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Updated: Jun 28, 2026
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Published on: August 23, 2007
Dissection and 2-photon imaging of peripheral lymph nodes in mice
Melanie P Matheu1, Ian Parker, Michael D Cahalan
1Department of Physiology and Biophysics, University of California, Irvine, CA, USA.
Insights
This study details methods for tracking T cell movement in lymph nodes using two-photon imaging. It demonstrates adoptive transfer and explanted lymph node preparation for observing CD4+ T cell motility.
Area of Science:
- Immunology
- Cell Biology
- Microscopy
Background:
- Lymph node cellular dynamics are crucial for immune responses.
- Two-photon microscopy enables in vivo visualization of cellular interactions.
- Understanding T cell motility is key to immune response initiation.
Purpose of the Study:
- To present detailed methods for imaging T cell motility in explanted lymph nodes.
- To demonstrate adoptive transfer and lymph node preparation techniques for two-photon imaging.
- To visualize naive CD4+ T cell behavior in a controlled ex vivo environment.
Main Methods:
- Adoptive transfer of fluorescently labeled T cells into recipient mice.
- Isolation and mounting of explanted lymph nodes for imaging.
- Two-photon microscopy with optimized conditions (oxygenation, temperature, laser power).
Main Results:
- Successful homing of transferred T cells to lymphoid organs within 15-30 minutes.
- Demonstration of explanted lymph node preparation for high-resolution imaging.
- Acquisition of 3D video images showing steady-state motility of CD4+ T cells at 37°C.
Conclusions:
- The presented methods facilitate the study of T cell migration and behavior within lymph nodes.
- Ex vivo two-photon imaging provides valuable insights into immune cell dynamics.
- This technique is essential for understanding immune responses at the cellular level.
Abstract:
Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response (1). Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 (2). Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37 degrees C.
