Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements
Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements
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DOI：
10.1002/cphc.200600638
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发表时间：
2007-02-19
期刊：
影响因子：
2.9
通讯作者：
Enderlein, Joerg
中科院分区：
文献类型：
--
作者：
Dertinger, Thomas;Pacheco, Victor;Enderlein, Joerg
我们提出了一种新的方法来测量绝对扩散系数在纳摩尔浓度与高精度。基于改进的荧光相关光谱（ECS）设置，该方法是通过引入外部标尺测量扩散时间，通过产生两个横向移动和重叠的失败者焦点在一个固定的和已知的距离。数据拟合是由一个新的双参数模型来描述的分子检测功能（ESTA）。我们提出了6个记录和显示良好的协议与拟合模型。我们在各种条件下测量红色荧光染料Atto 655的扩散系数，并将这些值与通过梯度脉冲场NMR（GPF NMR）实现的值进行比较。从这些测量中，我们得出结论，新的测量方案是强大的对光学和物理伪影是固有的标准FCS。使用双焦点FCS，Atto 655在25 ℃水中的扩散系数为4.26 x 10 - 6 cm（2）s（-1），与GPF NMR值4.28 x 10-6 cm（2）s（-1）相当。
We present a new method to measure absolute diffusion coefficients at nanomolar concentrations with high precision. Based on a modified fluorescence correlation spectroscopy (ECS)-setup, this method is improved by introducing an external ruler for measuring the diffusion time by generating two laterally shifted and overlapping loser foci at a fixed and known distance. Data fitting is facilitated by a new two-parameter model to describe the molecule detection function (MDF). We present 6 recorded MDF and show the excellent agreement with the fitting model. We measure the diffusion coefficient of the red fluorescent dye Atto655 under various conditions and compare these values with a value achieved by gradient pulsed field NMR (GPF NMR). From these measurements we conclude, that the new measurement scheme is robust against optical and photophysical artefacts which are inherent to standard FCS. With two-focus-FCS, the diffusion coefficient of 4.26 x 10(-6) cm(2) s(-1) for Atto655 in water at 25 degrees C compares well with the GPF NMR value of 4.28 x 10-6 cm(2) s(-1).