title: Autofluorescence multiphoton microscopy for visualization of tissue morphology and cellular dynamics in murine and human airways.
abstract: The basic understanding of inflammatory airway diseases greatly benefits from imaging the cellular dynamics of immune cells. Current imaging approaches focus on labeling specific cells to follow their dynamics but fail to visualize the surrounding tissue. To overcome this problem, we evaluated autofluorescence multiphoton microscopy for following the motion and interaction of cells in the airways in the context of tissue morphology. Freshly isolated murine tracheae from healthy mice and mice with experimental allergic airway inflammation were examined by autofluorescence multiphoton microscopy. In addition, fluorescently labeled ovalbumin and fluorophore-labeled antibodies were applied to visualize antigen uptake and to identify specific cell populations, respectively. The trachea in living mice was imaged to verify that the ex vivo preparation reflects the in vivo situation. Autofluorescence multiphoton microscopy was also tested to examine human tissue from patients in short-term tissue culture. Using autofluorescence, the epithelium, underlying cells, and fibers of the connective tissue, as well as blood vessels, were identified in isolated tracheae. Similar structures were visualized in living mice and in the human airway tissue. In explanted murine airways, mobile cells were localized within the tissue and we could follow their migration, interactions between individual cells, and their phagocytic activity. During allergic airway inflammation, increased number of eosinophil and neutrophil granulocytes were detected that moved within the connective tissue and immediately below the epithelium without damaging the epithelial cells or connective tissues. Contacts between granulocytes were transient lasting 3 min on average. Unexpectedly, prolonged interactions between granulocytes and antigen-uptaking cells were observed lasting for an average of 13 min. Our results indicate that autofluorescence-based imaging can detect previously unknown immune cell interactions in the airways. The method also holds the potential to be used during diagnostic procedures in humans if integrated into a bronchoscope.
authors.affiliation: Center for Structural and Cell Biology in Medicine, Institute of Anatomy, University of Lübeck, Lübeck, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kretschmer
authors.affiliation: Center for Structural and Cell Biology in Medicine, Institute of Anatomy, University of Lübeck, Lübeck, Germany.|Airway Research Center North, German Center for Lung Research (DZL), Lübeck, Germany.
authors.forename: Mario
authors.initials: M
authors.lastname: Pieper
authors.affiliation: Airway Research Center North, German Center for Lung Research (DZL), Lübeck, Germany.|Institute of Biomedical Optics, University of Lübeck, Lübeck, Germany.
authors.forename: Gereon
authors.initials: G
authors.lastname: Hüttmann
authors.affiliation: Institute of Anatomy II, University Hospital Jena, Jena, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Bölke
authors.affiliation: Clinic for Otolaryngology, University Clinic of Schleswig-Holstein, Campus Lübeck, Lübeck, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Wollenberg
authors.affiliation: Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.
authors.forename: Leigh M
authors.initials: LM
authors.lastname: Marsh
authors.affiliation: Institute for Laboratory Medicine and Pathobiochemistry, Member of the German Center for Lung Research (DZL), Philipps University of Marburg, Marburg, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Garn
authors.affiliation: Center for Structural and Cell Biology in Medicine, Institute of Anatomy, University of Lübeck, Lübeck, Germany.|Airway Research Center North, German Center for Lung Research (DZL), Lübeck, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: König
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000473:pathology; D001249:Asthma / Q000276:immunology / Q000473:pathology; D002465:Cell Movement; D004195:Disease Models, Animal; D005260:Female; D006098:Granulocytes / Q000276:immunology / Q000473:pathology; D006801:Humans; D007834:Lasers / Q000009:adverse effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009297:Nasal Mucosa / Q000098:blood supply / Q000276:immunology / Q000473:pathology; D061848:Optical Imaging / Q000379:methods; D010047:Ovalbumin / Q000009:adverse effects / Q000276:immunology; D020545:Respiratory Mucosa / Q000098:blood supply / Q000276:immunology / Q000473:pathology; D046509:Tissue Culture Techniques; D014132:Trachea / Q000098:blood supply / Q000276:immunology* / Q000473:pathology*
chemical_list: D010047:Ovalbumin