Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Intravital multiphoton imaging of immune responses in the mouse ear skin

Nature Protocols volume 7, pages 221–234 (2012)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic diagram of a custom-made ear skin stage for intravital multiphoton imaging.
Figure 2
Figure 3: Intradermal injection of mouse ear skin.
Figure 4: Ear preparation for multiphoton intravital imaging.
Figure 5: Optical sectioning by multiphoton microscopy.

Similar content being viewed by others

References

  1. Cahalan, M.D. & Parker, I. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. Immunol. 26, 585–626 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Coombes, J.L. & Robey, E.A. Dynamic imaging of host-pathogen interactions in vivo. Nat. Rev. Immunol. 10, 353–364 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Germain, R.N., Miller, M.J., Dustin, M.L. & Nussenzweig, M.C. Dynamic imaging of the immune system: progress, pitfalls and promise. Nat. Rev. Immunol. 6, 497–507 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Hickman, H.D., Bennink, J.R. & Yewdell, J.W. Caught in the act: intravital multiphoton microscopy of host-pathogen interactions. Cell Host Microbe 5, 13–21 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ng, L.G., Mrass, P., Kinjyo, I., Reiner, S.L. & Weninger, W. Two-photon imaging of effector T-cell behavior: lessons from a tumor model. Immunol. Rev. 221, 147–162 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Roediger, B., Ng, L.G., Smith, A.L., de St Groth, B.F. & Weninger, W. Visualizing dendritic cell migration within the skin. Histochem. Cell Biol. 130, 1131–1146 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Kissenpfennig, A. et al. Dynamics and function of Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from slower migrating Langerhans cells. Immunity 22, 643–654 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Nishibu, A., Ward, B.R., Boes, M. & Takashima, A. Roles for IL-1 and TNFalpha in dynamic behavioral responses of Langerhans cells to topical hapten application. J. Dermatol. Sci. 45, 23–30 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Nishibu, A. et al. Behavioral responses of epidermal Langerhans cells in situ to local pathological stimuli. J. Invest. Dermatol. 126, 787–796 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Auffray, C. et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science 317, 666–670 (2007).

    Article  CAS  PubMed  Google Scholar 

  11. Weninger, W. et al. Specialized contributions by alpha(1,3)-fucosyltransferase-IV and FucT-VII during leukocyte rolling in dermal microvessels. Immunity 12, 665–676 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Ng, L.G. et al. Migratory dermal dendritic cells act as rapid sensors of protozoan parasites. PLoS Pathog. 4, e1000222 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Hoeller, C. et al. In vivo imaging of cutaneous T-cell lymphoma migration to the skin. Cancer Res. 69, 2704–2708 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Sumaria, N. et al. Cutaneous immunosurveillance by self-renewing dermal {gamma}{delta} T cells. J. Exp. Med. 208, 505–518 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ng, L.G. et al. Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events. J. Invest. Dermatol. 131, 2058–2068 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Celli, S., Albert, M.L. & Bousso, P. Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy. Nat. Med. 17, 744–749 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Dudeck, A. et al. Mast cells are key promoters of contact allergy that mediate the adjuvant effects of haptens. Immunity 34, 973–984 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Matheu, M.P. et al. Imaging of effector memory T cells during a delayed-type hypersensitivity reaction and suppression by Kv1.3 channel block. Immunity 29, 602–614 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Peters, N.C. et al. In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 321, 970–974 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Graham, D.B. et al. ITAM signaling by Vav family Rho guanine nucleotide exchange factors regulates interstitial transit rates of neutrophils in vivo. PLoS ONE 4, e4652 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Zinselmeyer, B.H., Lynch, J.N., Zhang, X., Aoshi, T. & Miller, M.J. Video-rate two-photon imaging of mouse footpad—a promising model for studying leukocyte recruitment dynamics during inflammation. Inflamm. Res. 57, 93–96 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Amornphimoltham, P., Masedunskas, A. & Weigert, R. Intravital microscopy as a tool to study drug delivery in preclinical studies. Adv. Drug Deliv. Rev. 63, 119–128 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Sen, D., Forrest, L., Kepler, T.B., Parker, I. & Cahalan, M.D. Selective and site-specific mobilization of dermal dendritic cells and Langerhans cells by Th1- and Th2-polarizing adjuvants. Proc. Natl. Acad. Sci. USA 107, 8334–8339 (2010).

    Article  CAS  PubMed  Google Scholar 

  24. Hierck, B.P., Iperen, L.V., Gittenberger-De Groot, A.C. & Poelmann, R.E. Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies. J. Histochem. Cytochem. 42, 1499–1502 (1994).

    Article  CAS  PubMed  Google Scholar 

  25. Helft, J. & Merad, M. Isolation of cutaneous dendritic cells. Methods Mol. Biol. 595, 231–233 (2010).

    Article  PubMed  Google Scholar 

  26. Belkaid, Y., Jouin, H. & Milon, G. A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: a study in laboratory mice. J. Immunol. Methods 199, 5–25 (1996).

    Article  CAS  PubMed  Google Scholar 

  27. Saria, A. & Lundberg, J.M. Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues. J. Neurosci. Methods 8, 41–49 (1983).

    Article  CAS  PubMed  Google Scholar 

  28. Drobizhev, M., Makarov, N.S., Tillo, S.E., Hughes, T.E. & Rebane, A. Two-photon absorption properties of fluorescent proteins. Nat. Methods 8, 393–399 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Kim, J.V. & Dustin, M.L. Innate response to focal necrotic injury inside the blood-brain barrier. J. Immunol. 177, 5269–5277 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318 (2005).

    Article  CAS  Google Scholar 

  32. Zoumi, A., Yeh, A. & Tromberg, B.J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Beltman, J.B., Maree, A.F. & de Boer, R.J. Analysing immune cell migration. Nat. Rev. Immunol. 9, 789–798 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    Article  CAS  Google Scholar 

  35. Faust, N., Varas, F., Kelly, L.M., Heck, S. & Graf, T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96, 719–726 (2000).

    CAS  PubMed  Google Scholar 

  36. Townsend, D., Witkop, C.J. Jr. & Mattson, J. Tyrosinase subcellular distribution and kinetic parameters in wild type and C-locus mutant C57BL/6J mice. J. Exp. Zool. 216, 113–119 (1981).

    Article  CAS  PubMed  Google Scholar 

  37. Zhang, J. et al. CD41-YFP mice allow in vivo labeling of megakaryocytic cells and reveal a subset of platelets hyperreactive to thrombin stimulation. Exp. Hematol. 35, 490–499 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Lindquist, R.L. et al. Visualizing dendritic cell networks in vivo. Nat. Immunol. 5, 1243–1250 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Jung, S. et al. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell Biol. 20, 4106–4114 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Gray, E.E., Suzuki, K. & Cyster, J.G. Cutting edge: identification of a Motile IL-17-Producing {gamma}{delta} T cell population in the dermis. J. Immunol. 186, 6091–6095 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Unutmaz, D. et al. The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse. J. Immunol. 165, 3284–3292 (2000).

    Article  CAS  PubMed  Google Scholar 

Download references

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.

Author information

Author notes

  1. Jackson LiangYao Li and Chi Ching Goh: These authors contributed equally to this work.

Authors and Affiliations

  1. Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Biopolis, Singapore

    Jackson LiangYao Li, Chi Ching Goh, Jo L Keeble, Yilin Wang, Weng Keong Chew & Lai Guan Ng

  2. The Centenary Institute, Newtown, New South Wales, Australia

    Jim S Qin, Ben Roediger, Rohit Jain & Wolfgang Weninger

  3. Discipline of Dermatology, University of Sydney, Camperdown, New South Wales, Australia

    Wolfgang Weninger

  4. Department of Dermatology, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia

    Wolfgang Weninger

Authors
  1. Jackson LiangYao Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chi Ching Goh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jo L Keeble
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jim S Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ben Roediger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rohit Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yilin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Weng Keong Chew
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Wolfgang Weninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lai Guan Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding authors

Correspondence to Wolfgang Weninger or Lai Guan Ng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2011.438

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing