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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演讲 MO 荧光相关光谱（Magde 等人，1972 年，Appl. 物理。 莱特。 29:705）在共焦显微镜几何结构中长期以来 建立无创超灵敏方法来测定 荧光标记的扩散特性和相互作用动力学 生物分子，例如 DNA、RNA 和蛋白质。 将此方法应用到 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可能性 荧光团的大量光漂白。 最有前途的方法 抑制非特异性光并减少漂白是多光子 激发（Denk et al. 1990, Science 248:73），能够 仅在它们可以贡献的空间区域激发荧光团 到检测信号。 我们的研究将两种替代方案进行了比较 关于它们对细胞内 FCS 的适用性。 我们证明了 通常多光子技术的主要缺点是脉冲激发 与寿命相比，暗期较长（12ns） 荧光团的激发态，可以通过加倍或 将脉冲重复率提高四倍。 关键的测量 参数，从单次收集的平均光子数 荧光分子在物镜中停留的时间 从而可以将焦体积推至高于 2-3 的值， 足以执行可靠的超灵敏荧光 相关性分析。