Array Confocal Fluorescence Microscope

阵列共焦荧光显微镜

• 批准号: 8516070
• 负责人: Rongguang Liang
• 金额: $ 15.77万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8516070
• 关键词: Area; Biological; Biological Process; Biomedical Engineering; Budgets; Cells; Collaborations; Complex; Custom; Detection; Development; Devices; Diamond; Dimensions; Electronics; Elements; Engineering; Fiber; Fluorescence; Goals; Head; Image; Imaging Techniques; Individual; Lateral; Learning; Length; Life; Light; Lighting; Liquid substance; Measures; Microscope; Mus; Optics; Photobleaching; Phototoxicity; Plastics; Process; Research; Resolution; Resort; Sampling; Scanning; Scientist; Signal Transduction; Slide; Speed; Staging; System; Techniques; Testing; Tissue Sample; Toxic effect; Training; base; bioimaging; cellular imaging; clinical application; design; experience; fluorescence imaging; fluorescence microscope; imaging modality; improved; in vivo; interdisciplinary collaboration; lens; light emission; light intensity; meetings; millisecond; miniaturize; next generation; novel; optical imaging; portability; prototype; success; technology development

DESCRIPTION (provided by applicant): There is a fundamental limit to the imaging speed of a point-scanning confocal fluorescence microscope due to the limited amount of fluorescence signal that is emitted from a biological sample. To improve the speed of scanning, several techniques have been developed in the past, such as Nipkow spinning disk confocal microscope and line-scanning confocal microscope. However, to scan a large volume, their speed is still limited by the field of view (FOV) of the microscope objective. Additionally, they ae difficult to be miniaturized into portable handheld devices. We propose to develop an array confocal fluorescence microscope (ACFM) that can image large 3D volumes at a speed one order of magnitude faster than conventional confocal microscope over a large FOV. The proposed ACFM consists of an array of miniature high-NA confocal fluorescence objectives, each of which scans a small sub-FOV. Multiple point scanning and detection will not only increase the overall scanning speed dramatically, but also reduce photobleaching or phototoxicity significantly in live cells because it requires a lower level of liht intensity per unit area. The FOV of the proposed ACFM will not be limited by the FOV of individual objective; it will only be limited by the scan range of the scanning mechanism. Most importantly, instead of scanning stage or objective for depth imaging, the proposed ACFM will develop a novel tunable liquid plate located in the image space of the objective to perform high speed depth scan in the object space. An array of fibers, one for each channel in the ACFM, delivers the excitation illumination and collects the emitted fluorescence signal. The fibers will also act as the confocal pinholes to eliminate out-of-focus light. With this unique configuration the confocal head of the proposed ACFM can be very compact and scalable, particularly suitable for handheld clinical applications. In this proposed three-year effort, we will design, build, and test a compact high NA (NA=0.7) and large FOV ACFM with 5x5 confocal objectives. The objectives will be designed with optical plastics and fabricated using diamond turning techniques. We will calibrate the system, measure the lateral and axial resolution, and demonstrate system capabilities through imaging mouse tissue samples. This effort will require interdisciplinary collaboration of various areas in biomedical engineering optical engineering and fabrication, and system engineering and electronics. We will apply experience learned from developing bright-field array microscope to the proposed development of ACFM. If successful, the proposed ACFM will have significant impacts on biomedical imaging, especially in in-vivo clinical applications over large FOVs and whole slide imaging. The proposed ACFM can be used as a scalable platform for other imaging modalities, such as confocal Raman microscope, multiphoton microscope, and hyperspectral microscope. It will also be a platform for more advanced imaging applications, such as parallel depth imaging and multiple-band fluorescence imaging. The research will provide an excellent opportunity to train the next generation of interdisciplinary scientists and engineers, at both the undergraduate and graduate levels.

描述（由申请人提供）：由于从生物样品发射的荧光信号的量有限，点扫描共聚焦荧光显微镜的成像速度存在基本限制。为了提高扫描速度，过去发展了几种技术，如Nipkow旋转圆盘共聚焦显微镜和线扫描共聚焦显微镜。然而，为了扫描大体积，它们的速度仍然受到显微镜物镜的视场（FOV）的限制。此外，它们难以小型化成便携式手持设备。 我们建议开发一种阵列共聚焦荧光显微镜（ACFM），它可以在一个大的FOV比传统的共聚焦显微镜的速度快一个数量级的图像大的3D体积。所提出的ACFM由一系列微型高NA共焦荧光物镜组成，每个物镜扫描一个小的子FOV。多点扫描和检测不仅可以显著提高整体扫描速度，而且可以显著降低活细胞中的光漂白或光毒性，因为它需要较低的单位面积光强水平。所提出的ACFM的FOV将不受单个物镜的FOV的限制;它将仅受扫描机构的扫描范围的限制。最重要的是，而不是扫描阶段或目标的深度成像，建议ACFM将开发一种新的可调液体板位于目标的图像空间中进行高速深度扫描的对象空间。ACFM中的每个通道一个光纤阵列提供激发照明并收集发射的荧光信号。光纤还将充当共焦针孔以消除失焦光。通过这种独特的配置，所提出的ACFM的共焦头可以非常紧凑和可扩展，特别适合于手持临床应用。 在这项为期三年的计划中，我们将设计、建造和测试一个紧凑的高NA（NA=0.7）和大FOV ACFM，并配备5x 5共焦物镜。物镜将采用光学塑料设计，并使用金刚石车削技术制造。我们将校准系统，测量横向和轴向分辨率，并通过对小鼠组织样本进行成像来证明系统的能力。 这项工作需要生物医学工程、光学工程和制造、系统工程和电子学等各个领域的跨学科合作。我们将从开发明场阵列显微镜中吸取的经验应用于ACFM的拟议开发。 如果成功的话，所提出的ACFM将对生物医学成像产生重大影响，特别是在大FOV和整个载玻片成像的体内临床应用中。建议ACFM可用作其他成像模式，如共焦拉曼显微镜，多光子显微镜，和高光谱显微镜的可扩展平台。它还将成为更先进的成像应用平台，如并行深度成像和多波段荧光成像。这项研究将提供一个极好的机会，培养下一代跨学科的科学家和工程师，在本科和研究生水平。