Recently, the reconstruction of microvascular walls has been used to determine the nature of endothelial openings or gaps induced by various permeability-raising stimuli (Michel & Neal, 1999). Attempts to assess the gap or pore density of the wall using randomly chosen electron micrographs of transverse sections of microvessel have proved successful when the openings were at a high density (Clough & Michel, 1988). The application of vascular endothelial growth factor (VEGF) to Rana mesenteric microvessels produces an increase in permeability and induces transcellular gaps (Michel & Neal, 1999). These gaps often occurred in clusters and were difficult to find with conventional electron microscopy of serial sections because the reconstructed area of the vessel wall was only a small percentage of the whole wall over which permeability was estimated.

We addressed this problem by cutting ribbons of ultrathin serial sections (100 nm thick) of mesenteries of frogs that were humanely killed and mounting them on Formvar coated glass slides. After staining with Toluidine Blue, sections were rapidly scanned using a light microscope for the presence of endothelial gaps (endothelial breaks within the section). Areas of interest were delineated and the Formvar film with attached sections was floated off the glass and placed on a slot grid for observation in the electron microscope.

From resin-embedded vessels treated with pressure or calcium ionophore where gaps were known to be present, 503 serial sections showed that endothelial gaps were resolvable under oil immersion using a X 100 objective. From three resin-embedded vessels treated with VEGF 1, 206 sections were scanned for the presence of gap clusters. Four areas of gaps were delineated along the combined 120 µm length of the three vessels. The Formvar coating and attached sections separated easily from the glass allowing confirmation of the structures in the electron microscope.

This technique allows the rapid scanning of vast areas of vessel wall for endothelial gaps that occurred due to the action of permeability raising stimuli.

This work was supported by The Wellcome Trust and British Heart Foundation.