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

该项目的目标是进一步开发和评估一种新型荧光显微镜，该显微镜将足够快地提高图像的采集率和景深，并具有足够的深度分辨率，以研究活细胞动态过程，这些过程发生得太快，无法用现有的共聚焦或宽视场反褶积显微镜成像。这种新仪器被称为扩展焦深（EDF）显微镜，它基于一种被称为波前编码的新型光学设计方法，在这种方法中，一个特殊设计的光学元件被插入到标准的荧光显微镜中，对灵敏的CCD相机记录的图像进行编码。这个CCD图像然后解码使用一个特别设计的，非常快的数字滤波过程。如果提议的仪器完全成功，它将提供15到20倍的正常聚焦深度的图像，而不会损失图像质量。现有的商用显微镜需要从连续的聚焦平面获取多个图像，然后使用数字技术将它们组合起来。由于获得多幅图像所需的时间延长，样品暴露在强光下的时间要长得多。这加速了用于标记特定细胞成分的荧光染料的漂白速度，也增加了对活制剂的重大损害的可能性。因此，商业共聚焦、反卷积甚至双光子显微镜的用户在试图追踪移动的细胞成分时，往往只能获得少量图像。这种折衷意味着细胞内感兴趣的物体往往会迅速移出焦点而无法被记录。初步结果表明，新的EDF显微镜有潜力克服这些问题，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是锐利聚焦的。该研究将扩展这些初步结果，并开发新的波前编码光学元件，以最佳地将商用荧光显微镜转换为EDF系统。作为该项目的一部分，EDF结果将与使用反卷积和共聚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。显微镜的改进有待开发，应该产生一种仪器，可以执行现有的商业荧光系统在许多生物应用。

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.