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Fluorescence ptychographic microscopy for 4D live-cell imaging with high-spatiotemporal resolution and reduced phototoxicity

用于 4D 活细胞成像的荧光叠层照相显微镜，具有高时空分辨率和降低的光毒性

基本信息

批准号：
9332392
负责人：
Guoan Zheng
金额：
$ 7.39万
依托单位：
UNIVERSITY OF CONNECTICUT STORRS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2019-05-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY/ABSTRACT 4D fluorescence microscopy is considered the workhorse for biomedical research. It provides a window into the spatially complex, temporally evolving physiology of living specimens with high specificity. The knowledge gained from the 4D space-time data has also facilitated the discovery of informative biomarkers that can be used for the early detection of diseases and for the development of drugs directed at new therapeutic targets. However, many cellular structures and events are below the spatial resolution limit of traditional fluorescence microscope and happen on sub-second time scale, putting them beyond our ability to investigate in greater detail. In addition, observing the fast evolving dynamic process over the entire 3D volume involves inevitable tradeoffs on spatial resolution, temporal resolution, sample-induced aberrations, and phototoxicity. This last point is especially important for 4D live-cell imaging, where phototoxicity caused by the high level of free radicals would alter the physiological state of living specimens. In this project, we propose to develop a super-resolution imaging method, termed fluorescence ptychographic microscopy, for 4D live cell imaging with high spatiotemporal resolution and reduced phototoxicity. The proposed approach will be built upon the structured illumination microscopy (SIM) technique that combines multiple acquisitions under sinusoidal illumination patterns for super-resolution imaging. We aim to significantly shorten the acquisition time of the SIM approach by using a modified ptychographic recovery procedure developed in the PI’s lab. More importantly, we will also develop a ptychographic procedure that is able to correct for the unknown distortions of the sinusoidal patterns and compensate for the sample-induce wavefront aberrations. If successful, the proposed imaging procedure would provide a turnkey solution for 4D fluorescence microscopy with sub-diffraction spatial resolution and sub-second temporal resolution, with minimum invasiveness for the living specimen, and is able to compensate for sample-induced aberrations. Our long term goal is to translate advanced imaging technologies for the broad biomedical and clinical communities.

项目摘要/摘要 4D荧光显微镜被认为是生物医学研究的主力。它提供了一扇进入具有高度特异性的活体标本在空间上复杂的、随时间演变的生理学。这个从4D时空数据中获得的知识也促进了信息生物标志物的发现可用于疾病的早期检测和针对新的药物的开发治疗靶点。然而，许多蜂窝结构和事件都低于空间分辨率限制传统的荧光显微镜和发生在亚秒级的时间尺度，使它们超出了我们的能力更详细地调查。此外，在整个3D上观察快速演变的动态过程体积涉及到在空间分辨率、时间分辨率、样品诱导的像差、和光毒性。最后这一点对于4D活细胞成像尤其重要，在4D活细胞成像中，光毒性会导致高水平的自由基会改变活标本的生理状态。在这个项目中，我们建议开发一种超分辨率成像方法，称为荧光层析显微镜，用于 4D活细胞成像具有较高的时空分辨率和较低的光毒性。建议的方法将建立在结构照明显微镜(SIM)技术的基础上，该技术结合了多项采集在正弦照明模式下进行超分辨率成像。我们的目标是显著缩短使用改进的层析恢复程序获得SIM方法的采集时间派的实验室。更重要的是，我们还将开发一种能够纠正正弦图案的未知失真，并补偿样品诱导的波前像差。如果成功，拟议的成像程序将为4D荧光提供一个交钥匙解决方案具有亚衍射空间分辨率和亚秒时间分辨率的显微镜，最小对活体标本具有侵入性，并能够补偿样品引起的像差。我们的龙学期目标是将先进的成像技术转化为广泛的生物医学和临床社区。