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

描述（由申请人提供）：本提案旨在开发一种荧光光谱成像系统，用于同时对活细胞中的多个荧光探针进行高分辨率亚细胞显微镜观察。荧光探针、成像仪器和微加工技术的最新发展使得构建用于实时定量光谱成像的图像切片光谱仪（ISS）成为可能。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最新的大画幅CCD相机和制造技术结合起来开发ISS系统。ISS是一种宽视场方法，能够同时从每个像素获取全光谱信息。这种方法通过空间重定向图像区域来获得图像线之间的空间。接下来，通过使用衍射元件，ISS获得CCD相机上的波长扩展（有关系统原理的更多详细信息，请参见C和D节）。这样，我们明确地映射了x，y, ?；数据传输到二维图像传感器。该项目的具体目标是：(1)构建初始波长范围为450至700纳米的国际空间站；(2)在几种活细胞成像应用中，对图像切片光谱仪与目前可用的光谱成像系统进行测试。迄今为止，用于细胞成像的成像光谱仪的开发工作受到视场小、时空光谱分辨率有限、需要大量计算或光效有限的限制。这里提出的图像切片光谱仪是基于从天文学领域借鉴的概念，并解决了以前与快照光谱仪建设相关的主要困难。图像切片器将矩形视场（FoV）转换成一系列的小“狭缝”，并重新排列它们，以便在快照模式下为光谱扩展和采集创造足够的区域。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, ?；数据立方体。该系统的核心将是一个用金刚石车削技术制造的定制重定向镜。该仪器将采用4000 x 2624像素单元的滨松CCD相机，Peltier冷却和低噪声读数（C4742-98-24HR）。使用该大幅面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 <¿m。一旦系统建成并优化，我们将根据蔡司LSM510 META和光学洞察光谱DV系统对ISS的结果进行定量评估。这些评估将利用“标准”荧光团组合，从双色对开始，但包括更具挑战性的组合，如CFP/GFP/YFP/Fluo-4和mCherry/SNARF-1/Fura-Red。综上所述，ISS在细胞成像领域具有显著推进广泛应用的潜力。为了进一步扩大其影响，我们计划在未来将ISS与光学切片结合起来，使用结构化照明、尼普科夫盘共聚焦和/或空间反卷积。虽然超出了目前的应用范围，但一个四维成像系统(X， Y, ？，；)将进一步提高数据的信噪比，以及4d成像的速度。该项目的目标是开发一种称为图像切片光谱仪的现代光谱仪，实现实时高分辨率光谱成像。因此，研究人员将能够通过多种荧光对比快速推进活细胞的研究。该仪器的原理允许在不扫描的情况下获得整个图像的光谱信息，从而提高信噪比。它还允许对短暂的生物事件进行有效的调查。该项目中应用的技术及其低成本可能使更多的科学家能够使用光谱成像仪器。

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.