SNARF is one of the most commonly used pH indicators for biological applications, owing to its unique fluorescent characteristics. For intracellular applications, esterase-substrate derivatives of SNARF, such as acetate or acetoxymethyl esters, have been employed previously as a generally accepted strategy to increase cell permeability. Unfortunately such cell-permeable SNARF derivatives retain significant fluorescence in aqueous solution, a property which results in a low signal-to-noise ratio. This in turn can lead to incorrect intracellullar pH measurements. Here we describe UTX-40, a newly designed SNARF derivative that successfully addresses these problems. In aqueous solution, UTX-40 is devoid of fluorescence before ester hydrolysis because it exists in aqueous environment as nano-scaled aggregates. As UTX-40 is converted into SNARF by enzymatic hydrolysis inside the cell, the aggregates become diffused and monomeric SNARF displays its characteristic fluorescent properties. The results of our studies reported in this communication demonstrate clearly the benefits of UTX-40 as an intracellular pH indicator. Since intracellular localized fluorescence was observed without cell-washing, the efficient uptake of intracellular fluorescence was confirmed. In addition, the actual intracellular pH and changes in intracellular pH caused by drug addition were monitored. The low background noise produced by UTX-40 is a property of this new pH probe that should be particularly advantageous for in vivo usage because, for this application, it is difficult to wash out the redundant probe
