Nitric Oxide Measurements by Fluorescent Microscopy
Arteriolar nitric oxide production can be measured by fluorescent microscopy technique using 4,5 diaminofluorescein (DAF-2) diacetate (DAF-2DA). DAF-2DA is cell permeable and hydrolyzed to DAF-2 by cytosolic enzymes. In the presence of nitric oxide and oxygen, the non-fluorescent DAF-2 is converted to highly fluorescent DAF-2 triazole (DAF-2T). The slope of the increase in fluorescence over time is directly proportional to vascular NO levels. Prior to experiments, vessels are loaded with DAF-2DA. For imaging, the microvessel chamber is mounted on a stage of an inverted microscope fitted with 485 nm excitation and 530 nm emission filters, and a Coolsnap monochrome camera, and automated shutters for time-lapse imaging. The camera is connected to a personal computer equipped with video dimensioning software (Nikon Elements). With this setup a magnified image of the arteriolar segment is performed on the computer monitor, and fluorescent images are collected for each condition and the slope of the increase in fluorescent signals is used as an index of vascular NO production. Figure shows representative images demonstrating successful DAF-2DA loading and measurements of arteriolar diameter responses. This setup enables simultaneous measurements of NO production and vascular diameter.
A: Bright-light, B: autofluorescence before DAF-2DA loading, C: baseline after DAF-2DA loading, D: vasodilation and increase in signal by nitric oxide donor