title: Intravital multiphoton imaging of immune responses in the mouse ear skin.
abstract: Multiphoton (MP) microscopy enables the direct in vivo visualization, with high spatial and temporal resolution, of fluorescently tagged immune cells, extracellular matrix and vasculature in tissues. This approach, therefore, represents a powerful alternative to traditional methods of assessing immune cell function in the skin, which are mainly based on flow cytometry and histology. Here we provide a step-by-step protocol describing experimental procedures for intravital MP imaging of the mouse ear skin, which can be easily adapted to address many specific skin-related biological questions. We demonstrate the use of this procedure by characterizing the response of neutrophils during cutaneous inflammation, which can be used to perform in-depth analysis of neutrophil behavior in the context of the skin microanatomy, including the epidermis, dermis and blood vessels. Such experiments are typically completed within 1 d, but as the procedures are minimally invasive, it is possible to perform longitudinal studies through repeated imaging.
authors.affiliation: Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore.
authors.forename: Jackson LiangYao
authors.initials: JL
authors.lastname: Li
authors.forename: Chi Ching
authors.initials: CC
authors.lastname: Goh
authors.forename: Jo L
authors.initials: JL
authors.lastname: Keeble
authors.forename: Jim S
authors.initials: JS
authors.lastname: Qin
authors.forename: Ben
authors.initials: B
authors.lastname: Roediger
authors.forename: Rohit
authors.initials: R
authors.lastname: Jain
authors.forename: Yilin
authors.initials: Y
authors.lastname: Wang
authors.forename: Weng Keong
authors.initials: WK
authors.lastname: Chew
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Weninger
authors.forename: Lai Guan
authors.initials: LG
authors.lastname: Ng
mesh_terms: D000818:Animals; D004423:Ear / Q000033:anatomy & histology*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008815:Mice, Inbred Strains; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D009504:Neutrophils / Q000166:cytology* / Q000276:immunology; D012867:Skin / Q000166:cytology* / Q000648:ultrastructure