Novel Dual-Organelle-Targeting Probe (RCPP) for Simultaneous Measurement of Organellar Acidity and Alkalinity in Living Cells.

Many organelles, such as lysosomes and mitochondria, maintain a pH that is different from the cytoplasmic pH. These pH differences have important functional ramifications for those organelles. Many cellular events depend upon a well-compartmentalized distribution of H+ ions spanning the membrane for the optimal function. Cells have developed a variety of mechanisms that enable the regulation of organelle pH. However, the measurement of organellar acidity/alkalinity in living cells has remained a challenge. Currently, most existing probes for the estimation of intracellular pH show a single -organelle targeting capacity. Such probes provide data that fails to comprehensively reveal the pathological and physiological roles and connections between mitochondria and lysosomes in different species. Mitochondrial and lysosomal functions are closely related and important for regulating cellular homeostasis. Accordingly, the design of a single fluorescent probe that can simultaneously target mitochondria and lysosomes is highly desirable, enabling a better understanding of the crosstalk between these organelles. We report the development of a novel fluorescent sensor, rhodamine-coumarin pH probe (RCPP), for detection of organellar acidity/alkalinity. RCPP simultaneously moves between mitochondrion and lysosome subcellular locations, facilitating the simultaneous monitoring of pH alterations in mitochondria and lysosomes.

Targeted fluorescent probes for detection of oxidative stress in the mitochondria.

Mitochondrial oxidative stress has been implicated in aging, neurodegenerative diseases, diabetes, stroke, ischemia/reperfusion injury, age-related macular degeneration (AMD) and cancer. Recently, we developed two new mitochondria-targeting fluorescent probes, MitoProbes I/II, which specifically localize in mitochondria and employed both in vivo and in vitro for detection of mitochondrial oxidative stress. Here, we report the design and synthesis of these agents, as well as their utility for real-time imaging of mitochondrial oxidative stress in cells.

Highly stable and sensitive fluorescent probes (LysoProbes) for lysosomal labeling and tracking.

We report the design, synthesis and application of several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to commercially available lysosome markers since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We predict that labeling of cancer cells and solid tumor tissues with LysoProbes will provide an important new tool for monitoring the role of lysosome trafficking in cancer invasion and metastasis.

Determination of intracellular pH using sensitive, clickable fluorescent probes.

We synthesized and evaluated a series of acidic fluorescent pH probes exhibiting robust pH dependence, high sensitivity and photostability, and excellent cell membrane permeability. Titration analyses indicated that probe 3 could increase its fluorescence intensity 800-fold between pH 8.0 and 4.1. Additionally, its pK(a) value is optimal for intracellular probing of acidic organelles. Fluorescent imaging of HepG2 and Hela cells further revealed that probe 3 demonstrates outstanding capacity for monitoring of intracellular [H(+)] levels. The easily accessible terminal alkyne/azido function groups of these probes offer the possibility of rapidly constructing sensor molecule libraries using 'click' chemistry.

New rhodamine nitroxide based fluorescent probes for intracellular hydroxyl radical identification in living cells.

The synthesis, characteristics, and biological applications of a series of new rhodamine nitroxide fluorescent probes that enable imaging of hydroxyl radicals (•OH) in living cells are described. These probes are highly selective for •OH in aqueous solution, avoiding interference from other reactive oxygen species (ROS), and they facilitate •OH imaging in biologically active samples. The robust nature of these probes (high specificity and selectivity, and facile synthesis) offer distinct advantages over previous methods for •OH detection.