Representative tumour data from a hyperpolarized 13C measurement (coloured) in an axial slice overlayed on anatomical proton images (greyscale). A calibration phantom containing 13C urea and the catheter used for injection are visible. Both (a) ZA and (b) urea accumulate in the MAT B III tumour. (c) The mean pH map shows a lower pH value in the tumour compared to the surrounding tissue. For all 13C images, intensity windows are based on sufficiently high signal levels for either (intensity images) or both (pH images) ZA and urea. Spectra from tumour voxels (d, shown for one representative animal) are best fitted with two pairs of ZA peaks (red, blue) and consistently show different pH values in the tumour in comparison to the vena cava, demonstrated in five animals (e, individual data points and mean±s.d.) and compared with three other interstitial/extracellular pH measurements in four animals, namely 31P MRS in vivo using 3-APP, a needle-type optical sensor in vivo and an ex vivo tissue electrode pH measurement. Scale bars, 1 cm.