Image Slicing Spectrometer (ISS) for high resolution sub-cellular microscopy

用于高分辨率亚细胞显微镜的图像切片光谱仪 (ISS)

基本信息

批准号：
7572583
负责人：
TOMASZ S TKACZYK
金额：
$ 19.75万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7572583
关键词：
3-Dimensional Address Adopted Area Astronomy Biological Biological Preservation Cells Collection Color Custom Data Development Devices Diamond Electronics Elements Evaluation Event Exploratory/Developmental Grant Facility Construction Funding Category Fluo 4 Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Probes Four-dimensional Future Goals Image Investigation Ions Lasers Life Light Lighting Maps Mechanics Methods Microscope Microscopy Noise Optics Process Proteins Range Research Research Personnel Research Project Grants Resolution Scanning Scientist Series Side Signal Transduction Slice Speed Standards of Weights and Measures Structure System Techniques Technology Testing Time Work base cellular imaging charge coupled device camera concept cost design desire digital fluorescence imaging fluorophore follow-up fura red imaging probe improved insight instrument instrumentation lens novel strategies parallel computing prevent research study sensor

项目摘要

DESCRIPTION (provided by applicant): This proposal is directed toward development of a fluorescent spectral imaging system for simultaneous high resolution sub-cellular microscopy of multiple fluorescence probes in living cells. Recent developments in fluorescent probes, imaging instrumentation and micro- fabrication now permit building for an Image Slicing Spectrometer (ISS) for real time quantitative spectral imaging. We propose to combine our expertise in microscopy, optical design, fabrication, and imaging with newly available large format CCD cameras and fabrication techniques to develop an ISS system. ISS is a widefield method that is capable of acquiring full spectral information simultaneously from every pixel. This approach works by spatially redirecting image zones to obtain space between image lines. Next, by using a diffractive element, ISS obtains wavelength spread on the CCD camera (for more details on the system principle see Sections C and D). In this way, we unambiguously map x,y, ?; data onto the 2-D image sensor. The specific aims of the project are: (1) to construct the ISS with an initial wavelength range of 450 to 700 nm and (2) to test the Image Slicing Spectrometer against currently available spectral imaging systems in several live cell imaging applications. Work on development of imaging spectrometers for cellular imaging has thus far been hampered either by small fields of view, limited temporal-spatial-spectral resolution, requirement of extensive computations, or limited light efficiency. The Image Slicing Spectrometer proposed here is based on a concept borrowed from the astronomy field, and addresses the major difficulties previously connected with construction of a snapshot spectrometer. The Image Slicer transforms a rectangular Field of View (FoV) into a series of mini "slits", and rearranges them to create sufficient area for spectral spread and acquisition in the snapshot mode. No complicated processing is necessary and only simple remapping is sufficient to obtain a complete x,y, ?; data cube. The core of the system will be a custom-made redirecting mirror fabricated with diamond turning technology. The instrument will employ a Hamamatsu CCD camera with 4000 x 2624 pixel elements, Peltier cooling, and low-noise readout (C4742-98-24HR). Using this large format CCD, the final image data cube will be 400 x 260 x 50 (X, Y, ?) with a spectral resolution of 5 nm and ~0.5 < ¿m spatial resolution. Once the system is built and optimized we will quantitatively evaluate the results from the ISS against the Zeiss LSM510 META, and an Optical Insights Spectral DV system. These evaluations will utilize "standard" fluorophore combinations, starting with two-color pairs, but including more challenging combinations such as CFP/GFP/YFP/Fluo-4, and mCherry/SNARF-1/Fura-Red. In summary the ISS has the potential to significantly advance a wide range of applications in area of cellular imaging. To further its impact, we plan to combine the ISS with optical sectioning in the future, using structured illumination, Nipkow disk confocal, and/or spatial deconvolution. Although it is beyond the scope of the present application, a 4-dimensional imaging system (X, Y, ?, ;) would further improve the S/N of the data, as well as speed of 4-D imaging. The project targets the development of a modern spectrometer called Image Slicing Spectrometer enabling high resolution spectral imaging in real time. In consequence researchers will be able to rapidly advance the investigation of live cells with multiple fluorescent contrasts. The instrument's principle allows obtaining spectral information for entire image without scanning and thus improve signal to noise ratio. It also allows also efficient investigation of transient biological events. Technologies applied in the project and their low cost may potentially allow access of larger group of scientists to spectral imaging instrumentation.

描述（由申请人提供）：该提案旨在开发一种荧光光谱成像系统，用于活细胞中多个荧光探针的同时高分辨率亚细胞显微镜检查，荧光探针、成像仪器和微制造的最新发展现在允许构建。我们建议将我们在显微镜、光学设计、制造和成像方面的专业知识与新推出的大幅面 CCD 相机和制造技术相结合，以实现实时定量光谱成像的图像切片光谱仪 (ISS)。 ISS 是一种宽场方法，能够同时从每个像素获取完整的光谱信息。该方法通过空间重定向图像区域来获取图像线之间的空间，然后通过使用衍射元件获得波长扩展。在 CCD 相机上（有关系统原理的更多详细信息，请参阅第 C 节和第 D 节）。是：(1) 构建初始波长范围为 450 至 700 nm 的 ISS，以及 (2) 在多种活细胞成像应用中针对当前可用的光谱成像系统测试图像切片光谱仪。迄今为止，成像一直受到视场小、时空光谱分辨率有限、需要大量计算或光效率有限的阻碍。这里提出的图像切片光谱仪是基于借用的概念。图像切片器将矩形视场 (FoV) 转换为一系列迷你“狭缝”，并重新排列它们以创建足够的光谱扩展区域。快照模式下的采集不需要复杂的处理，只需简单的重新映射即可获得完整的x，y，?；系统的核心将是定制的重定向镜像。该仪器将采用具有 4000 x 2624 像素元素、Peltier 冷却和低噪声读出功能的 Hamamatsu CCD 相机（C4742-98-24HR），使用这种大幅面 CCD，最终的图像数据立方体将为 400 个。 x 260 x 50 (X, Y, ?)，光谱分辨率为 5 nm，~0.5 < ¿一旦系统建成并优化，我们将根据 Zeiss LSM510 META 和 Optical Insights Spectral DV 系统定量评估 ISS 的结果，这些评估将利用“标准”荧光团组合，从两种颜色对开始。，但包括更具挑战性的组合，例如 CFP/GFP/YFP/Fluo-4 和 mCherry/SNARF-1/Fura-Red 总之，国际空间站具有显着的潜力。为了进一步扩大其影响，我们计划在未来将国际空间站与光学切片相结合，使用结构照明、尼普科圆盘共焦和/或空间反卷积。在本申请的范围内，4维成像系统（X、Y、？、；）将进一步提高数据的信噪比以及4维成像的速度。现代光谱仪称为图像切片光谱仪可实现实时高分辨率光谱成像，因此研究人员将能够利用多种荧光对比快速推进活细胞的研究，无需扫描即可获得整个图像的光谱信息，从而提高信噪比。它还允许对项目中应用的瞬态生物事件进行有效的研究，并且其低成本可能允许更多的科学家使用光谱成像仪器。