Development of near-field optical microscopy for hydrogen ion concentration and membrane potential using environmental sensitivity dye

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In this study, a dual-wavelength pH-sensitive dye was excited by two-photon absorption initiated using afemtosecond 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 thisproposed method, a pH calibration curve was obtained from the fluorescent intensity ratio of the dyesolution, and temporal pH variations with 0.1 s time resolution following the addition of acid wereobserved. Moreover, mitochondrial activity on the basis of the pH changes was successfully observed in three different mitochondrial densities.