A hyperspectral microscope optimized for spectrally multiplexed live cell imaging

描述(申请人提供)：该项目是一种高光谱荧光显微镜的开发。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30赫兹的全光谱成像，并将基于商用倒置显微镜，以允许标准配件和实践用于活细胞成像。 高光谱分辨率将允许分离多达8个光谱不同的量子点，由于其宽广的激发光谱，可以用488 nm激光同时激发。光谱分离与单粒子跟踪能力相结合，可以将单个粒子定位到10 nm以下，从而能够在接近10 nm的长度尺度上观察到异种或同种蛋白质-蛋白质相互作用，远远低于荧光显微镜250 nm的分辨率。 该项目将从重新设计Sandia高光谱显微镜开始，以并入行扫描以实现最佳速度。将开发分析技术，以利用关于单个粒子的知识，衍射受限的探测器的空间特征，以加强光谱分离和定位。第一个应用将是研究膜蛋白FceRI之间的动态同位相互作用。该显微镜将被开发并安置在新墨西哥大学物理系的实验室中，建造完成后，预计将被新墨西哥州大学癌症中心的研究人员大量使用。 公共卫生相关声明：该项目将资助开发一种‘荧光高光谱显微镜’，它将利用新的荧光探针(量子点)和光谱多路复用来观察10纳米尺度上蛋白质-蛋白质相互作用的动力学。利用该仪器产生的数据有望为细胞信号通路提供新的见解，包括那些涉及免疫系统和癌症的信号通路。

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.