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Uta Gehlsen, Regina Orzekowsky-Schroeder, Marta Szaszák, Norbert Koop, Sebastian E. Siebelmann, Gereon Huettmann, Philipp Steven; Non-Invasive Multi-Dimensional Two-Photon Microscopy Distinguishes Activation States Of Antigen Presenting Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4271.
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To characterize inflammation at the ocular surface in vivo on the cellular level, functional non-invasive imaging is mandatory. Our group demonstrated that non-invasive two-photon microscopy can be used to identify cells and tissue structures at the ocular surface based on their intrinsic autofluorescence. As activated antigen presenting cells (APCs) are one of the major key players during inflammatory processes we tested the feasibility of multi-dimensional two-photon microscopy for optical differentiation of activated from non-activated APCs.
A modified DermaInspect two-photon microscope (Jenalab, Germany) was used. Macrophage cell suspensions were placed in a heated chamber under the microscope and investigated in vitro, using excitation wavelengths from 710-850nm. Multidimensional imaging of adherent and non-adherent cells included detection of total autofluorescence intensity and emission spectrum, fluorescence lifetime measurements (FLIM) and the ratio of free to protein bound NAD(P)H. Macrophages were activated by phorbol-myristate-acetate (PMA).
Adherent macrophages differ markedly from non-adherent macrophages by morphology, fluorescence lifetime, autofluorescence intensity and emission spectrum, and ratio of free to protein bound NAD(P)H. In addition, PMA activated macrophages demonstrated distinct morphological and optical differences in comparison to non-activated adherent and non-adherent macrophages in vitro.
Multidimensional two-photon microscopy enables to distinguish and detect specifically activated and non-activated macrophages. This is a further step towards characterizing inflammation at the ocular surface in vivo through non-invasive characterization of activation states of individual immune cells that are involved in diseases such as ocular allergy, infection and dry-eye.
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