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• 批准号: 6349475
• 负责人: WATT W WEBB
• 金额: $ 0.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6349475
• 关键词: bioengineering /biomedical engineering; biomedical resource; education; technology /technique

Talk at the Biophysical Society Meeting, Feb 22-26 in Kansas City MO Fluorescence correlation spectroscopy (Magde et al. 1972, Appl. Phys. Lett. 29:705) in confocal microscope geometries has long been established as noninvasive ultrasensitive method to determine diffusion properties and interaction kinetics of fluorescently labeled biomolecules such as DNA, RNA and proteins. To apply this method to living cells, a highly promising aim, however, confocal discrimination of off-focus light like autofluorescence or scattering may not be sufficient for the required signal-to-background ratios. Beyond that, small cellular volumes and slow diffusion increase the probability of massive photobleaching of the fluorophores. The most promising method to suppress nonspecific light and reduce bleaching is multiphoton excitation (Denk et al. 1990, Science 248:73), with the ability to excite fluorophores only in spatial regions where they can contribute to the detection signal. Our study compares both alternatives with respect to their suitability for intracellular FCS . We show that the major drawback of usual multiphoton techniques, pulsed excitation with long dark periods (12ns) compared to the lifetimes of the fluorophore's excited state, can be overcome by doubling or quadrupling the pulse repetition rate. The crucial measurement parameter, the average number of photons collected from single fluorescent molecules during the residence time in the objective's focal volume can thereby be pushed to values higher than 2-3, sufficient for performing reliable ultrasensitive fluorescence correlation analysis.

2月22日至26日在堪萨斯城举行的生物物理学会会议上的讲话 MO荧光相关光谱法（Magde等人1972，Appl. Phys. Lett. 29：705）在共焦显微镜几何结构中长期以来一直是 建立了一种非侵入性的超灵敏方法， 荧光标记物的扩散性质和相互作用动力学 生物分子，如DNA、RNA和蛋白质。 将这种方法应用于 活细胞，一个非常有前途的目标，然而，共焦歧视 像自发荧光或散射这样的离焦光可能不会 足以满足所需的信号背景比。 除此之外， 小细胞体积和缓慢扩散增加了 荧光团的大量光漂白。 最有前途的方法 抑制非特异性光和减少漂白是多光子 激发（Denk等人，1990，Science 248：73），具有 仅在它们可以贡献的空间区域中激发荧光团 检测信号。 我们的研究比较了这两种选择， 就其对细胞内FCS的适用性而言。 我们证明了 通常的多光子技术的主要缺点，脉冲激发， 长的黑暗时期（12纳秒）相比，寿命的 荧光团的激发态，可以通过加倍或 使脉冲重复率提高四倍。 关键的测量 参数，从单个 荧光分子在物镜中的停留时间期间 焦点体积因此可以被推到高于2-3的值， 足以进行可靠的超灵敏荧光 相关分析