A hyperspectral microscope optimized for spectrally multiplexed live cell imaging

针对光谱多重活细胞成像进行优化的高光谱显微镜

• 批准号: 7761186
• 负责人: Keith A Lidke
• 金额: $ 17.52万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7761186
• 关键词: Affinity; Algorithms; Arts; Behavior; Biological; Cancer Center; Cell Surface Receptors; Cells; Color; Custom; Data; Data Analyses; Data Set; Development; Electrons; Environment; Fluorescence; Fluorescent Probes; Funding; Goals; Homo; Housing; IgE; IgE Receptors; Image; Immune system; Integral Membrane Protein; Knowledge; Label; Laboratories; Lasers; Length; Life; Light; Malignant Neoplasms; Membrane; Membrane Proteins; Microscope; Motion; New Mexico; Optics; Performance; Photobleaching; Physics; Process; Proteins; Quantum Dots; Reagent; Receptor Activation; Reporting; Research Personnel; Resolution; Sampling; Scanning; Signal Pathway; Signal Transduction; Spatial Distribution; Speed; System; Techniques; Time; Universities; base; cellular imaging; charge coupled device camera; computing resources; crosslink; design; fluorescence microscope; improved; insight; instrument; particle; protein protein interaction; public health relevance; receptor

DESCRIPTION (provided by applicant): This project is the development of a fluorescence hyperspectral microscope. A hyperspectral microscope collects a broad region of the spectrum with high spectral resolution in every sampled spatial volume. This microscope will improve and optimize the design of a Sandia National Laboratories designed hyperspectral microscope that incorporates a high throughput prism spectrometer and state of the art electron multiplied CCD cameras. Optimizations will include a focus on speed, with a target of full spectral imaging at 30 Hz, and will be based on a commercial inverted microscope to allow standard accessories and practices used for live cell imaging. High spectral resolution will allow separation of up to eight spectrally distinct quantum dots, which due to their broad excitation spectra can be excited simultaneously with a 488 nm laser. Spectral separation combined with the ability of single particle tracking to localize single particles to less than 10 nm will allow the observation of either hetero or homo protein-protein interactions at length scales close to 10 nm, much less than the 250 nm resolution of a fluorescence microscope. The project will begin with a redesign of the Sandia hyperspectral microscope to incorporate line scanning for optimal speed. Analysis techniques will be developed to utilize knowledge about the single particle, diffraction limited spatial signature of the probes to enhance spectral separation and localization. The first application will be to study the dynamic homo-interaction between the membrane protein FceRI. The microscope will be developed and housed in a laboratory in the University of New Mexico physics department, and after construction, is expected to be heavily used by researchers of the UNM cancer centers. Public Health Relevance Statement: This project will fund the development of a 'fluorescence hyperspectral microscope' that will make use of new fluorescent probes (quantum dots) and spectral multiplexing to observe the dynamics of protein-protein interactions at the 10 nm scale. Data generated with this instrument is expected to give new insight into cellular signaling pathways, including those involved in the immune system and cancer.

描述（由申请人提供）：该项目是荧光高光谱显微镜的开发。高光谱显微镜在每个采样的空间体积中以高光谱分辨率收集广泛的光谱区域。该显微镜将改进和优化桑迪亚国家实验室设计的高光谱显微镜的设计，该显微镜结合了高通量棱镜光谱仪和最先进的电子倍增 CCD 相机。优化将包括对速度的关注，目标是在 30 Hz 下进行全光谱成像，并将基于商用倒置显微镜，以允许用于活细胞成像的标准配件和实践。 高光谱分辨率将允许分离多达八个光谱不同的量子点，由于其宽激发光谱，可以用 488 nm 激光同时激发。光谱分离与单粒子追踪能力相结合，可将单个粒子定位到小于 10 nm，将允许在接近 10 nm 的长度尺度上观察异源或同源蛋白质-蛋白质相互作用，远低于荧光显微镜的 250 nm 分辨率。 该项目将从重新设计桑迪亚高光谱显微镜开始，将线扫描纳入最佳速度。将开发分析技术，利用有关探针的单粒子、衍射限制空间特征的知识来增强光谱分离和定位。第一个应用是研究膜蛋白 FceRI 之间的动态同源相互作用。该显微镜将在新墨西哥大学物理系的实验室中开发和安置，建成后预计将被新墨西哥州癌症中心的研究人员大量使用。 公共健康相关性声明：该项目将资助“荧光高光谱显微镜”的开发，该显微镜将利用新的荧光探针（量子点）和光谱多重技术来观察 10 nm 尺度上蛋白质-蛋白质相互作用的动态。使用该仪器生成的数据预计将为细胞信号传导途径提供新的见解，包括那些涉及免疫系统和癌症的信号传导途径。