Cell Biophysics Laboratory, Department of Physics and Astronomy, Optical Sciences Centre, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, Melbourne 3122, Australia.
Int J Mol Sci. 2023 Jul 31;24(15):12271. doi: 10.3390/ijms241512271.
The dynamics of condensed matter can be measured by the time-dependent Stokes shift of a suitable fluorescent probe. The time-dependent spectral correlation function is typically described by one or more spectral relaxation correlation times, which, in liquid solvents, characterize the timescales of the dipolar relaxation processes around the excited-state probe. The phasor plot provides a powerful approach to represent and analyze time and frequency-domain data acquired as images, thus providing a spatial map of spectral dynamics in a complex structure such as a living cell. Measurements of the phase and modulation at two emission wavelength channels were shown to be sufficient to extract a single excited-state lifetime and a single spectral relaxation correlation time, supplying estimates of the mean rate of excited-state depopulation and the mean rate of spectral shift. In the present contribution, two more issues were addressed. First, the provision of analytic formulae allowing extraction of the initial generalized polarization and the relaxed generalized polarization, which characterize the fluorescence spectrum of the unrelaxed state and the fully relaxed state. Second, improved methods of model discrimination and model parameter extraction for more complex spectral relaxation phenomena. The analysis workflow was illustrated with examples from the literature.
凝聚态物质的动力学可以通过合适的荧光探针的时间相关斯托克斯位移来测量。时间相关的光谱相关函数通常由一个或多个光谱弛豫相关时间来描述，在液体溶剂中，这些时间常数表征了激发态探针周围偶极弛豫过程的时间尺度。相量图提供了一种强大的方法来表示和分析以图像形式获得的时间和频域数据，从而为诸如活细胞等复杂结构中的光谱动力学提供了空间图谱。已经证明，测量两个发射波长通道的相位和调制足以提取单个激发态寿命和单个光谱弛豫相关时间，从而提供激发态去激发的平均速率和光谱位移的平均速率的估计值。在本贡献中，还解决了另外两个问题。首先，提供了分析公式，可以提取初始广义极化和弛豫的广义极化，它们分别表征未弛豫状态和完全弛豫状态的荧光光谱。其次，对于更复杂的光谱弛豫现象，提出了改进的模型判别和模型参数提取方法。通过文献中的示例说明了分析工作流程。
