Images were acquired using a Nikon Eclipse E600 microscope with a C1 confocal module (Nikon Instruments, Melville, NY). In order to image the whole window chamber, a Nikon 1X/0.04NA CFI Plan UW objective was used. The pinhole of the confocal microscope was opened as far as possible to a diameter of 150 μm (corresponding to 75 μm in object space) to maximize the photon-collection efficiency. With the objective lens and pinhole setting above, the system was expected to have a lateral resolution of 9 μm and an axial section thickness of approximately 2 mm. Scattering will lead to an even smaller section thickness at the surface. There is potential for partial volume effects (mixing of cancerous and noncancerous tissue in the section) and the results represent the average pH within the measured volume. GFP images were captured with Ar-ion-laser (488 nm, 250 μW) excitation and a bandpass (channel 1) emission filter. Carboxy SNARF-1 was excited using a green He-Ne laser (543 nm, 240 μW). To collect the emitted fluorescence from SNARF-1, two emission filters, bandpass (channel 2) and 640 longpass (channel 3), were used. The mouse was placed on a custom holder, which was mounted on the microscope stage. Isoflurane was used to anaesthetize the animal during the imaging experiment. The GFP signal from channel 1 and background fluorescence from channel 2 and 3, and , were recorded before the injection of carboxy SNARF-1. 200 μl of 1-mM carboxy-SNARF-1 solution in phosphate-buffered saline (PBS) was injected via a tail-vein catheter. The emission signals from channel 2 and 3, and , increased slowly over a period of about 30 min following the injection. Images with a pixel resolution of over a field of view of were collected at 5, 15, and 30 min after injection. The PMT gains for the three channels were set to fixed values of 60, 90, and 110, respectively, to ensure consistency in all experiments. The dwell time per pixel was 6.96 μs, yielding a single-image collection time of 2.82 sec. Due to the low photon-emission rate from the sample, 10 images were taken successively and averaged to improve the signal-to-noise ratio (SNR) at each data-collection time point. There was no noticeable decay in signal level observed due to photobleaching at the exposures used. The imaging parameters described above were applied to both calibration and imaging processes.