We proposed a novel pH measurement method based on two-photon fluorescence excitation of a dual-wavelength pHsensitive
dye combined with scanning near-field optical microscopy (SNOM) that can be used to evaluate mitochondrial
activity. Mitochondria produce ATP using a proton concentration (pH) gradient generated between both sides of their
inner membrane. Thus, pH distribution around mitochondria can change with time when mitochondria produce ATP.
This pH distribution has attracted interest because of its influence on necrotic cell death. Because ATP depletion causes
necrotic cell death, measurement of pH distribution around mitochondria is expected to lead to clarification of the
mechanism underlying necrotic cell death. However, it is very difficult to accurately measure pH around mitochondria
using conventional pH measurement methods. In this study, a dual-wavelength pH-sensitive dye was excited locally
using two-photon fluorescence excitation. In addition, collection-mode SNOM was used to avoid reabsorption by
collecting the fluorescent light directly from a florescence point. Using this method, we successfully calibrated pH and
observed temporal variations in pH after dropwise addition of acid. Moreover, mitochondrial activity was successfully
observed based on these pH changes.