Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2): implications for cellular NO imaging

FEBS Lett. 2001 Mar 2;491(3):227-32. doi: 10.1016/s0014-5793(01)02206-2.


The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Artifacts
  • Calcium / metabolism*
  • Calcium / pharmacology
  • Cations, Divalent / pharmacology
  • Cell Line
  • Dose-Response Relationship, Drug
  • Dose-Response Relationship, Radiation
  • Fluorescein / chemistry*
  • Fluorescein / metabolism*
  • Fluorescence
  • Fluorometry / methods
  • Humans
  • Intracellular Fluid / drug effects
  • Intracellular Fluid / metabolism
  • Intracellular Fluid / radiation effects
  • Isotonic Solutions / metabolism
  • Kidney / cytology
  • Kidney / drug effects
  • Kidney / metabolism
  • Kidney / radiation effects
  • Light
  • Nitric Oxide / analysis*
  • Nitric Oxide / metabolism
  • Nitric Oxide Donors / pharmacology
  • Photochemistry / methods*
  • Reproducibility of Results
  • Ringer's Solution
  • Sensitivity and Specificity


  • 4,5-diaminofluorescein
  • 4,5-diaminofluorescein diacetate
  • Cations, Divalent
  • Isotonic Solutions
  • Nitric Oxide Donors
  • Nitric Oxide
  • Ringer's Solution
  • Calcium
  • Fluorescein