The limitations of nonlinear fluorescence effect in super resolution saturated structured illumination microscopy system

Classically, optical systems are considered to have a fundamental resolution limit due to diffraction. Many strategies for improving both axial and lateral resolutions are based on a priori information about the input signal. These strategies lead to a numerical aperture improvement. However these are still limited by the wave nature of light. By using fluorescence technique one theoretically can reach unlimited resolution. The key point is to use the nonlinear dependence of the fluorescence emission rate on the intensity of the applied illumination. In this paper we present simulation as well as experimental results which show the advantage and the problems of using the nonlinear fluorescence effect in super resolution systems as well as discussing the nonlinear phenomena concerning the fluorescence process. The results show that the nonlinear fluorescence effect is accompanied by severe quenching, bleaching and saturation phenomena. As consequence, super resolution using saturated structured illumination method in living biological samples becomes severely restricted.