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Updated: Jun 12, 2026
A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva
Published on: May 17, 2020
Intravital multidimensional real-time imaging of the conjunctival immune system
Insights
Visualizing immune cell migration in the conjunctiva requires advanced microscopy. Two-photon microscopy offers deep tissue imaging and detailed cellular analysis, enhancing diagnostics for ocular surface inflammation.
Area of Science:
- Ophthalmology
- Immunology
- Microscopy
Background:
- The conjunctiva relies on immune cell migration for immune responses.
- Direct visualization of these cellular dynamics is limited by technical constraints.
- Investigating cellular migration requires intravital, 3D, time-resolved imaging with tissue characterization.
Purpose of the Study:
- To explore advanced microscopy techniques for visualizing conjunctival immune cell dynamics.
- To assess the potential of two-photon microscopy for studying ocular surface inflammation.
Main Methods:
- Confocal laser scanning microscopy for superficial imaging and rapid migration.
- Intravital two-photon microscopy for deep tissue analysis, cell tracking, and structural characterization using autofluorescence, fluorescence lifetime, and second harmonic generation.
- Investigating cellular migration in three spatial dimensions over time.
Main Results:
- Confocal microscopy enables high-resolution imaging of superficial immune cell migration.
- Two-photon microscopy allows detailed tracking and characterization of individual cells and structures in deep conjunctival tissue.
- Autofluorescence, fluorescence lifetime, and second harmonic generation provide unique optical features for analysis.
Conclusions:
- Two-photon microscopy is a powerful tool for basic research and clinical practice in ocular surface studies.
- This technique can significantly improve the diagnosis and analysis of inflammatory processes.
- Detailed investigation of common diseases like allergy and dry eye, and their treatment progress, is feasible.
Abstract:
The conjunctiva, as a peripheral mucosal surface, is dependent on the migration of immune cells to facilitate an orchestrated immune response. So far, only limited data to visualize these dynamics directly have been obtained, mainly due to technical and experimental restrictions. To investigate migration on a cellular level, the following conditions need to be met: (1) intravital investigations need to be facilitated by suitable microscopic techniques; (2) tissues need to be investigated in three spatial dimensions and over time; (3) data need to contain detailed information about the tissue character. Whereas the use of confocal laser scanning microscopy allows high-resolution imaging of the superficial conjunctival immune system and enables the recording of rapid cellular migration, intravital two-photon microscopy further enables tracking of individual cells and characterization of cells and structures with unique optical features using autofluorescence detection, fluorescence lifetime measurements and second harmonic generation in deep tissue. Based on current results and experimental studies, two-photon microscopy has the potential for general use in basic research and clinical practice, and would greatly enhance possibilities for diagnosing and analyzing inflammatory processes of the ocular surface. In particular, inflammation in common diseases, such as allergy and dry eye, and its progress under treatment could be investigated in detail.
