Compound 2 displayed significant fluorescence intensity starting from the peripheral region 4 and increasing toward the center of the spheroid -region 1. Region 1 exhibited a 3.7-fold increase in fluorescence intensity compared to the peripheral region 6 (Figure ). As compound 3 was seen to have a turn on response at <2% O2, to obtain a larger hypoxic core and increased likelihood of a visible signal, spheroids were grown to a diameter of ~700 µm in diameter Compound 3 displayed a less linear increase in fluorescence toward the center of the spheroids, but a 1.5-fold increase was measured in region 1 compared to the peripheral region of the spheroid 6 (Figure ). As compound 4 was seen to have a turn on response at the most severe levels of hypoxia, to obtain a larger hypoxic core and increased likelihood of a visible signal, spheroids were grown to a diameter of ~700 µm in diameter. Compound 4 showed a steady increase in fluorescence intensity toward the center of the spheroidregion 1, with a maximum 1.7-fold increase observed in the central region of the spheroid (Figure ). These data demonstrate compounds 2, 3, and 4 can detect regions of hypoxia within spheroids, with compounds 2 and 4 able to image different levels of hypoxia in this setting. Compound 2 can detect milder hypoxia, similar to that imaged by pimonidazole, while compound 4 detects only extreme hypoxia as is also the case for EF5. Having established the use of compound 2 and 4 to image different levels of oxygen in both 2D and 3D cell culture systems individually, the probes were used in combination on spheroids to further demonstrate their ability to measure different levels of oxygen. Spheroids were grown to an average diameter of 600 µm. For reference staining with pimonidazole was also carried out (Figure ). Pimonidazole staining of HCT116 spheroids shows a large central region of hypoxia, suggesting in this model, regions of <2% O2 are not restricted to a region within the core of the spheroid and extend far out into the peripheral regions (Figure ). Compound 2 displayed significant fluorescence intensity starting from the peripheral region 5 and increasing toward the center of the spheroid -region 1. Compound 4 showed a steady increase in fluorescence intensity starting from the peripheral region 3 toward the center of the spheroid -region 1 (Figure ).