Development of An Extended-Depth-of-Focus Microscope to Enable Fast Fluorescence Imaging of 3D Live-Cell Dynamics

开发扩展焦深显微镜以实现 3D 活细胞动力学的快速荧光成像

• 批准号: 0096723
• 负责人: Carol Cogswell
• 金额: $ 40.84万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0096723&HistoricalAwards=false
• 关键词: Development; Extended; Depth; Focus; Microscope

The goal of this project is to further develop and evaluate a new kind of fluorescence microscope that will have sufficiently improved the acquisition rate and depth of field of images fast enough, and with enough depth resolution, to investigate live-cell dynamic processes that occur too rapidly to be imaged with existing confocal or widefield deconvolution microscopes. The new instrument, called an extended-depth-of-focus (EDF) microscope, is based on a novel approach to optical design known as wavefront coding, in which a specially designed optical element is inserted into a standard fluorescence microscope to encode the image recorded by a sensitive CCD camera. This CCD image is then decoded using a specially designed, very fast digital filtering process. If the proposed instrument is fully successful, it will provide an image with 15 to 20 times the normal depth of focus, with no loss in image quality. Existing commercial microscopes need to acquire multiple images from successive planes of focus and then combine them using digitally. Because of the extended time required to acquire the multiple images, the sample is exposed to intense light for a much longer period. This accelerates the rate of bleaching of the fluorescent dyes used for labeling specific cell components and also increases potential for significant damage to living preparations. Thus, users of commercial confocal, deconvolution or even two-photon microscopes often acquire only a few images when trying to track moving cell components over time. This compromise means that objects of interest within a cell often move out of focus too rapidly to be recorded. Preliminary results show that the new EDF microscope has the potential to overcome these problems and will be able to rapidly produce images (potentially at video rates) in which all regions throughout the three-dimensional cell volume are sharply focused. The proposed research will expand on these preliminary results and develop new wavefront coding optical elements to optimally convert a commercial fluorescence microscope into an EDF system. As part of the project, the EDF results will be compared to images of the same test objects and biological preparations obtained with deconvolution and confocal microscopes to ensure rigorous evaluation of the results. The microscope improvements to be developed should produce an instrument that can out perform existing commercial fluorescence systems in many biological applications.

该项目的目标是进一步开发和评估一种新的荧光显微镜，它将以足够快的速度和足够的深度分辨率充分提高图像的获取速度和景深，以研究发生得太快而无法用现有的共焦或宽场去卷积显微镜成像的活细胞动态过程。这种名为扩展焦深(EDF)显微镜的新仪器基于一种名为波前编码的新光学设计方法，即在标准荧光显微镜中插入一个特殊设计的光学元件，对由灵敏的CCD相机记录的图像进行编码。然后，使用专门设计的、非常快速的数字滤波过程对这幅ccd图像进行解码。如果建议的仪器完全成功，它将提供15到20倍于正常焦深的图像，图像质量不会有任何损失。现有的商用显微镜需要从连续的焦平面获取多幅图像，然后使用数字方式将它们组合在一起。由于获取多个图像所需的时间延长，样品暴露在强光下的时间要长得多。这加快了用于标记特定细胞成分的荧光染料的漂白速度，也增加了对活性制剂造成重大损害的可能性。因此，商用共焦、去卷积甚至双光子显微镜的用户在试图跟踪随时间移动的细胞成分时，通常只获得几个图像。这种折衷意味着单元格中感兴趣的对象经常以太快的速度移出焦点而无法记录。初步结果表明，新的EDF显微镜具有克服这些问题的潜力，并将能够快速产生图像(可能以视频速率)，其中整个三维细胞体积的所有区域都被清晰地聚焦。这项拟议的研究将在这些初步结果的基础上扩展，并开发新的波前编码光学元件，以最佳地将商业荧光显微镜转换为EDF系统。作为该项目的一部分，EDF结果将与用去卷积和共焦显微镜获得的相同测试对象和生物制剂的图像进行比较，以确保对结果进行严格评估。即将开发的显微镜改进应该会产生一种仪器，在许多生物应用中，它可以超越现有的商业荧光系统。