break down other substances, they require high internal acidity in order to successfully perform their intended function. In contrast to the relatively low pH inside lysosomes and phagocytes, the mitochondrial matrix has an internal pH of around 8.0, which is approximately 0.9 pH units higher than that of inside intermembrane space. Since oxidative phosphorylation must occur inside the mitochondria, this pH discrepancy is necessary to create a gradient across the membrane. This membrane potential is ultimately what allows for the mitochondria to generate large quantities of ATP. Measurement There are several common ways in which intracellular pH (pHi) can be measured including with a microelectrode, dye that is sensitive to pH, or with nuclear magnetic resonance techniques. For measuring pH inside of organelles, a technique utilizing pH-sensitive green fluorescent proteins (GFPs) may be used. Overall, all three methods have their own advantages and disadvantages. Using dyes is perhaps the easiest and fairly precise, while NMR presents the challenge of being relatively less precise. Furthermore, using a microelectrode may be challenging in situations where the cells are too small, or the intactness of the cell membrane should remain undisturbed. GFPs are unique in that they provide a noninvasive way of determining pH inside different organelles, yet this method is not the most quantitatively precise way of determining pH. Microelectrode The microelectrode method for measuring pHi consists of placing a very small electrode into the cell’s cytosol by making a very small hole in the plasma membrane of the cell. Since the microelectrode has fluid with a high H+ concentration inside, relative to the outside of the electrode, there is a potential created due to the pH discrepancy between the inside and outside of the electrode. From this voltage difference, and a predetermined pH for the fluid inside the electrode, one can determine the intracellular pH (pHi) of the cell of interest. Fluorescence spectroscopy Another way to measure Intracellular pH (pHi) is with dyes that are sensitive to pH, and fluoresce differently at various pH values. This technique, which makes use of fluorescence spectroscopy, consists of adding this special dye to the cytosol of a cell. By exciting the dye in the cell with energy from light, and measuring the wavelength of light released by the photon as it returns to its native energy state, one can determine the type of dye present, and relate that to the intracellular pH of the