Array Confocal Fluorescence Microscope

描述(申请人提供)：由于生物样品发出的荧光信号量有限，点扫描共聚焦荧光显微镜的成像速度受到基本限制。为了提高扫描速度，过去已经开发了几种技术，如尼普考旋转盘共焦显微镜和线扫描共焦显微镜。然而，对于大体积的扫描，它们的速度仍然受到显微镜物镜的视场(FOV)的限制。此外，它们很难被微型化成便携式手持设备。 我们建议开发一种阵列共焦荧光显微镜(ACFM)，它可以在大视场下以比传统共焦显微镜快一个数量级的速度成像大3D体积。建议的ACFM由一组微型高NA共焦荧光物镜组成，每个物镜扫描一个小的亚视场。多点扫描和检测不仅可以显著提高整体扫描速度，而且可以显著减少活细胞中的光漂白或光毒性，因为它需要较低水平的单位面积LiHT强度。提出的ACFM的视场不受单个目标视场的限制，只受扫描机构扫描范围的限制。最重要的是，与扫描工作台或物镜进行深度成像不同，所提出的ACFM将开发一种位于物镜成像空间的新型可调液板，以在物镜空间进行高速深度扫描。ACFM中的每个通道对应一个光纤阵列，提供激励照明并收集发射的荧光信号。光纤还将充当共焦针孔，以消除散焦光。有了这种独特的配置，建议的ACFM的共焦头可以非常紧凑和可扩展，特别适合手持临床应用。 在这项拟议的三年工作中，我们将设计、建造和测试具有5x5共焦物镜的紧凑型高NA(NA=0.7)和大视场ACFM。物镜将用光学塑料设计，并使用钻石车削技术制造。我们将校准系统，测量横向和轴向分辨率，并通过成像小鼠组织样本来展示系统的能力。 这一努力将需要生物医学工程、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。我们将把发展明视场阵列显微镜的经验应用到ACFM的拟议发展中。 如果成功，提出的ACFM将对生物医学成像产生重大影响，特别是在大视野和全载玻片成像的活体临床应用中。所提出的ACFM可以用作其他成像方式的可扩展平台，例如共焦拉曼显微镜、多光子显微镜和高光谱显微镜。它还将成为更先进的成像应用的平台，如并行深度成像和多波段荧光成像。这项研究将为培养下一代本科生和研究生水平的跨学科科学家和工程师提供一个极好的机会。

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.