Optical Sciences Centre, Department of Physics and Astronomy, School of Science, Computing and Engineering Technologies, Swinburne University of Technology, Hawthorn, Melbourne, VIC 3122, Australia.
Int J Mol Sci. 2024 Oct 17;25(20):11165. doi: 10.3390/ijms252011165.
Fluorescence lifetime imaging microscopy is sensitive to molecular interactions and environments. In homo-dyne frequency-domain fluorescence lifetime imaging microscopy, images of fluorescence objects are acquired at different phase settings of the detector. The detected intensity as a function of detector phase is a sinusoidal function that is sensitive to the lifetime of the fluorescent species. In this paper, the theory of phase-sensitive fluorescence image correlation spectroscopy is described. In this version of lifetime imaging, image correlation spectroscopy analysis (i.e., spatial autocorrelation) is applied to successive fluorescence images acquired at different phase settings of the detector. Simulations of different types of lifetime distributions reveal that the phase-dependent density of fluorescent objects is dependent on the heterogeneity of lifetimes present in the objects. We provide an example of this analysis workflow to a cervical cancer cell stained with a fluorescent membrane probe.
荧光寿命成像显微镜对分子相互作用和环境敏感。在同频域荧光寿命成像显微镜中，荧光物体的图像在探测器的不同相位设置下获取。作为探测器相位函数的检测强度是对荧光物质寿命敏感的正弦函数。本文描述了相敏荧光图像相关光谱学的理论。在这种寿命成像中，图像相关光谱分析（即空间自相关）应用于在探测器的不同相位设置下获取的连续荧光图像。对不同类型的寿命分布的模拟表明，与相位相关的荧光物体的密度取决于物体中存在的寿命异质性。我们提供了一个使用荧光膜探针染色的宫颈癌细胞的分析工作流程示例。
