Department of Chemical Engineering and Biotechnology, University of Cambridge, Philippa Fawcett Drive, Cambridge, CB3 0AS, United Kingdom.
Methods Appl Fluoresc. 2017 Dec 19;6(1):014004. doi: 10.1088/2050-6120/aa872e.
Fluorescence anisotropy imaging microscopy (FAIM) measures the depolarization properties of fluorophores to deduce molecular changes in their environment. For successful FAIM, several design principles have to be considered and a thorough system-specific calibration protocol is paramount. One important calibration parameter is the G factor, which describes the system-induced errors for different polarization states of light. The determination and calibration of the G factor is discussed in detail in this article. We present a novel measurement strategy, which is particularly suitable for FAIM with high numerical aperture objectives operating in TIRF illumination mode. The method makes use of evanescent fields that excite the sample with a polarization direction perpendicular to the image plane. Furthermore, we have developed an ImageJ/Fiji plugin, AniCalc, for FAIM data processing. We demonstrate the capabilities of our TIRF-FAIM system by measuring [Formula: see text]-actin polymerization in human embryonic kidney cells and in retinal neurons.
荧光各向异性成像显微镜（FAIM）测量荧光染料的退偏特性，以推断其环境中的分子变化。要成功进行 FAIM，必须考虑几个设计原则，并且彻底的系统特定校准协议至关重要。一个重要的校准参数是 G 因子，它描述了光的不同偏振状态下系统引起的误差。本文详细讨论了 G 因子的确定和校准。我们提出了一种新的测量策略，特别适用于在 TIRF 照明模式下工作的具有高数值孔径物镜的 FAIM。该方法利用倏逝场以与图像平面垂直的偏振方向激发样品。此外，我们还为 FAIM 数据处理开发了一个 ImageJ/Fiji 插件 AniCalc。我们通过测量人胚肾细胞和视网膜神经元中的 [Formula: see text]-肌动蛋白聚合来演示我们的 TIRF-FAIM 系统的功能。
