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

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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.