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

The concentration of NO, a key mediator of microvascular oxygen (O 2 ) delivery both through its influence on vascular tone and mitochondrial O 2 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] & [O 2 ], and thus with [O 2 ] 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