Before evaluating the bioreduction of the probes in cells we determined that none of the probes 1-4 nor their parent fluorophores 5-7 were toxic at concentrations of up to 40 µM in an MTT assay conducted in A549 lung cancer cells over 24 h (Figure ). We also showed that the parent fluorophores (5-7) could be observed, using confocal microscopy, in A549 cells at concentrations ranging from 10-40 µM (Figure ). To assess the cellular bioreduction of probes 2-4, A549 cells were treated with the probe (40 µM) at oxygen concentrations of 21% O2, 4%, 2% 1%, 0.5% or <0.1% O2 for 2 hours. All compounds showed a hypoxia-dependent increase in fluorescence, with maximum fluorescence observed at <0.1% O2. However, the oxygen level at which fluorescence was initially detected differs between the compounds and correlates with our cyclic voltammetry and enzyme assay data (vide supra). Compound 4 displayed increasing fluorescence intensity starting at 1% O2, with a ~1.8-fold increase in intensity when exposed to 0.5% O2 (p≤0.05), and a maximal 3-fold increase when exposed to <0.1% O2 (p<0.001) compared to normoxic controls. No significant increase in fluorescent intensity was observed at the higher oxygen concentrations (2, 4 % O2) tested (Figure ). Compound 3 displayed a significant increase in fluorescent intensity starting at 2% O2 (p<0.5), and at 1% (p<0.001), 0.5% (p<0.001) and <0.1% O2 (p<0.001). Maximum intensity was observed at <0.1% O2, with a 3.5-fold increase in fluorescence compared to the normoxic control observed (Figure ). A 5-fold increase in fluorescence intensity was observed when compound 2 was exposed to 4% O2, the fluorescence increase was similar at 2% O2, but increased further, with a 10-fold increase at 1% O2 (p<0.01), a 15-fold increase at 0.5% O2 (p<0.001) and a 30-fold increase when exposed to <0.1% O2 (p<0.001) compared to normoxic controls (Figure ). These data confirm that compound 2 releases resorufin at higher oxygen concentrations than compounds 4 releases Me-Tokyo green, while compound 3 has intermediate oxygen sensitivity.