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Spatio‐temporal mapping of nitric oxide gradients in the microvasculature via a fluorescent detector
Author(s) -
Nugent William,
Golub Aleksander S,
Pittman Roland N
Publication year - 2007
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.21.5.a484-a
Subject(s) - fluorescence , membrane , nafion , bead , microelectrode , distilled water , chemistry , substrate (aquarium) , fluorescence microscope , suspension (topology) , materials science , chromatography , analytical chemistry (journal) , chemical engineering , electrode , composite material , electrochemistry , biochemistry , optics , oceanography , mathematics , engineering , homotopy , pure mathematics , geology , physics
Nitric oxide (NO) plays a key role in microcirculatory oxygen transport via its effects on vascular tone and mitochondrial O 2 consumption. Localized microelectrode measurements have greatly advanced the description of NO's effects, but accurate spatio‐temporal quantification of its production and wide scale in vivo distribution would enhance our understanding. We developed a technique using ~3 μm silica gel microspheres coated with a fluorescent NO indicator (DAF‐2), embedded within a Nafion membrane. These non‐porous beads were first incubated for 15 min in 10 μM DAF‐2, washed three times, and suspended in distilled water. Nafion was then added to the suspension in a 65:35 Nafion to bead ratio. A precise amount of the mixture was then placed on a glass coverslip. Once the beads settled onto the glass surface, the assembly was cured at ~120 °C for 15 min. This yielded a 10 μm thick Nafion membrane encasing a NO sensitive monolayer of closely packed beads. In vitro experiments indicated that the bead fluorescence increases rapidly when exposed to a solution containing NO, showing that NO freely crosses the membrane as expected. Calibration involves exposure to solutions of different [NO] while measuring fluorescence rate of change. Application to a tissue using fluorescence microscopy should resolve not only the sources of NO production, but also yield accurate quantification of NO dynamics over a wide spatial domain. Supported by NIH Grant HL18292