title: Non-Invasive Multi-Dimensional Two-Photon Microscopy enables optical fingerprinting (TPOF) of immune cells.
abstract: Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non-invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two-photon microscopy to non-invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence-based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients. Two photon based autofluorescence features of immune cells enables non-invasive differentiation.
authors.affiliation: Department of Ophthalmology, University of Cologne, Kerpenerstr. 62, 50937 Cologne, Germany. uta.gehlsen@uk-koeln.de
authors.forename: Uta
authors.initials: U
authors.lastname: Gehlsen
authors.forename: Marta
authors.initials: M
authors.lastname: Szaszák
authors.forename: Andreas
authors.initials: A
authors.lastname: Gebert
authors.forename: Norbert
authors.initials: N
authors.lastname: Koop
authors.forename: Gereon
authors.initials: G
authors.lastname: Hüttmann
authors.forename: Philipp
authors.initials: P
authors.lastname: Steven
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000473:pathology; D001854:Bone Marrow Cells / Q000166:cytology / Q000473:pathology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000473:pathology; D004195:Disease Models, Animal; D005123:Eye / Q000276:immunology / Q000473:pathology; D005260:Female; D005453:Fluorescence; D008264:Macrophages / Q000166:cytology* / Q000473:pathology; D008807:Mice, Inbred BALB C; D008853:Microscopy / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology / Q000473:pathology; D013154:Spleen / Q000166:cytology / Q000473:pathology; D013601:T-Lymphocytes / Q000166:cytology* / Q000473:pathology; D046509:Tissue Culture Techniques
keywords: antigen presenting cells; in vivo; intravital two-photon microscopy; non invasive; ocular surface