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Measurement of NO using microscopic imaging of DAF‐2T fluorescence
Author(s) -
Carvalho Helena,
Sellergren Andrew,
Murray LaTisha C,
Pittman Roland N
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a698-a
Subject(s) - fluorescence , fluorescence lifetime imaging microscopy , biophysics , chemistry , biomedical engineering , materials science , optics , physics , biology , medicine
Nitric oxide (NO) is an important molecule with multiple physiologic functions (e.g., control of vasomotor responses and mitochondrial respiration). Previously, direct, local measurements of NO concentration, [NO], in the microcirculation have been carried out using microsensors. Extending these measurements to larger spatial regions, by use of a widely dispersed fluorescent indicator, would aid in understanding NO’s diverse functions. The measurement of [NO] in vivo should have good spatial (μm’s) and temporal (10’s of sec or better) resolution in order to quantify the changes in [NO] that accompany various physiologic stimuli. In recent years visual detection of NO has been reported using the fluorescent indicator 4,5‐diaminofluorescein (DAF‐2) in vitro, in cultured cells and in vivo. After reaction with an auto‐oxidation product of NO, DAF‐2 is converted into the highly fluorescent DAF‐2T. We have carried out in vitro experiments in which [NO] was related to the rate of change of DAF‐2T fluorescence. Solutions containing 10 μM DAF‐2 were mixed with solutions containing a range of [NO] (100 – 1500 nM). Different NO concentrations were produced by using either dissolved NO gas or by adding sodium nitrite to an acidified solution in the presence of potassium iodide. Sequences of fluorescence images (at 10‐sec intervals over 300 sec) were acquired with a digital camera in conjunction with measurements using a NO microsensor. Over the above range of [NO], we found that the rate of change of DAF‐2T fluorescence was proportional to the square of [NO], as predicted by a model that incorporated the reaction kinetics of NO and DAF‐2 in the presence of oxygen. These results will be used as a basis to extend the measurement of [NO] to microcirculatory preparations.