Given the unexpected behavior of compound 1 in the enzyme assay, we wanted to investigate whether compounds 1 and the DDAO fluorophore were bioreduced in a cellular setting. A549 cells were incubated with compound 1 (40 µM) and exposed to <0.1% O2 for 2 hours. Then, cells were either fixed inside the hypoxia chamber, or exposed to 5, 10, 20, 30, or 60 mins reoxygenation to 21% O2. Minimal fluorescent intensity was observed when cells were fixed inside the hypoxia chamber or after 5 mins reoxygenation. In contrast, maximal fluorescence intensity was reached following reoxygenation for just 10 mins, with a reduction in fluorescence intensity observed after this time. This observation indicates that DDAO is reduced in cells at <0.1% O2, but as in the enzyme assay, is rapidly re-oxidized upon exposure to 21% O2 (Figure ). Consequently, a 10 min of reoxygenation step was incorporated into experiments to assess the hypoxia-dependent reduction of compound 1 in cells. A549 cells were treated with compound 1 (40 µM) exposed to 8%, 6%, 4%, 2%, or <0.1% O2 for 2 hours, followed by fixing inside the hypoxia chamber or exposure to 10 minutes reoxygenation at 21% O2 followed by fixation. Like the other probes, compound 1 showed maximum fluorescence intensity at <0.1% O2, with a 6.7-fold increase in fluorescence observed. A 2.5-fold fluorescence increase was observed at 8% O2, indicating that the lower pKa value of DDAO enables this probe to image very mild levels of hypoxia (Figure ). However, the need for a reoxygenation step means that, while this compound can be used in a cellular setting, it is not suitable for application to more complex systems such as spheroids. Having established the suitability of compounds 2, 3, and 4 to measure different levels of oxygen in a 2D cellular setting, we investigated the ability of these probes to measure oxygen levels in the more complex and physiologically relevant spheroid 3D cell model. HCT116 colorectal cancer cells were chosen for the spheroid model due to their propensity to form spheroids. The presence of hypoxic cores in HCT116 spheroids were confirmed using positive pimonidazole and EF5 staining (Figure ). To investigate the bioreduction of compounds in this setting, spheroids were incubated with compounds 2, 3, or 4 (40 µM) for 1.5 hours. This length of incubation was chosen as it is comparable to the time required to visualize a pimonidazole signal (Figure ). To calculate the mean fluorescence intensity at different depths within the spheroid, CellProfiler was used to identify individual cells and determine their distance from the center of the spheroid. Region 1 represents the central core of the spheroid, and region 6 represents the periphery of the spheroid (Figure ). As compound 2 was seen to have a turn on response at <4% O2, spheroids were grown to an average size of 300 µm in diameter.