We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.
Abstract
Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.
We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.
Materials
Alcian Blue, 1% in 3% Acetic Acid
Sigma
B8438-250ML
Hematoxylin, Gill 1
Sigma
GHS116-500ML
Papanicolaou stain, OG-6
Sigma
HT40116-500 ml
Papanicolaou stain, Modified EA
Sigma
HT40232-1L
Live/Dead Viability/Cytotoxicity Kit
Life Technologies
L-3224
contains ethidium homodimer-1 and Calcein AM dyes
Alcaine (0.5% proparacaine hydrochloride)
Alcon
Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 µm
Muntz, A., van Doorn, K., Subbaraman, L. N., Jones, L. W. Impression Cytology of the Lid Wiper Area. J. Vis. Exp. (114), e54261, doi:10.3791/54261 (2016).