The alkalization of intracellular pH (pH,) advances together with enhancement of aerobic glycolysis within tumor cells (the Warburg effect), and that is responsible for the progression of tumor malignancy together with hypoxia and angiogenesis. But how they correlate each other during tumor growth is poorly understood, partly due to the lack of suitable imaging methods. In present study, we propose a novel method to visually determine the pH(in), of tumor xenograft model from fluorescent image ratios. We utilized tandemly-linked two fluorescent proteins as a pH indicator; yellow fluorescent protein (YFP, pH sensitive) as an indicator, and red fluorescent protein (RFP, pH insensitive) as a reference. This method can eliminate the influence of optical factors from tissue as well as of the diverse expression level of pH indicator in the grafted cells. In addition, that can be operated by filter-based fluorescent imagers that are generally used in small animal study. The efficacy of the pH indicator, RFP-YFP, was confirmed by studies using recombinant protein in vitro and HeLa cells expressing RFP-YFP in vivo. Furthermore, we prepared nude mice subcutaneously xenografted HeLa cells expressing RFP-YFP cells as tumor model. The image ratios (YFP/RFP) of the tumor at the day 5 after surgery clearly showed the heterogeneous distribution of diverse pH in cells in the tumor tissue. Concomitantly acquired angiography using near-infrared fluorescence (680 nm for emission) also indicated that the relative alkaline pH in cells located in the region far from tumor vessels in which tumor aerobic glycolysis would be facilitated by progression of hypoxia and nutrient starvation. Applying the present method for a multi-wavelength imaging concerning pO(2) and/or nutrient starvation states in addition to pH(in) and angiogenesis would provide valuable information about complicated alteration of tumoral cell states during tumorigenesis. (C) 2015 Elsevier Inc. All rights reserved.