Experimental Testing and Validation of a Quantum Dot FRET Calcium Sensor

项目摘要 这项应用计划测试钙量子点(Qdot)的细胞属性和功能- 我们研制的荧光能量转移(FRET)传感器。这些探头是用 目的提供快速、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。有许多有效的方法 基因编码的钙指示剂存在，但表达所需的时间(通常为几天) 排除了它们在我们所针对的那些急性实验中的使用。有机染料钙指示剂可以 被批量装载到孵化期相对较短但发射光谱较宽的细胞中， 这防止了由于光谱重叠而检测到多个信号，显著的光漂白 即使是短曝光，空间分辨率也有限。在这里，我们建议测试一种新的快速加载的急性 具有坚固和改进的光学性能的钙指示器。我们预计其他公司也会有类似的需求 调查人员并相信，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，我们将研究量子点的物理色散和分布- 生物学条件下的FRET探针，并使用该探针对星形胶质细胞神经中的钙信号进行成像 神经胶质细胞。

Project Summary 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 distribution of our quantum dot- FRET probes under biological conditions and use the probes to image calcium signaling in astrocyte neural glial cells.