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P. Steven, J. Rupp, G. Huettmann, N. Koop, H. Laqua; Two-Photon Real-Time Imaging of Conjunctiva-Associated Lymphoid Tissue (CALT). Invest. Ophthalmol. Vis. Sci. 2007;48(13):201.
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Immunological real-time analysis of conjunctiva-associated lymphoid tissue (CALT) is challenging at state. For the first time, two-photon microscopy, a new optical method, is evaluated in its use to analyze morphology and function of CALT.
Conjunctiva of female Balb/c mice is challenged with Chlamydia trachomatis serovar C (ChtC) or ovalbumin and choleratoxin subunit B (OVA/CTB) for CALT induction. A two-photon microscope equipped with a near infrared femtosecond-laser and a fluorescence-lifetime detector is used for ex-vivo analysis of unfixed and unstained ocular tissue with additional application of fluorescent microspheres to demonstrate transepithelial particle transport.
Challenge with ChtC or OVA/CTB induce all CALT components (lymphoepithelium, follicles, blood and lymphatic vessels), that are demonstrated in cellular and subcellular resolution by means of autofluorescence imaging. Wavelength adaptation allows specific differentiation of cellular and acellular components. Fluorescence-lifetime detection permits differentiation of cellular subsets (e.g. lymphocytes and macrophages). Application of fluorescent microspheres demonstrates transepithelial particle transport and detection within intracellular vesicles.
Two-photonmicroscopy is an innovative optical technique to analyse morphological and functional features of CALT. Detection of transepithelial particle transport and its impact on conjunctival immunological processes can be visualized in real-time. Future in-vivo experiments with suitable animal models would allow detailed analysis of CALT in a clinical context e.g. corneal transplant rejection, keratoconjunctivitis sicca and follicular conjunctivitis.
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