Calcium-imaging with Fura-2 in isolated cerebral microvessels

Cerebral microvessels were isolated from rat hippocampus with a modified, mild collagenase digestion and loaded with the calcium fluoroprobe, Fura-2. Using a fluorescence microscope with quartz optics and an image analyser, it was possible to measure the intracellular concentration of free calcium ions [Ca2+]i in single microvessels for the first time. A resting level of (90 +/- 28) nmol/l (+/- SD, n = 24) was calculated which immediately rose after ionomycin application. The temporal resolution for [Ca2+]i of our set-up was 0.7 s. By image processing, sequences of digitized fluorescence images of single microvessels were colour-coded in terms of [Ca2+]i with a spatial resolution up to 1.5 microns (pixel size). Both the temporal and spatial resolution make our system suitable for investigation of calcium-mobilizing receptors of the blood-brain barrier.