Cell Biophysics Laboratory, Centre for Micro-Photonics, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Victoria 3122, Australia.
Methods Appl Fluoresc. 2016 May 23;4(2):024005. doi: 10.1088/2050-6120/4/2/024005.
The anisotropy decay of a fluorescently-labelled macromolecule provides information on the internal and global dynamics of the macromolecule. Weber was a pioneer of fluorescent probes, polarization and polarized phase-modulation methods and revealed the power of combining or comparing these methods to disentangle complex modes of emission depolarization. In this paper we take a similar course and show that when measurements of dynamic depolarization are combined with steady-state anisotropy, complex anisotropy decays can be deduced from measurements at a single modulation frequency. Specifically, a double exponential anisotropy decay can be resolved by combining one of the polarized emission phasors with the steady-state anisotropy. The key is the polarized phasor ellipse plot which provides a convenient visualisation aid and reduces the dimensionality of the minimisation problem from three variables to one variable. We illustrate these concepts with an experimental measurement of the anisotropy decay of a small cytoplasmic fluorescent probe in live cells.
荧光标记的生物大分子的各向异性衰减提供了关于生物大分子内部和整体动力学的信息。Weber 是荧光探针、偏振和偏振相位调制方法的先驱，他揭示了结合或比较这些方法以解耦复杂发射去偏振模式的威力。在本文中，我们采取了类似的方法，并表明当动态去偏振测量与稳态各向异性结合时，可以从单个调制频率的测量中推断出复杂的各向异性衰减。具体来说，通过将极化发射相量之一与稳态各向异性相结合，可以分辨出双指数各向异性衰减。关键是极化相量椭圆图，它提供了一个方便的可视化辅助工具，并将最小化问题的维数从三个变量减少到一个变量。我们用活细胞中小细胞质荧光探针的各向异性衰减的实验测量来说明这些概念。
