A mirror-based 2D isotropic illumination in total internal reflection fluorescence microscopy

Abstract

In a total internal reflection fluorescence microscope, there is a 3D anisotropy in intensity of the excitation light. Using a mirror in a prism-based total internal reflection fluorescence microscope, we propose a very low-cost method to decrease the anisotropic property of the excitation light. Theoretically, we obtained the electric field of the transmitted light inside the sample as a function of the rotational angle of the mirror, polarization degree of the incident beam, and the refractive indices of the prism, immersion oil, cover glass, and sample. By using the refractive indices that are commercially available, proper practical combinations of the angle of mirror and polarization degree of the incident light are suggested to provide a merely 2D isotropic excitation light at the sample plane of a total internal reflection fluorescence microscope.