Total internal reflection fluorescence (TIRF) microscopy because of usage at imaging from surface with 100-200 nm in thickness is a good method for investigation of biological processes in/out of cells, specially in cell membrane proximity. In this paper a prism-based TIRF microscope is designed and operated. For optimizing the experimental setup, a beam expander for collimating light and expanding the illumination surface of sample, and the immersion oil for decreasing the loss due to scattering and absorption are employed. Also, the polarizer and analyzer are used to control the intensity and polarization of light and investigation of the laser power stability in different time and distance intervals. The experimental set up is used for total internal reflection fluorescence imaging of rhodamine (Rd6G) dyes.