Molecular mapping deep within a living human organ: analysis of microvessel function on the timescale of seconds and with sub-micrometre spatial resolution.

Visualising vascular endothelial cell function in individual blood microvessels allows elucidation of molecular interactions at the vascular wall, the first barrier between blood-borne therapeutic agent and its target. Functional analysis in situ requires sub-micrometer spatial resolution and tagged molecules generating contrast in living blood vessels. Light microscopy fulfills these requirements, particularly if fluorescent tags deliver the contrast. However, vascular arborisations in living organs defy morpho-functional analysis, filling tissues with closely meshed three-dimensional networks which are inaccessible to optical imaging. We protocol here successful morpho-functional analysis of microvascular processing in a living organ, the human placental cotyledon. Fluorescence-tagged tracer was positionally fixed by snap-freezing, frozen sections were cut, freeze-dried and heat-fixed. A brief histochemical procedure then labelled all vascular elements in the sections, providing fluorescence contrast in two colour channels. Mosaic monochromatic images acquired in both channels delivered high-resolution maps of centimeter-wide tissue areas. Quantitative analysis of the images' greyscale histograms defined objectifiable, reproducible thresholds, used to reduce the images to colour-coded wide-area functional maps tracking placental vascular processing of the tagged molecules. Rapid positional fixing of tracer with reduction of images to maps was combined with ultrastructural tracking to elucidate vascular processing at scales of nanometres and seconds.