Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.

荧光检测线性二色性显微镜可以观察活细胞中的各种分子过程，并获得与细胞特征相关的荧光分子方向的定量信息。这些信息可以提供对蛋白质结构的深入了解，帮助开发基因编码探针，并允许确定脂质膜特性。然而，定量和解释生物系统中的线性二色性一直是费力且不可靠的。在这里，我们展示了一套基于 ImageJ 的开源软件工具，即使在生命系统中，也可以快速轻松地进行线性二色性可视化和定量，以及提取分子方向的定量信息。这些工具在合成脂质囊泡模型上进行了测试，并应用于各种生物系统，包括使用荧光蛋白观察活细胞中 G 蛋白信号传导过程中的构象变化。我们的结果表明，我们的工具和模型系统适用于广泛的分子和偏振分辨显微镜技术，并且代表着使偏振显微镜成为生物成像主流工具的重要一步。