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：
7695567
负责人：
TOMASZ S TKACZYK
金额：
$ 18.55万
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 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7695567
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 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 Research Research Personnel Research Project Grants Resolution Scanning Scientist Series Side Signal Transduction Slice Speed Structure System Techniques Technology Testing Time Work active method base biological systems cellular imaging charge coupled device camera cost design digital fluorescence imaging fluorophore follow-up fura red imaging modality 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（由适用提供）：该提案针对荧光光谱成像系统的开发，用于用于活细胞中多个荧光探针的简单高分辨率亚细胞显微镜。荧光探针，成像仪器和微制造的最新发展现在允许用于实时定量光谱成像的图像切片光谱仪（ISS）。我们建议将显微镜，光学设计，制造和成像的专业知识与新的大格式CCD摄像机和制造技术相结合，以开发ISS系统。 ISS是一种广阔的方法，可以简单地从每个像素中获取全光谱信息。该方法通过空间重定向图像区域来获得图像线之间的空间来起作用。接下来，通过使用衍射元件，ISS在CCD摄像头上获得了波长（有关系统原理的更多详细信息，请参见C和D节）。这样，我们明确地映射x，y，？;数据数据到2-D图像传感器。该项目的具体目的是：（1）构建具有450至700 nm和（2）的初始波长范围和（2）的ISS，以测试在几个活细胞成像应用中当前可用的光谱成像系统的图像切片光谱仪。迄今为止，用于细胞成像的成像光谱仪开发的工作已经受到小型视野，有限的临时空间 - 光谱分辨率，广泛计算的要求或有限的光效率的阻碍。此处提出的图像切片光谱仪是基于从天文学领域借来的概念，并解决了先前与快照光谱仪结构相关的主要困难。图像切片器将矩形视野（FOV）转换为一系列迷你“缝隙”，并重新布置它们以在快照模式下创建足够的区域以进行光谱传播和获取。不需要复杂的处理，只有简单的重新映射就足以获得完整的x，y，？数据立方体。系统的核心将是用钻石转弯技术制造的定制重定向镜。该仪器将采用带有4000 x 2624像素元素的Hamamatsu CCD摄像头，Peltier冷却和低噪声读数（C4742-98-24HR）。使用此大格式CCD，最终图像数据立方体将为400 x 260 x 50（x，y，？），光谱分辨率为5 nm，空间分辨率为〜0.5 <€。构建和优化系统后，我们将针对Zeiss LSM510 Meta和光学见解光谱DV系统进行定量评估ISS的结果。这些评估将利用“标准”荧光团组合，从两色对开始，但包括更多的挑战组合，例如CFP/GFP/YFP/YFP/Fluo-4，以及MCHERRY/SNARF-1/FURA-RED。总之，ISS有可能在细胞成像区域显着推进广泛的应用。为了进一步影响其影响，我们计划将来使用结构化照明，Nipkow磁盘共焦和/或空间反卷积将ISS与光学截面相结合。尽管它超出了本应用的范围，但四维成像系统（x，y，？,,;）将进一步改善数据的S/N，以及4-D成像的速度。该项目针对现代光谱仪的开发，称为图像切片光谱仪，实时实现高分辨率光谱成像。因此，研究人员将能够快速推进具有多种荧光对比度的活细胞投资。该仪器的原理允许在不扫描的情况下获取整个图像的光谱信息，从而改善了信号与噪声比。它还可以有效地投资瞬态生物事件。在项目中应用的技术及其低成本可能会允许更多的科学家进入光谱成像仪器。