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Updated: Jul 6, 2026
Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice
Published on: August 23, 2007
Experimental induction and three-dimensional two-photon imaging of conjunctiva-associated lymphoid tissue
Philipp Steven1, Jan Rupp, Gereon Hüttmann
1Eye-Hospital, University of Lübeck, Lübeck, Germany. psteven@gmx.de
Insights
A new mouse model was developed to study conjunctiva-associated lymphoid tissue (CALT) function. This model, using topical stimulation and two-photon microscopy, allows for detailed 3-D imaging of immune responses on the ocular surface.
Area of Science:
- Ocular immunology
- Immunological research techniques
- Animal modeling
Background:
- Conjunctiva-associated lymphoid tissue (CALT) is crucial for ocular surface adaptive immunity.
- Functional studies of CALT have been limited due to a lack of appropriate models.
- Investigating CALT's role in adaptive immunity requires advanced imaging techniques.
Purpose of the Study:
- To establish a novel animal model for functional analysis of CALT.
- To evaluate two-photon microscopy for in vivo examination of CALT immunologic interactions.
- To enable three-dimensional (3-D) and four-dimensional (4-D) imaging of CALT immune mechanisms.
Main Methods:
- BALB/c mice conjunctiva were repeatedly challenged with topical *Chlamydia trachomatis* or ovalbumin/cholera toxin B.
- Explanted, unfixed, and unstained eyes with nictitating membranes were analyzed.
- Two-photon microscopy was utilized for high-resolution 3-D imaging of CALT components.
Main Results:
- CALT was successfully generated in the nictitating membrane in 70-73% of animals after stimulation.
- CALT comprised B cells (CD45R/B220+), T cells (CD4+, CD8+), dendritic cells, and macrophages.
- Two-photon microscopy enabled detailed 3-D visualization of CALT structures up to 65 micrometers deep.
Conclusions:
- A reliable mouse model for CALT functional studies was established.
- Topical stimulation effectively induces CALT in the nictitating membrane.
- Two-photon microscopy provides unprecedented capabilities for 3-D and future 4-D intravital imaging of CALT.
Purpose:
Conjunctiva-associated lymphoid tissue (CALT) is assumed to be a key location for the generation of adaptive immune mechanisms of the ocular surface, but functional studies of CALT are still lacking. The purpose of this study was to establish an animal model that enables functional analysis of immune mechanisms going on within CALT. In addition, the use of two-photon microscopy, a new optical method, was evaluated for examining complex immunologic interactions of CALT by volume (three-dimensional [3-D]) and time-dependence (four-dimensional [4-D]) in vivo.
Methods:
The conjunctiva of female BALB/c mice was repeatedly challenged with topical Chlamydia trachomatis serovar C or a solution of ovalbumin and cholera toxin B. Two-photon microscopy was conducted on explanted, unfixed, and unstained eyes with adjacent nictitating membranes.
Results:
After three to five stimulations, CALT was detected exclusively in the nictitating membrane of 73% (C. trachomatis) or 70% (ovalbumin/ cholera toxin) of the animals. CALT mainly consisted of CD45R/B220+ B cells and CD4+ and CD8+ T cells. Electron microscopy showed intraepithelial lymphocytes and follicles consisting of lymphocytes, dendritic cells, and macrophages. Two-photon microscopy based on tissue autofluorescence allowed all components of CALT to be detected three dimensionally. High-resolution images were generated in tissue depths of 65 microm below the mucosal surface.
Conclusions:
This study introduces a novel mouse model for functional investigations of CALT. Topical stimulation with C. trachomatis or ovalbumin/cholera toxin B reliably leads to CALT generation at the nictitating membrane. The use of two-photon microscopy enables groundbreaking 3-D and, in the future, intravital 4-D investigations of immunologic processes initiated in CALT.
