Qin Qin, Laub Shannon, Shi Yiwen, Ouyang Mingxing, Peng Qin, Zhang Jin, Wang Yingxiao, Lu Shaoying
Department of Bioengineering, Institute of Engineering in Medicine, University of California, San Diego, San Diego, CA, United States.
Department of Mathematics, Center of Computational Mathematics, University of California, San Diego, San Diego, CA, United State.
Front Phys. 2019 Oct;7. doi: 10.3389/fphy.2019.00154. Epub 2019 Oct 23.
Spatiotemporal regulation of molecular activities dictates cellular function and fate. Investigation of dynamic molecular activities in live cells often requires the visualization and quantitation of fluorescent ratio image sequences with subcellular resolution and in high throughput. Hence, there is a great need for convenient software tools specifically designed with these capabilities. Here we describe a well-characterized open-source software package, , customized to visualize pixelwise ratiometric images and calculate ratio time courses with subcellular resolution and in high throughput. also provides group statistics and kinetic analysis functions for the quantified time courses, as well as 3D structure and function visualization for ratio images. The application of is demonstrated by the ratiometric analysis of intensity images for several single-chain Förster (or fluorescence) resonance energy transfer (FRET)-based biosensors, allowing efficient quantification of dynamic molecular activities in a heterogeneous population of single live cells. Our analysis revealed distinct activation kinetics of Fyn kinase in the cytosolic and membrane compartments, and visualized a 4D spatiotemporal distribution of epigenetic signals in mitotic cells. Therefore, provides an integrated environment for ratiometric live-cell image visualization and analysis, which generates high-quality single-cell dynamic data and allows the quantitative machine-learning of biophysical and biochemical computational models for molecular regulations in cells and tissues.
分子活动的时空调节决定了细胞的功能和命运。对活细胞中动态分子活动的研究通常需要以亚细胞分辨率和高通量对荧光比率图像序列进行可视化和定量分析。因此，迫切需要专门设计具有这些功能的便捷软件工具。在这里，我们描述了一个经过充分表征的开源软件包，它经过定制，可用于可视化逐像素比率图像，并以亚细胞分辨率和高通量计算比率时间进程。该软件包还为量化的时间进程提供组统计和动力学分析功能，以及比率图像的三维结构和功能可视化。通过对几种基于单链福斯特（或荧光）共振能量转移（FRET）的生物传感器的强度图像进行比率分析，展示了该软件包的应用，从而能够有效地量化单个活细胞异质群体中的动态分子活动。我们的分析揭示了Fyn激酶在细胞质和膜区室中的不同激活动力学，并可视化了有丝分裂细胞中表观遗传信号的四维时空分布。因此，该软件包为比率活细胞图像可视化和分析提供了一个集成环境，可生成高质量的单细胞动态数据，并允许对细胞和组织中分子调控的生物物理和生化计算模型进行定量机器学习。
