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.
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Acknowledgements
We thank T. Graf for providing Lysozyme-GFP mice and M. Nussenzweig for providing CD11c-YFP mice. We thank L. Renia and L. Robinson for their careful reading and critical comments on the manuscript. This work was supported by the Agency of Science, Technology and Research (A*STAR), Singapore. This study was also supported by the Australian National Health and Medical Research Council (NHMRC) project grants 570769 and 632706 and a grant from the New South Wales Office of Science and Medical Research (OSMR) to W.W.
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J.L.L., C.C.G., J.L.K. and L.G.N. wrote the first draft and prepared the figures. J.L.L., B.R., J.S.Q., R.J., Y.W. and W.K.C. conducted experiments and further improved the technique. L.G.N. and W.W. developed the methodology for the ear skin imaging.
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Supplementary information
Supplementary Fig. 1
Close-up image of mouse with an intact left ear (correctly prepared for imaging) and an injured right ear (incorrectly prepared for imaging). Mechanical damage induced during ear preparation can be detected by the leakage of Evans blue. All experiments dealing with live animals were performed in accordance with the relevant animal use and care guidelines and regulations. (TIFF 2894 kb)
Supplementary Fig. 2
Recommended filter and dichroic mirror setups for imaging: Set A: GFP, SHG and Evans blue; Set B: tdTomato, SHG and Evans blue and Set C: YFP, SHG and Evans blue. (EPS 459 kb)
Supplementary Fig. 3
A representative image of a typical setup of an anesthetized mouse on the custom-built intravital mouse ear skin stage. (TIFF 4585 kb)
Supplementary Fig. 4
Multiphoton imaging of immune cells in the skin. Maximal intensity projection of the CD11c-YFP dorsal mouse ear showing (a) Langerhans cells in the epidermis (16 µm stack) and (b) a network of dendritic cells within the highly vascularized dermis (57 µm stack). YFP (yellow, epidermal Langerhans cells and dermal dendritic cells), SHG (blue, collagen fibers) and Evans blue (red, blood vessels), excited at 950 nm with a Ti:Sapphire laser. Imaging of the epidermal layer was performed without hair removal to illustrate the autofluorescence from hairs. The collagen-rich dermis can also be differentiated from the epidermis by the presence of SHG signal. Scan field is 500 × 500 µm and 400 × 400 µm for the epidermis and dermis respectively. Scale bar: 100 µm. All experiments dealing with live animals were performed in accordance with the relevant animal use and care guidelines and regulations. (EPS 14992 kb)
Supplementary Movie 1
Migration of GFP+ cells in the dermis after laser induced injury. A time-lapse sequence of maximum projection (500 x 500 µm scan field, 51 µm stack, 3 µm step size) depicting the migratory patterns of GFP+ cells in the dermis of lysozyme-GFP mice after laser injury. GFP+ cells infiltrate the laser injury site as indicated by intense red signals from the Evans blue leakage. GFP (green, neutrophils), SHG (blue, collagen) and Evans blue (red, blood vessels) were excited with 950 nm, 20 mW at sample, using a tunable Ti:Sapphire laser. The laser injury was induced with 200 mW laser power at 800 nm, 75 × 75 µm scanfield, 242 × 242 pixels, 8.86 µs pixel-1 dwell time. Scale bar: 100 µm. All experiments dealing with live animals were performed in accordance with the relevant animal use and care guidelines and regulations. Elapsed time shown is hh:mm:ss. (MOV 8323 kb)
Supplementary Movie 2
Effects of speckling in pigmented skin. A time-lapse maximum projection of a 3D volume (500 × 500 µm scan field, 48 µm stack, 3 µm step size, 4.31 µs/pixel dwell time). In the pigmented mouse (left), speckling can be first observed within 2.5 minutes of imaging. After 20 minutes, neutrophils start to infiltrate the site where speckling has occurred. This phenomenon was absent in the albino mouse (right). GFP (green, neutrophils), SHG (blue, collagen) and Evans blue (red, blood vessels), were excited with 950 nm, 20 mW at sample, using a tunable Ti:Sapphire laser. Scale bar: 100 µm. All experiments dealing with live animals were performed in accordance with the relevant animal use and care guidelines and regulations. (MOV 6381 kb)
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Li, J., Goh, C., Keeble, J. et al. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat Protoc 7, 221–234 (2012). https://doi.org/10.1038/nprot.2011.438
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DOI: https://doi.org/10.1038/nprot.2011.438
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