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首页> 外文会议>Confocal, Multiphoton, and Nonlinear Microscopic Imaging III; Progress in Biomedical Optics and Imaging; vol.8 no.43; Proceedings of SPIE-The International Society for Optical Engineering; vol.6630 >Determination of the Confocal Volume for Quantitative Fluorescence Correlation Spectroscopy
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Determination of the Confocal Volume for Quantitative Fluorescence Correlation Spectroscopy

机译：定量荧光相关光谱法测定共聚焦体积

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Single molecule detection methods, in particular those based on fluorescent labels offer the possibility to gain not only qualitative but also quantitative insight into the function of complex biological systems. Fluorescence Correlation Spectroscopy (FCS) is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness in the pico- to nano-Molar concentration range, with broad applications in Biology and Chemistry. Although FCS in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts FCS to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by FCS-curve fitting, a FCS dilution series and confocal bead scanning. The results are compared in the view of quantitative FCS measurement and analysis.
机译：单分子检测方法，尤其是那些基于荧光标记的方法，不仅可以对复杂的生物系统的功能进行定性而且可以定量的了解。荧光相关光谱法（FCS）是确定浓度，扩散常数以及皮摩尔级至纳米摩尔浓度范围内分子亮度的常用技术之一，在生物学和化学领域具有广泛的应用。尽管FCS原则上具有测量绝对浓度和扩散系数的潜力，但是了解共焦体积的确切大小和形状的必要性常常会妨碍获得定量结果的可能性，并将FCS主要限制在相对测量范围内。共焦体积的原位测定很困难，因为它对光学标本和像差，光学饱和度以及在生物样本中观察到的折射率变化敏感。在目前的贡献中，我们比较了不同的技术来表征共焦体积并通过FCS曲线拟合，FCS稀释系列和共焦珠扫描获得共焦参数。从定量FCS测量和分析的角度比较了结果。

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