Abstract
A novel method combining dual wavelength fluorescent ratiometry with scanning near-field optical microscopy (SNOM) is proposed and developed to measure the concentration and distribution of protons in the vicinity of biological samples. This method involves immersing mitochondria in a pH-sensitive fluorescent dye solution instead of injecting the dye into the surface of the mitochondrial membrane. It uses a dual emission pH-sensitive dye and SNOM with a thermally pulled and metal-coated optical fiber probe to improve the spatial resolution. The time dependence of the fluorescence intensity ratio (FIR) under acid addition and the response of mitochondria to nutritional supplementation were studied by using this method. Activation of mitochondria and a distance-dependent delay in the FIR response were observed. The results confirmed that mitochondrial activity could be observed by using this method.
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