Deep vascular imaging in wounds by two-photon fluorescence microscopy.

Area of Science:

• Biomedical Imaging
• Tissue Engineering
• Dermatology

Background:

• Deep tissue imaging at micrometer resolution is crucial for understanding biological processes.
• Two-photon fluorescence microscopy (2PFM) enables deep tissue interrogation of fluorophores.
• Integrins are cell-adhesion proteins found on angiogenic blood vessels, making them targets for imaging.

Purpose of the Study:

• To utilize a two-photon absorbing fluorescent probe targeting integrins to image vasculature and fibroblasts in mouse skin wound models.
• To visualize the 3D architecture of new blood vessels and fibroblast distribution within regenerating wound tissue.
• To explore the potential of this technique for analyzing whole-organ level biological events like angiogenesis.

Main Methods:

• Employing a two-photon absorbing fluorescent probe with integrin-targeting capabilities.
• Performing deep tissue imaging (up to ≈ 1600 μm) in mouse skin wound models using 2PFM.
• Reconstructing 3D images to analyze vascular plexus and fibroblast distribution.

Main Results:

• Successfully imaged new vasculature and fibroblasts up to approximately 1600 μm deep within mouse wound tissue.
• Revealed the three-dimensional (3D) architecture of the vascular plexus during wound regeneration.
• Identified a fibroblast bed surrounding the newly formed capillaries.

Conclusions:

• 2PFM with integrin-targeting probes allows for deep, high-resolution imaging of wound healing processes.
• The technique provides novel tools for 3D, whole-organ level analysis of angiogenesis and fibroblast presence.
• This approach has significant potential for biomedical applications in studying tissue regeneration.