A mitochondrion has a pH gradient between the two sides of its inner membrane in order to produce adenosine triphosphate (ATP). Because ATP depletion causes numerous diseases, the measurement of the pH value around the mitochondrion is expected to clarify the mechanism of these diseases. In this study, a dual-wavelength pH-sensitive dye was excited by two-photon absorption initiated using a femtosecond pulse laser. In addition, fluorescence from the dye was directly collected from the fluorescent point using the collection-mode probe of a scanning near-field optical microscope. By this proposed method, a pH calibration curve was obtained from the fluorescent intensity ratio of the dye solution, and temporal pH variations with 0.1 s time resolution following the addition of acid were observed. Moreover, mitochondrial activity on the basis of the pH changes was successfully observed in three different mitochondrial densities.
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.
pH measurements enable the direct monitoring and evaluation of mitochondrial activity. We constructed a scanning near-field optical microscopy system with multioptical fiber probes using the two-photon absorption of a pH-sensitive fluorescent dye, SNARF-4F, to measure the activity difference of mitochondrial aggregates. pH can be monitored through the fluorescence intensity ratio (FIR) of SNARF-4F. We derived a calibration curve of the FIR as a function of pH. The FIR dynamic responses were measured by adding hydrochloric acid to the buffer solution. Using the developed system, we simultaneously measured the pH changes at two different locations in the SNARF-4F solution. Mitochondrial samples were prepared using optical tweezers to control the number and position of mitochondria. Mitochondrial pH changes (ΔpH) between 0.05 and 0.57 were observed after adding a nutritional supplement (malate and glutamate). In addition, in the comparative experiment on the activities of two mitochondrial populations, the obtained result suggested that the activity differs depending on the difference in the number of mitochondria.
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