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Updated: May 15, 2026
Non-invasive Imaging of Leukocyte Homing and Migration in vivo
Published on: December 5, 2010
Visualizing leukocyte trafficking in the living brain with 2-photon intravital microscopy
Saparna Pai1, Karyn J Danne, Jim Qin
1Immune Imaging Program, The Centenary Institute Newtown, NSW, Australia ; Sydney Medical School, University of Sydney Sydney, NSW, Australia.
Insights
This study details a two-photon brain imaging protocol for tracking immune cells in mice. The method allows high-resolution visualization of leukocytes and brain structures, aiding CNS disease research.
Area of Science:
- Neuroscience
- Immunology
- Microscopy
Background:
- Central nervous system (CNS) diseases involve complex cellular and molecular interactions.
- Intravital imaging offers a powerful method to study these interactions in vivo.
- A standardized protocol is needed for non-specialists to utilize this technique.
Purpose of the Study:
- To present a detailed, step-by-step protocol for two-photon brain imaging in mice.
- To enable visualization of immune cells, microvasculature, and extracellular matrix in the brain.
- To provide a versatile method adaptable for various CNS-related research questions.
Main Methods:
- A two-part protocol involving cranial window implantation and continuous superfusion with artificial cerebrospinal fluid (aCSF).
- Simultaneous high-resolution imaging of fluorescently labeled leukocytes, pial microvasculature, and collagen fibers.
- Methodology optimized over 10 years, integrating experience from multiple laboratories and disease models.
Main Results:
- The protocol enables non-specialists to establish a two-photon brain imaging model.
- High spatial and temporal resolution visualization of immune cell dynamics within the brain vasculature.
- Successful tracking of leukocytes in transgenic mice under steady-state conditions.
Conclusions:
- This protocol provides a robust and adaptable tool for studying CNS inflammatory and infectious diseases.
- It facilitates the dissection of cellular and molecular mechanisms in neurological disorders.
- The method supports extended imaging sessions under physiological conditions.
Abstract:
Intravital imaging of the superficial brain tissue in mice represents a powerful tool for the dissection of the cellular and molecular cues underlying inflammatory and infectious central nervous system (CNS) diseases. We present here a step-by-step protocol that will enable a non-specialist to set up a two-photon brain-imaging model. The protocol offers a two-part approach that is specifically optimized for imaging leukocytes but can be easily adapted to answer varied CNS-related biological questions. The protocol enables simultaneous visualization of fluorescently labeled immune cells, the pial microvasculature and extracellular structures such as collagen fibers at high spatial and temporal resolution. Intracranial structures are exposed through a cranial window, and physiologic conditions are maintained during extended imaging sessions via continuous superfusion of the brain surface with artificial cerebrospinal fluid (aCSF). Experiments typically require 1-2 h of preparation, which is followed by variable periods of immune cell tracking. Our methodology converges the experience of two laboratories over the past 10 years in diseased animal models such as cerebral ischemia, lupus, cerebral malaria, and toxoplasmosis. We exemplify the utility of this protocol by tracking leukocytes in transgenic mice in the pial vessels under steady-state conditions.
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