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P. Steven, J. Rupp, G. Huettmann, R. Orzekowsky, N. Koop, A. Gebert; Intravital Two-Photon Imaging of Conjunctiva-Associated Lymphoid Tissue - A New Window to Visualize Immune Mechanisms of the Ocular Surface. Invest. Ophthalmol. Vis. Sci. 2008;49(13):435. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Two-photon microscopy, a new optical imaging technique has been demonstrated to be a suitable method to investigate conjunctiva-associated lymphoid tissue (CALT) in a newly established mouse model. Here we present data from leadoff intravital real-time imaging of murine CALT that feature the potential for further functional investigations.
Balb/c mice were topically challenged with C. trachomatis serovar C or ovalbumin/choleratoxin B to induce CALT in the nictitating membrane of the eye. A two-photon microscope equipped with a tuneable near infrared femtosecond-laser was used. Mice were deeply anesthetized for intravital analysis of CALT with additional application of fluorescent microspheres to demonstrate particle uptake or i.v. application of Hoechst nuclear dye to enhance cellular contrast.
High resolution autofluorescence images of lymphoepithelium, follicles and adjacent vessels were obtained in tissue depths up to 80 µm. 3-D time-lapse image series of CALT within the nictitating membrane demonstrate that macrophages and lymphocytes rapidly migrate within the subepithelial space. Macrophages move along elastic fibres. Vascular blood flow is detectable. Hoechst dye injection enables single cell tracking at highly reduced laser intensities.
For the first time two-photon microscopy enables intravital real-time investigations of immune cells related to CALT. This new optical method could be a useful tool to evaluate the physiological function of CALT and to elucidate its functional role under pathological conditions such as inflammation or auto-immune disease.
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