Immunology JoVE (Open Access) Citable · DOI

Intravital Imaging of the Mouse Popliteal Lymph Node

Name: Intravital Imaging of the Mouse Popliteal Lymph Node
Uploaded: 2026-08-05
Duration: 8 min 41 s
Description: Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

DOI: 10.3791/3720-v

What you'll learn

✓ Prepare mouse and imaging apparatus for intravital lymph node microscopy
✓ Perform surgical exposure of the popliteal lymph node
✓ Acquire and process 2-photon microscopy images of live lymphocytes
✓ Analyze lymphocyte migration dynamics in real-time imaging data

Protocol

Biopharma Insights Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Difficulty

advanced

Total time

~2–3 hours per mouse (including surgery, imaging acquisition, and post-acquisition analysis)

Model organism

Mouse (C57BL/6 or transgenic GFP+ lymphocyte line)

Biosafety

BSL-1

Steps

Assemble mouse holder for imaging stage

Construct and secure the mouse holder apparatus that will position the anesthetized animal on the microscope stage. This ensures stable positioning of the popliteal lymph node during image acquisition.

▶ 01:13

Prepare mouse and induce anesthesia

Anesthetize the mouse using standard inhalational or injectable anesthetic protocols and position the animal in the prepared holder. Maintain body temperature and physiologic stability throughout the procedure.

▶ 01:58

Surgically expose popliteal lymph node

Perform surgical incision and dissection to expose the popliteal lymph node in the hind limb while maintaining sterile technique and hemostasis. Position the lymph node for direct optical access to the microscope objective.

▶ 03:27

Acquire 2-photon microscopy image stacks

Configure the 2-photon microscope for GFP and structural imaging, set appropriate laser power and detector gain, and capture time-lapse image series of the exposed lymph node.

▶ 04:55

Analyze lymphocyte migration in image sequences

Process acquired image stacks using image analysis software to track GFP+ lymphocyte positions across time frames and calculate migration velocities and patterns within the lymph node microenvironment.

▶ 05:55

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