Opt Lett. 2020 Jun 1;45(11):3001-3004. doi: 10.1364/OL.394116.
Being the established imaging tool for cell membrane-associated studies, total internal reflection fluorescence microscopy (TIRFM) still has some limitations. The most important one is the inhomogeneous evanescent excitation field mainly caused by the large-angle and fixed-azimuth illumination scheme, which can be eliminated by using ring-shaped illumination (ring TIRFM). However, it is challenging in assembling a ring TIRFM system with precise parameter control that works well. Here we emphasize the quantification of the ring TIRFM system and introduce a robust calibration routine to simultaneously rectify the asymmetry of the spinning light beam and determine the crucial experimental parameter, i.e., the incident angle. The calibration routine requires no specific sample preparation and is entirely based on the automatic back focal plane manipulation, avoiding possible errors caused by the sample difference and manual measurement. Its effectiveness is experimentally demonstrated by both the qualitative and quantitative comparisons of the images acquired using different samples, illumination schemes, and calibration approaches. These characteristics should enable our approach to greatly improve the practicability of TIRFM in life sciences.
作为细胞膜相关研究的既定成像工具，全内反射荧光显微镜（TIRFM）仍然存在一些限制。最重要的限制是由大角度和固定方位照明方案引起的非均匀消逝场，这可以通过使用环形照明（环形 TIRFM）来消除。然而，组装具有良好工作性能的精确参数控制的环形 TIRFM 系统具有挑战性。在这里，我们强调了环形 TIRFM 系统的定量，并介绍了一种强大的校准程序，该程序可以同时校正旋转光束的不对称性并确定关键的实验参数，即入射角。该校准程序不需要特定的样品制备，完全基于自动后焦面操作，避免了由于样品差异和手动测量引起的可能误差。通过使用不同的样品、照明方案和校准方法获得的图像的定性和定量比较，实验证明了该方法的有效性。这些特性应该使我们的方法大大提高 TIRFM 在生命科学中的实用性。
