The application of total internal reflection fluorescence microscopy in single fluorophore molecules axial imaging

Here we present a method of modeling fluorophore molecules as spheres in evanescent fields mathematically instead of the assumption of top-hat function distribution. Based on this model, we discuss that it is possible to calculate relative separation distances of the molecules, and their axial positions and diameters with the total internal reflection fluorescence microscopy (TIRFM) due to its unique intensity pattern in the z direction. In the whole process, we need merely to change the incident angle once, that is to get only two frame imaging of TIRFM.