Ratiometric imaging of tumor hypoxia after i.v. injection of the nanoprobe. A, Nude mice bearing HeLa tumor (6 mm) on the right rear thigh were i.v. injected with RHyLI (2 mg/kg) and imaged by ratiometric imaging over time. B, The tumor signal intensities in the 700 nm channel in A show the accumulation and retention of the nanoprobe in the tumor tissues over 7 days. C, Plot of the values of the mean ratio (the mean signal intensity of all the pixels in the tumor region) for the tumor in the ratiometric images in (A) versus time. D-I, Ratiometric imaging of hypoxia in tumors with indicated size or type. Nude mice bearing (D) HeLa tumors with a size of 2, 6, 10, or 16 mm (N = 3 for each group), or (E) HeLa, H460, or H1299 tumor with a size of 8 mm (N = 3 for each group) were i.v. injected with the nanoprobe (2 mg/kg) and imaged at 24 h p.i. F,G, The values of mean ratio of tumors in the ratiometric images. H,I, Partial oxygen pressure (PO2) in tumors in (D,E) measured by the needle-type oxygen microelectrode. J,K, Reversible response of the nanoprobe to hypoxia. Nude mice bearing HeLa tumor (2 mm) were i.v. injected with the nanoprobe (2 mg/kg). At 24 h p.i., the tumor tissue on the right thigh (red arrow) or the normal tissue on the left thigh (white arrow) was tied with a string to induce the temporal increase of hypoxia and then untied for the recovery of blood circulation. (J) Mice were monitored by ratiometric imaging and (K) tumor hypoxia in the ratiometric images was quantified by the values of mean ratio. Results are presented as Mean ± SD (N = 3). ** p < 0.01, when comparing the mean ratio values of the tied state with those of loosen states by one-way ANOVA with Geisser-Greenhouse correction. L, Correlation of the reciprocal of the mean ratio with the corresponding oxygen partial pressure measured by the oxygen microelectrode. The values of mean ratio and PO2 used for correlation are the measurement results of HeLa tumors at various tumor sizes by ratiometric imaging (F,G) and oxygen microelectrode (H,I).