Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland ,Galway, University Road, Galway, H91 CF50, Ireland.
J Fluoresc. 2021 May;31(3):835-845. doi: 10.1007/s10895-021-02719-y. Epub 2021 Mar 19.
Fluorescence Correlation Spectroscopy (FCS) studies of the interaction of polymers or proteins in solution are strongly affected by the viscosity and refractive index of the medium, and the effects are likely to be more significant with the use of short wavelength excitation (e.g., 405 nm diode lasers). Failing to account for these issues can lead to incorrect measurement of average size, conformational changes, and dynamic behaviour of polymers and proteins. Steady-state, time-resolved, and FCS measurements of Alexa 405 in glycerol:water mixtures were performed to determine its suitability for FCS measurements with 405 nm excitation. The effects of the refractive index and viscosity on the diffusion coefficient and photophysical parameters (lifetime and relative quantum yield) of the fluorophore were determined. Alexa 405 lifetime decreased from 3.55 ns in water to 3.25 ns in a 50:50 glycerol:water mixture, while its diffusion coefficient dropped from 333 ± 16 to 44 ± 1 µms. Lifetime data collected from micromolar solutions of Alexa 405 did however also suggest that as solvent polarity decreased, aggregates (excimers) were formed as evidenced by the appearance of a rising edge in the decay plots. The interdependence between lifetime, refractive index, and diffusion coefficient could be accurately fitted by a simple polynomial function indicating that the probe is well behaved and predictable in the glycerol:water model system. Overall, Alexa 405 is a most promising and reliable probe for FCS measurement using violet laser diode excitation sources.
荧光相关光谱（FCS）研究溶液中聚合物或蛋白质的相互作用强烈受到介质粘度和折射率的影响，并且在使用短波长激发（例如，405nm 二极管激光器）时，这些影响可能更为显著。如果不考虑这些问题，可能会导致对聚合物和蛋白质的平均大小、构象变化和动态行为的不正确测量。为了确定其在 405nm 激发下进行 FCS 测量的适用性，在甘油：水混合物中对 Alexa 405 进行了稳态、时间分辨和 FCS 测量。确定了折射率和粘度对荧光团扩散系数和光物理参数（寿命和相对量子产率）的影响。Alexa 405 的寿命从水中的 3.55ns 降低到 50:50 甘油：水中的 3.25ns，而其扩散系数从 333±16µms 降低到 44±1µms。然而，从微摩尔浓度的 Alexa 405 溶液中收集的寿命数据也表明，随着溶剂极性的降低，形成了聚集体（激基复合物），这表现为衰减曲线中出现上升边缘。寿命、折射率和扩散系数之间的相互依赖性可以通过简单的多项式函数准确拟合，这表明探针在甘油：水模型系统中表现良好且可预测。总的来说，Alexa 405 是一种非常有前途且可靠的用于使用紫激光二极管激发源进行 FCS 测量的探针。
