Stray light rejection by structured illumination

Structured illumination microscopy (SIM) combines confocal microscopy and wide field microscopy. By illuminating the specimen with a grid which is shifted in phase, it is possible to discriminate out of focus information without lateral scanning. A problem of structured illumination is that the visibility of the illumination pattern drops significantly for high numerical apertures (NA). The reason for low visibilities is the modulation transfer function (MTF) of the objectives which reveals the ability of transferring a modulation frequency of an object to an image. The MTF for incoherent illumination is not as good as for coherent light. For high NA imaging, a fiber based method to create the illumination grid by interference of two plane waves was developed. The illumination grid is projected on the specimen and not as in classical structured illumination microscopy imaged onto it. The visibilities of the grid are by far higher with this technique.