1f:T40b,The homeostasis of mitochondrial pH (pH_m) is crucial in cell physiology. Developing small-molecular fluorescent sensors for the ratiometric detection of pH_m fluctuation is highly demanded yet challenging. A ratiometric pH sensor, Mito-pH, was constructed by integrating a pH-sensitive FITC fluorophore with a pH-insensitive hemicyanine group. The hemicyanine group also acts as the mitochondria targeting group due to its lipophilic cationic nature. Besides its ability to target mitochondria, this sensor provides two ratiometric pH sensing modes, the dual excitation/dual emission mode (D_ex/D_em) and dual excitation (D_ex) mode, and its linear and reversible ratiometric response range from pH 6.15 to 8.38 makes this sensor suitable for the practical tracking of pH_m fluctuation in live cells. With this sensor, stimulated pH_m fluctuation has been successfully tracked in a ratiometric manner via both fluorescence imaging and flow cytometry.20:T4f6,Small-molecule fluorescent probes are powerful tools for chemical biology; however, despite the large number of probes available, there is still a need for a simple fluorogenic scaffold, which allows for the rational design of molecules with predictable photophysical properties and is amenable to concise synthesis for high-throughput screening. Here, we introduce a highly modular quinoline-based probe containing three strategic domains that can be easily engineered and optimized for various applications. Such domains are allotted for (1) compound polarization, (2) tuning of photophysical properties, and (3) structural diversity. We successfully synthesized our probes in two steps from commercially available starting materials in overall yields of up to 95%. Facile probe synthesis was permitted by regioselective palladium-catalyzed cross-coupling, which enables combinatorial development of structurally diverse quinoline-based fluorophores. We have further applied our probes to live-cell imaging, utilizing their unique two-stage fluorescence response to intracellular pH. These studies provide a full demonstration of our strategy in rational design and stream-lined probe discovery to reveal the diverse potential of quinoline-based fluorescent compounds.21:T4fa,We have measured the intracellular pH of human peripheral blood lymphocytes by means of high-resolution 19F NMR spectroscopy using D,L-2-amino-3,3-difluoro-2-methylpropanoic acid (F2MeAla) as a probe. Lymphocytes readily took up the methyl ester of F2MeAla, and endogenous esterase hydrolyzed the ester to the free amino acid inside the cell. This alpha-methyl amino acid is not metabolized by the cell, and its 19F NMR spectrum exhibits large pH-dependent shifts as the alpha-amino group is protonated. The size of the 19F shifts, the high sensitivity of 19F NMR, and the favorable pKa of the alpha-amino group of F2MeAla (pKa = 7.3) allowed us to measure intracellular pH of lymphocytes at 25-30 degrees C with approximately 5-min acquisition times. Measurements at various external pH values demonstrated that human peripheral blood lymphocytes regulate their internal pH, a process requiring expenditure of metabolic energy. In the pH range between 6.8 and 7.4, lymphocytes maintain a constant internal pH of 7.17 +/- 0.06 pH unit. Outside this range, intracellular pH changes with extracellular pH. The accuracy of this 19F pH probe has been confirmed by independent measurements of intracellular pH using equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione.22:T564,

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