High-throughput high-content single cell analysis by multichannel stimulatedRaman flow cytometry

通过多通道受激拉曼流式细胞术进行高通量高内涵单细胞分析

• 批准号: 9080637
• 负责人: Ji-Xin Cheng
• 金额: $ 42.87万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9080637
• 关键词: Amplifiers; Biological Markers; Cell Separation; Cells; Cellular Metabolic Process; Characteristics; Chemicals; Classification; Cytometry; Data; Data Analyses; Detection; Development; Disease; Doctor of Philosophy; Energy Transfer; Event; Flow Cytometry; Fluorescence; Foundations; Geometry; Glucose; Goals; Image; Label; Lead; Light; Machine Learning; Malignant neoplasm of ovary; Mammalian Cell; Measurement; Measures; Metabolic; Microfluidic Microchips; Microfluidics; Microscope; Microscopy; Modeling; Molecular; Molecular Profiling; Nanostructures; Optics; Phase; Polymers; Population; Principal Component Analysis; Process; Property; Quartz; Reporting; Sampling; Side; Signal Transduction; Specificity; Speed; Stream; Surface; System; Techniques; Time; Tube; base; biological systems; cancer cell; cancer stem cell; data acquisition; design; design and construction; detector; electric impedance; high throughput analysis; instrumentation; light scattering; novel strategies; particle; photomultiplier; public health relevance; single cell analysis; small molecule; spectroscopic imaging; tool; treatment strategy; vibration

 DESCRIPTION (provided by applicant): Flow cytometry is one of the most important tools for high-throughput single cell analysis. Fluorescent labeling acts as the primary approach for cellular analysis in flow cytometry. Nevertheless, fluorescent tags are not applicable to all cases especially small molecules (e.g. metabolites) for which labeling may significantly perturb their properties. Raman spectroscopic signals arising from inherent molecular vibrations provide a key approach to detect specific molecules inside cells and to differentiate cellular state. Raman-based microfluidic devices have been reported. However, the very small cross section of spontaneous Raman scattering results in low Raman signal level and consequently long data acquisition time, which is not compatible with the high- speed flow condition. The long-term goal of the proposed project is to establish a high-throughput high-content single cell analysis platform using molecular fingerprint vibrations as contrast. The specific objective of current application is to develop a vibrational spectroscopic cytometer based on the stimulated Raman scattering (SRS) process. Several recent advances in the Ji-Xin Cheng (PI) lab, including the highly sensitive femtosecond SRS imaging, lock-in free SRS signal detection and a tuned amplifier array for multiplex SRS imaging, pave the foundation for the planned instrumentation. The PI has assembled an interdisciplinary team for the proposed study. Dr. J. Paul Robinson (co-PI) is a leader in development and applications of fluorescence-based flow cytometer and he will bring expertise to the design of fluidics and multichannel detection systems. Dr. Bartek Rajwa (co-PI) will provide expertise for spectroscopic cytometry data analysis and machine learning. The team will design and construct a SRS flow cytometer by multichannel detection of dispersed SRS signal (Aim 1), construct a tandem system able to collect SRS and fluorescence data (Aim 2), develop spectral un-mixing and machine-learning analysis tools able to combine the information obtained from SRS spectra and labeled biomarkers for functional classification of cells (Aim 3), and validate the capability of SRS flow cytometer for label-free detection of single-cell metabolism (Aim 4). With a speed of analyzing thousands of cells per second, SRS flow cytometer will enable high-throughput analysis of single-cell chemical content which is beyond the reach by fluorescence-based flow cytometer.

 描述（适用提供）：流式细胞仪是高通量单细胞分析的最重要工具之一。荧光标记是流式细胞仪中细胞分析的主要方法。然而，荧光标签并不适用于所有情况，尤其是小分子（例如代谢产物），其标记可能会显着扰动其性质。由继承分子振动引起的拉曼光谱信号提供了检测细胞内部特定分子并区分细胞状态的关键方法。已经报道了基于拉曼的微流体设备。但是，赞助拉曼散射的非常小的横截面导致拉曼信号水平低，因此数据采集时间很长，这与高速流量条件不兼容。拟议项目的长期目标是使用分子指纹振动作为对比度建立高通量的高含量单细胞分析平台。当前应用的具体目标是基于刺激的拉曼散射（SRS）工艺开发振动光谱细胞仪。 JI-XIN Cheng（PI）实验室的最新进展，包括高度敏感的飞秒SRS成像，锁定自由SRS信号检测和用于多重SRS成像的调谐放大器阵列，为计划的仪器铺平了基础。 PI已组建了一个跨学科团队进行拟议的研究。 J. Paul Robinson博士（Co-Pi）是基于荧光的流式细胞仪开发和应用领域的领导者，他将为流体和多通道检测系统的设计带来专业知识。 Bartek Rajwa博士（CO-PI）将为光谱细胞仪数据分析和机器学习提供专业知识。 The team will design and construct a SRS flow cytometer by multichannel detection of dispersed SRS signal (Aim 1), construct a tandem system can to collect SRS and fluorescence data (Aim 2), develop spectral un-mixing and machine-learning analysis tools can to combine the information obtained from SRS spectra and labeled biomarkers for functional classification of cells (Aim 3), and validate the capability of SRS flow cytometer用于无标签的单细胞代谢（AIM 4）。 SR流式细胞仪每秒分析了数千个细胞的速度，将实现对单细胞化学含量的高通量分析，而基于荧光的流式细胞仪，这超出了范围。