Experimental Testing and Validation of a Quantum Dot FRET Calcium Sensor

量子点 FRET 钙传感器的实验测试和验证

• 批准号: 8620447
• 负责人: Gabriel A Silva
• 金额: $ 22.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8620447
• 关键词: Acute; Address; Astrocytes; Biological; Biosensing Techniques; Calcium; Calcium Oscillations; Calcium Signaling; Cell Signaling Process; Cells; Cerebrospinal Fluid; Color; Complex; Cytosol; Data; Data Analyses; Detection; Dyes; Energy Transfer; Environment; Event; Feedback; Fluorescence; Fluorescence Resonance Energy Transfer; Hour; Image; Ions; Measures; Methods; Nanotechnology; Neurobiology; Neuroglia; Neurons; Optics; Phosphate Buffer; Photobleaching; Physiological; Property; Protocols documentation; Publications; Quality Control; Quantum Dots; Relative (related person); Reporting; Research Personnel; Resolution; Saline; Signal Transduction; System; Testing; Time; Validation; Water; Work; calcium indicator; comparative; cytotoxicity; design; improved; novel; particle; prevent; public health relevance; relating to nervous system; research study; sensor

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.

项目摘要 本申请提出测试钙量子点（qdot）的细胞性质和功能性， 荧光能量转移（FRET）传感器。这些探头的设计 目的是提供快速，高空间分辨率的荧光报告细胞中的钙信号传导， 在实验条件下，在4到6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（通常为几天） 排除了它们在急性实验中的应用比如我们要找的那些有机染料钙指示剂可 可以以相对短的孵育期大量装载到细胞中，但是遭受宽的发射光谱， 其由于光谱重叠而防止了多个信号的检测，显著的光漂白， 甚至短曝光，并且具有有限的空间分辨率。在这里，我们建议测试一种新的快速加载急性 钙指示剂具有稳定和改进的光学特性。我们期待其他类似的需求。 我们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性。 具体来说，在这个应用程序中，我们将研究我们的量子点的物理色散和分布- FRET探针在生物学条件下的作用，并使用该探针对星形胶质细胞神经细胞中的钙信号传导进行成像。 神经胶质细胞