Experimental Testing and Validation of a Quantum Dot FRET Calcium Sensor

描述(由申请人提供)：本申请建议测试我们开发的钙量子点(QDot)-荧光能量转移(FRET)传感器的细胞属性和功能。这些探针的设计意图是提供快速、高空间分辨率的细胞内钙信号的荧光报告。 实验条件，在即刻的四到六个小时的窗口内。有许多有效的基因编码的钙指示剂存在，但表达所需的时间(通常是几天)排除了它们在我们所针对的急性实验中的使用。有机染料钙指示剂可以批量装载到细胞中，孵育期相对较短，但发射光谱较宽，这阻止了由于光谱重叠而检测到的多个信号，甚至在短时间曝光时也会出现严重的光漂白，并且空间分辨率有限。在这里，我们建议测试一种新型的快速加载急性钙指示器，具有坚固和改进的光学特性。我们预计其他调查人员也会有类似的需求，并相信我们正在开发的探测器将具有重要的广泛价值和实用价值。具体地说，在这个应用中，我们将研究我们的量子点-弗雷特探针在生物学条件下的物理分散和分布，并使用这些探针对星形胶质细胞神经胶质细胞中的钙信号进行成像。

DESCRIPTION (provided by applicant): 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.