Related Experiment Video
Updated: May 28, 2026
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Published on: August 23, 2007
Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels
Philipp Steven1, Felix Bock, Gereon Hüttmann
1Department of Ophthalmology, University of Lübeck, Lübeck, Germany. philipp.steven@uk-koeln.de
Insights
This study visualizes immune cell migration into lymphatic vessels in real-time using advanced microscopy. This provides new insights into immune cell transport and lymphatic vessel interactions in vivo.
Area of Science:
- Immunology
- Vascular Biology
- Microscopy
Background:
- Lymphatic vessels play crucial roles in transplantation and cancer metastasis.
- Understanding immune cell interactions with lymphatics is vital for disease research.
Purpose of the Study:
- To develop and apply a novel non-invasive method for visualizing immune cell transmigration into lymphatic vessels in vivo.
- To investigate immune cell migration dynamics within corneal lymphatic vessels.
Main Methods:
- Developed a non-invasive two-photon microscopy (TPM) technique.
- Utilized a mouse model of suture-induced corneal neovascularization.
- Employed intravital staining of lymphatic vessels with LYVE-1 antibody and autofluorescence imaging.
Main Results:
- Achieved simultaneous visualization of lymphatic vessels and immune cells in vivo.
- Observed and tracked immune cell migration into lymphatic vessels in real-time.
- Quantified immune cell immigration times and velocities within corneal lymphatics.
Conclusions:
- Demonstrated the first in vivo evidence of real-time immune cell transmigration into lymphatic vessels.
- Highlighted the utility of intravital autofluorescence TPM for studying immune cell-lymphatic interactions.
- Provided a powerful model for investigating immune and tumor cell interactions with lymphatic vessels under physiological conditions.
Background:
The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.
Methodology/Principal Findings:
We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.
Conclusions:
To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.
