HPTS is a non-toxic, water-soluble fluorescent pyrene derivative bearing three sulfonate groups at positions 1, 3, and 6 and a hydroxyl group at position 8 (Figure ). HPTS has been employed as a dye and chemosensor, as a tool to develop environmental sensors, to monitor protein structural transitions, and as a fluorometric pH indicator. HPTS is sensitive to pH changes from the protonation/deprotonation of the hydroxyl group and has a pKa of ~7.3 (although this depends on the experimental conditions and will be discussed in the calibration section). This pKa value is close to physiological pH, making HPTS an ideal candidate to monitor pH variations in biological media. HPTS has been used to measure intracellular pH and, as indicated above, intravesicular pH. Absorption wavelengths of the protonated and deprotonated forms of this probe are different, but they both emit at 510 nm. If the sample is excited at 460 nm (the excitation wavelength of the deprotonated form) and at excitations, it is possible to determine the I460/I403 ratio (Figure , Equation (1)). Also, if there is a variation of the I460/I403 quotient occurring with time, it means that the intravesicular pH is also changing; if it remains constant, the inner pH is not disturbed.