Calibration of a fluorescent membrane for use in mapping microvascular nitric oxide (NO) gradients

Abstract

The concentration of NO, a key mediator of microvascular oxygen (O2) delivery both through its influence on vascular tone and mitochondrial O2 consumption, has been measured locally around microvessels using microelectrodes. To extend these measurements beyond in vivo point‐sampling, we have developed a wide‐field, spatio‐temporal sensor using ~3 μm silica gel microspheres that are coated with an NO‐sensitive fluorescent indicator (DAF‐2) and embedded in a 10 μm thick layer of Nafion. To provide for accurate quantification, the membrane is undergoing in vitro calibration tests using a commercially available flow chamber with either bolus or continuous (20 μl/s) perfusion of solutions containing known [NO]'s. Upon taking a baseline series of images using low‐light epi‐illumination—to minimize photobleaching—along with image intensification, the NO perfusate is added without image time‐course interruption. DAF‐2's conversion to a highly fluorescent form is dependent on both [NO] & [O2], and thus with [O2] accounted for, the rate of rise of fluorescence can be linked to a specific [NO]. Over time, the bead field is expected to develop like a photograph when exposed to the heterogeneous distribution of [NO] in vivo, yielding quantifiable information regarding NO dynamics in the microcirculation. Supported by NIH Grants HLI8292&HL79087