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

Abstract

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 cells.

Nitric Oxide Sensors for Biological Applications

“…Chemosensors 2018, 6, x 5 of 13 when exposed to dehydroascorbic acid (DHA) and ascorbic acid (AA), which often co-localize with NO synthase, and a sensitivity to pH [83][84][85]. The Shear research group developed a new NO sensor in 2010, named NO550, that has a high specificity, does not react to co-localized proteins, and is not sensitive to pH differences, which is advantageous since various intracellular compartments have different pH values [86][87][88].…”

Profiling metabolites and peptides in single cells

“…To address this issue, either markers for the compounds or selective sensors can be positioned nearby the cell of interest before its lysis. 51 …”

“…51,60 It is feasible to design a system that utilizes both NMR spectroscopy and fluorescent probes to simultaneously follow the concentration changes of several metabolites of different abundances. There are a number of chemical imaging methods that allow metabolite detection in live cells, even inside an organism.…”

Review of recent advances in analytical techniques for the determination of neurotransmitters

“…DAFs often experience interference from dehydroascorbice acid and ascorbic acid. Sweedler and collegues demonstrated this interference could be avoided through the introduction of ascorbic acid oxidase,[288] by use of dual labeling with two fluorophores with different excitation wavelengths,[293] and by diffusive mixing of NO and the fluorophore in frozen blocks of sample and fluorophore, excluding interfering species which cannot diffuse through the solid phase to the fluorophore. [292] Yang et al .…”