This application proposes to test the cellular properties and functionality of a calcium quantum dot (qdot)- fluorescence energy transfer (FRET) sensor we have developed. These probes have been designed with the intent to provide fast, high spatial resolution fluorescence reporting of calcium signaling in cells in acute experimental conditions, within an immediate four to six hour window. There are a number of efficient genetically encoded calcium indicators that exist, but the time required for expression (typically several days) precludes their use in acute experiments like the ones we are targeting. Organic dye calcium indicators can be bulk loaded into cells with relatively short incubation periods but suffer from broad emission spectra, which prevents the detection of multiple signals due to the spectral overlap, significant photobleaching to even short exposures, and have limited spatial resolution. Here we propose testing a novel fast loading acute calcium indicator with robust and improved optical properties. We anticipate similar needs from other investigators and believe the probes we are developing will be of significant widespread value and utility. Specifically, in this application we will investigate physical dispersion and distribtion of our quantum dot- FRET probes under biological conditions and use the probes to image calcium signaling in astrocyte neural glial cells.
Fast spatially localized transient calcium changes underlie key functional cell signaling processes. These changes however are very difficult to measure directly and study because they occur quickly and are restricted to small volumes within a cell. This project proposes to develop a highly sensitive nanotechnology biosensing probe called a quantum dot fluorescence resonance energy transfer (FRET) sensor in order measure tiny calcium transients not detectable by other methods.
