Developing real-time, non-invasive, high-resolution chemical imaging of live mammalian cells, with high sensitivity and selectivity, is the objective. The tools to be employed are the smallest ever chemical and biochemical nanosensors, cell- implantable spheres, with a radius of 10-100 nm, called PEBBLEs (probes encapsulated by biologically localized embedding). Based on fluorophore molecules embedded in a variety of biocompatible matrices, the suggested methodologies will use, in tandem, optically silent ionophores and enzymes, so as to apply these sensors to a wide range of intracellular ions, free radicals and molecules, simultaneously, with high speed, sensitivity and selectivity. The list of subcellular analytes includes potassium, sodium, magnesium, zinc, copper, chloride, nitrite, carbonate and phosphate ions; oxygen, glucose and other enzyme substrates and reactive oxygen species -- singlet oxygen, nitric oxide, hydrogen peroxide, superoxide and hydroxyl radicals. Methods for controlling the size, shape and biocompatibility will be developed, as well as for steering the nanosensors (e.g., magnetically) and for targeting them to specific cell locations, with the help of molecular recognition. Validation and application examples of the subcellular real-time chemical imaging will be carried out on neural, liver, macrophage and other mammalian cell lines. Such refined chemical imaging is expected to accelerate biomedical research in many areas.
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