title: The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells.
abstract: BACKGROUND
  Small molecule fluorescent probes are vital tools for monitoring reactive oxygen species in cells.
  
  
  METHODS
  The types of probe available, the extent to which they are specific or quantitative and complications in interpreting results are discussed.
  
  
  CONCLUSIONS
  Most commonly used probes (e.g. dihydrodichlorofluorescein, dihydrorhodamine) have some value in providing information on changes to the redox environment of the cell, but they are not specific for any one oxidant and the response is affected by numerous chemical interactions and not just increased oxidant generation. These probes generate the fluorescent end product by a free radical mechanism, and to react with hydrogen peroxide they require a metal catalyst. Probe radicals can react with oxygen, superoxide, and various antioxidant molecules, all of which influence the signal. Newer generation probes such as boronates act by a different mechanism in which nucleophilic attack by the oxidant on a blocking group releases masked fluorescence. Boronates react with hydrogen peroxide, peroxynitrite, hypochlorous acid and in some cases superoxide, so are selective but not specific. They react with hydrogen peroxide very slowly, and kinetic considerations raise questions about how the reaction could occur in cells.
  
  
  CONCLUSIONS
  Data from oxidant-sensitive fluorescent probes can provide some information on cellular redox activity but is widely misinterpreted. Recently developed non-redox probes show promise but are not generally available and more information on specificity and cellular reactions is needed. We do not yet have probes that can quantify cellular production of specific oxidants. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
authors.affiliation: Centre for Free Radical Research, Department of Pathology, University of Otago Christchurch, Christchurch, New Zealand. Electronic address: Christine.winterbourn@otago.ac.nz.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Winterbourn
mesh_terms: D000818:Animals; D002468:Cell Physiological Phenomena; D005456:Fluorescent Dyes; D006801:Humans; D017382:Reactive Oxygen Species / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species
keywords: Boronate probe; DCF; Dichlorofluorescein; Fluorescent probe; GFP; Hydrogen peroxide; Reactive oxygen species detection; SOD; Superoxide; dichlorofluorescein; green fluorescent protein; superoxide dismutase