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.
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