Ray tracing analysis of inclined illumination techniques

The reduction of out of focus signal is a general task in fluorescence microscopy and is especially important in the recently developed super-resolution techniques because of the degradation of the final image. Several illumination methods have been developed to provide decreased out of focus signal level relative to the common epifluorescent illumination. In this paper we examine the highly inclined and the total internal reflection illumination techniques using the ray tracing method. Two merit functions were introduced for the quantitative description of the excitation of the selected region. We studied the feasibility of illumination methods, and the required corrections arising from the imperfections of the optical elements.