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: 8738669
• 负责人: Gabriel A Silva
• 金额: $ 17.91万
• 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/8738669
• 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

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.

描述（由申请人提供）：本申请提出测试我们开发的钙量子点（qdot）-荧光能量转移（FRET）传感器的细胞特性和功能。 设计这些探针的目的是提供快速、高空间分辨率的荧光报告，用于急性心肌梗死患者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。 存在许多有效的遗传编码钙指标，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使用，如我们所针对的。 有机染料钙指示剂可以以相对短的孵育期批量装载到细胞中，但是遭受宽的发射光谱，这由于光谱重叠而阻止了多个信号的检测，甚至短曝光也有显著的光漂白，并且具有有限的空间分辨率。 在这里，我们提出测试一种新的快速加载急性钙指示剂具有强大的和改进的光学性能。 我们预计其他研究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性。 具体而言，在本申请中，我们将研究我们的量子点- FRET探针在生物条件下的物理分散和分布，并使用探针来成像星形胶质细胞神经胶质细胞中的钙信号。