Simultaneous nitric oxide and dehydroascorbic acid imaging by combining diaminofluoresceins and diaminorhodamines

Spatial measurements of nitric oxide (NO) production are important to understand the function and metabolism of this molecule. The reagent, 4,5-diaminofluorescein (DAF-2) and several structurally similar probes are widely used for detection and imaging of NO. However, DAF-2 also reacts with dehydroascorbic acid (DHA) in biological samples, with both products having nearly indistinguishable fluorescence spectra. Measurements using fluorimetry and fluorescence microscopy cannot easily differentiate NO-related fluorescent signals from DHA-related signals. While DAFs and the structurally related diaminorhodamines (DARs) both react with NO and DHA, they do so to different extents. We report a multiderivatization method to image NO and DHA simultaneously by using both DAF and DAR. Specifically, DAF-2 and DAR-4M are used to image NO and DHA concentrations; after reaction, the solutions are excited, at 495 nm to measure fluorescence emission from DAF-2, and at 560 nm to measure fluorescence emission from DAR-4M. Using the appropriate calibrations, images are created that depend either on the relative NO or the relative DHA concentration, even though each probe reacts to both compounds. The method has been validated by imaging NO production in both undifferentiated and differentiated pheochromocytoma (PC12) cells.