Error analysis of ratiometric imaging of extracellular pH in a window chamber model

Ratiometric fluorescence-imaging technique is commonly used to measure extracellular pH in tumors and surrounding tissue within a dorsal skin-fold window chamber. Using a pH-sensitive fluorophore such as carboxy SNARF-1 one can measure pH distributions with high precision. However, it is often observed that the measured pH is lower than expected, with a bias that varies from one image to another. A comprehensive analysis of possible error sources is presented. These error sources include photon noise, estimator bias, instrument errors, temperature, and calibration errors from biological factors.