High accuracy 4D cell tracking into explanted skin using two-photon excitation microscopy

Area of Science:

• Immunology
• Microscopy
• Bioimaging

Background:

• Two-photon excitation microscopy (2PEM) is crucial for analyzing large explanted organs.
• Tissue movement during imaging causes lateral and axial drifts, complicating T cell tracking.
• Existing methods for drift correction are often laborious and less accurate.

Purpose of the Study:

• To develop and validate a robust method for accurate T cell tracking in explanted murine skin using 2PEM.
• To minimize sample drift during extended imaging sessions.
• To provide a free, efficient alternative to commercial software for drift correction.

Main Methods:

• Compared various explanted skin mounting techniques to identify optimal setups for minimizing drift.
• Developed ImageJ-based algorithms for lateral and axial drift correction using collagen network registration.
• Applied the developed macro to track fluorescent T cells in explanted skin samples.

Main Results:

• The developed two-step approach significantly improved the accuracy of T cell tracking.
• The ImageJ macro demonstrated superior efficiency in shift correction compared to existing software.
• Velocity calculations for T cells were more accurate using the new method.

Conclusions:

• The novel ImageJ macro provides a practical and efficient solution for drift correction in 2PEM of explanted skin.
• This approach enhances the reliability of T cell migration studies in skin imaging.
• The free availability of this tool facilitates broader adoption in research.