HIGH RESOLUTION 4-DIMENSIONAL FLUORESCENCE MICROSCOPY

高分辨率 4 维荧光显微镜

• 批准号: 6387272
• 负责人: WALTER A. CARRINGTON
• 金额: $ 27.13万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387272
• 关键词: bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; cell components; charge coupled device camera; computer program /software; computer system design /evaluation; fluorescence microscopy; green fluorescent proteins; image enhancement; video recording system

The Green Fluorescent Protein (GFP) has presented great opportunities for the use of fluorescence for the study of dynamic cellular processes in living cells. It has also presented great challenges for imaging technologies. This Bioengineering Research Grant proposes to develop a high resolution, high-speed fluorescence microscope for imaging single molecules or small aggregates of molecules moving at the speeds of molecular motors. This instrument will provide 3-D movies with the high temporal and spatial resolution that is needed for live cell imaging. It will be based on a fast, sensitive, low noise 640 x 480 CCD camera, wide field illumination and fast focus change. Spatial resolution of 100 nanometers (transverse) will be obtained by the use of computational methods called image restoration or deconvoluion. It will obtain images at rates of 94 frames per second for full frames images and faster for smaller images (188 frames/second) with 65 percent quantum efficiency and 8.5 electron readout noise. It will obtain 3-D images at rates up to 36 high-resolution 3-D images per second. High pixel rates are needed to provide an image at the speeds needed to follow motion at speeds up to 1 to 2 microns per second. This camera operates at about 30 Megapixels/sec, but since the whole sample is illuminated for the full frame time, fluorescence saturation does not limit signals. Signal levels in scanning confocal microscopes are severely limited by fluorescence saturation at these scanning rates. Sensitivity will be high enough to detect and image single molecules in a cultured cell's cytoplasm and to detect and image small clusters of proteins in parts of the cell with higher auto-fluorescence. Fluorescence will be calibrated so that small numbers of fluorescent proteins can be accurately counted. Imaging protocols will be developed and tested for optimizing the tasks of detecting, imaging and counting small numbers of proteins whether stationary or in motion at the speeds of molecular motors. This system will produce one gigabyte of data in less than 20 seconds. Software will be developed for proving nearly instantaneous feedback to the experimenter on the quality of this large stream of data. Sophisticated image restoration, volume visualization and data analysis software will be provided at the microscope for this purpose.

绿色荧光蛋白（GFP）表现出色 使用荧光进行动态细胞研究的机会 活细胞中的过程。它也提出了成像的巨大挑战 技术。这项生物工程研究补助金建议发展高 分辨率，高速荧光显微镜，用于成像单分子或 小分子的小聚集体以分子电动机的速度移动。这 乐器将提供3-D电影的时间和空间 活细胞成像所需的分辨率。它将基于快速 敏感，低噪声640 x 480 CCD摄像头，宽场照明和快速 重点更改。 100纳米（横向）的空间分辨率将是 通过使用称为图像修复的计算方法或 Deconvoluion。它将以每秒94帧的速率获得图像 框架图像和更快的较小图像（188帧/秒），速度为65％ 量子效率和8.5电子读数噪声。它将在 每秒最多36个高分辨率3-D图像的速率。 需要高像素率才能以遵循所需的速度提供图像 以每秒1到2微米的速度运动。该相机在大约 30百万像素/秒，但由于整个样品为全帧照亮 时间，荧光饱和度不会限制信号。扫描中的信号水平 共聚焦显微镜受到这些荧光饱和的严重限制 扫描率。 灵敏度将足够高以检测和图像单分子 培养的细胞的细胞质并检测和图像蛋白质的小簇 具有较高自动荧光的细胞部分。荧光将是 经过校准，因此可以准确地进行少量的荧光蛋白 计数。将开发和测试成像协议以优化 检测，成像和计数少量蛋白质的任务是否存在 静止或以分子电动机的速度运动。 该系统将在不到20秒的时间内生成一GB的数据。软件 将开发用于证明对实验者的几乎瞬时反馈 关于这一大数据流的质量。复杂的图像修复， 数量可视化和数据分析软件将在 为此目的的显微镜。