Study Of New Fluorescent PEBBLE Nanosensors For Intracellular Calcium

Abstract Changes in intracellular ion concentrations accompany many processes in living cells, including transport, signaling, and enzyme function. One of the most widely studied ion, calcium, has been found to be ubiquitous in these and other cellular processes. Several methods have been developed for studying intracellular calcium. Optical fluorescence methods for analyzing intracellular ion concentrations have become widespread biological tools. Injection of fluorescent indicator dyes into a cell combined with dualwavelength (ratiometric) imaging, confocal microscopy, twophoton fluorescence, or fluorescence lifetime imaging has provided important insights into the concentration and spatial location of ions throughout single cells. One challenge for these optical methods has been that each dye must be chosen carefully and assessed for its ability to provide accurate and reliable information from within a cell. Factors such as toxicity to the cell, intracellular sequestration, protein binding, and leakage of the dye from the cell frequently complicate the interpretation of the results and must be considered. While there are indicator dyes, such as fura-2, that work very well within various cells, many fluorescent probes suffer from the problems listed above, thus limiting the number of dyes available for reliable intracellular measurements.The probes encapsulated by biologically localized embedding (PEBBLE) sensor was devised in order to provide a fluorescence based method for intracellular analysis that circumvents many of the above-mentioned problems. The PEBBLE is a submicrometer, spherical polymer that contains one or more fluorescent dyes that are trapped within the pores of the matrix. The polymer provides a protective coating for the dyes, so that they are not affected by the cell, thereby minimizing error from protein binding and membrane/organelle sequestration. Thus, PEBBLEs can be calibrated in vitro and used in vivo, with the response of the sensors remaining consistent regardless of environment. Multiple dyes can be combined in a single PEBBLE sensor, so that a reference standard can be used to minimize errors from uneven dye distributions throughout the cell. Another advantage of PEBBLEs is that they are small enough to be biocompatible, yet large enough to be retained in the cytoplasm of the cell with no leakage of the sensor out of the cell, even after several days. Furthermore, many toxic effects of the dye are eliminated.