Intravital Visualization of Gamma Delta Intraepithelial Lymphocytes

Gamma delta intraepithelial lymphocytes, γδ IELs, are specialized migratory T cells expressing gamma delta T cell receptors. These cells are predominantly found within the intestinal epithelium and patrol the epithelium to limit pathogen translocation across the intestine.

To visualize γδ IELs within a living organism, begin with an anesthetized transgenic mouse with a partially exposed small intestine. The transgenic mouse is genetically modified to express reporter proteins — enhanced green fluorescent proteins — exclusively in γδ IELs, enabling their tracking.

Using an electrocautery, create a perforation line over the exposed small intestine along the antimesenteric surface, preventing excessive bleeding.

Make a longitudinal incision over the perforation line to expose the intestinal mucosa, showing multiple finger-like projections — villi. Each villus contains epithelial cells with residing γδ IELs that protrude into the intestinal lumen.

Position the mouse with the exposed mucosal surface directly in contact with the fluorescent dye, imparting red fluorescence to the lumen.

Image the mouse under a confocal microscope. Locate a villus and image at different focal planes along the z-axis, starting from the villus tip epithelium. Combine these optical sections to produce a 3D image.

Intravital imaging shows the dynamic movement of green fluorescence γδ IELs along the epithelial cell's basement membrane near the red lumen.