVCM (HRT3, Heidelberg Engineering, Germany) were performed on 13 healthy participants aged 31±5 years (9 females and 4 males). To explore the overall morphology of the eyelid margin, montages (1600µm × 1600µm) were created in the centre upper eyelid margin of 3 participants. To determine feasibility of IVCM, single frames (400µm × 400µm) were imaged (X, Y and Z scan applied) in the centre upper eyelid margin of 10 participants. Single frames of rete ridges were analysed for density, area and perimeter using ImageJ. Attempts to image Meibomian openings at 20µm depth intervals from 30µm to 130µm were conducted. The areas of the openings were measured using ImageJ.

Results : In the montages, structures of rete ridges, Meibomian openings and the mucocutaneous junction were observed from the top to the bottom. In single frames, the maximum length of X, Y and Z scan to capture clear frames were approximately 5200µm, 1500µm and 150µm, respectively. This scanning area included 9 non-overlapped frames of rete ridges and 5 Meibomian openings (Figure 1). The shape of the structures displayed a similar range of depths. The mean density, area and perimeter of rete ridges of 9 non-overlapped frames were 73±5/mm², 2504±403µm² and 250±33µm, respectively. Sampling analysis determined a minimum of 5 non-overlapped frames were necessary for analysing the rete ridges in the central upper eyelid margin per person. Increasing the numbers of frames during analysis had no effect on the variation in the measures. In consideration of time limitations and the tolerance of the participants, 3 Meibomian openings were imaged at set depths per person. The mean areas of the openings were 784±785µm², 963±1036µm², 1071±950µm², 954±848µm², 831±737µm², 743±735µm², respectively from 30µm to 130µm at 20µm depth intervals.

Conclusions : Future studies that image upper eyelid margin structures with IVCM should include at least 5 non-overlapping single frames of rete ridges, and at least 3 Meibomian openings at 20µm depth intervals between 30µm to 130µm.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Figure 1: A montage of in vivo confocal microscopy single frames in the central upper eyelid margin at a depth of 59µm.