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Updated: May 25, 2026
Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses
Published on: December 22, 2016
Intravital multiphoton imaging of immune responses in the mouse ear skin
Jackson LiangYao Li1, Chi Ching Goh, Jo L Keeble
1Singapore Immunology Network, Agency for Science, Technology and Research, Biopolis, Singapore.
Insights
Multiphoton microscopy offers high-resolution in vivo imaging of skin immune cells and vasculature. This protocol details intravital imaging of mouse ear skin for studying cutaneous inflammation and neutrophil behavior.
Area of Science:
- Immunology
- Microscopy
- Dermatology
Background:
- Traditional methods for assessing skin immune cell function include flow cytometry and histology.
- Multiphoton (MP) microscopy provides direct in vivo visualization of immune cells, extracellular matrix, and vasculature in tissues.
- MP microscopy offers high spatial and temporal resolution, surpassing limitations of conventional techniques.
Purpose of the Study:
- To provide a detailed protocol for intravital multiphoton microscopy of mouse ear skin.
- To demonstrate the application of this technique in studying cutaneous inflammation.
- To enable in-depth analysis of immune cell behavior within the skin microenvironment.
Main Methods:
- Intravital multiphoton microscopy of mouse ear skin.
- Step-by-step protocol for experimental procedures.
- Characterization of neutrophil response during cutaneous inflammation.
Main Results:
- Successful visualization of immune cells, extracellular matrix, and vasculature in vivo.
- Detailed analysis of neutrophil behavior in the epidermis, dermis, and blood vessels during inflammation.
- Demonstration of the technique's adaptability for various skin-related research questions.
Conclusions:
- Intravital MP microscopy is a powerful tool for studying skin immunity and inflammation.
- The provided protocol is minimally invasive, allowing for longitudinal studies.
- This technique facilitates a comprehensive understanding of immune cell dynamics in the skin microanatomy.
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.
