Transmission structured illumination microscopy with tunable frequency illumination using tilt mirror assembly

Abstract

AbstractWe present experimental demonstration of tilt-mirror assisted transmission structured illumination microscopy (tSIM) that offers a large field of view super resolution imaging. An assembly of custom-designed tilt-mirrors are employed as the illumination module where the sample is excited with the interference of two beams reflected from the opposite pair of mirror facets. Tunable frequency structured patterns are generated by changing the mirror-tilt angle and the hexagonal-symmetric arrangement is considered for the isotropic resolution in three orientations. Utilizing high numerical aperture (NA) objective in standard SIM provides super-resolution compromising with the field-of-view (FOV). Employing low NA (20X/0.4) objective lens detection, we experimentally demonstrate $$\sim$$ ∼ (0.56 mm$$\times$$ × 0.35 mm) size single FOV image with $$\sim$$ ∼ 1.7- and $$\sim$$ ∼ 2.4-fold resolution improvement (exploiting various illumination by tuning tilt-mirrors) over the diffraction limit. The results are verified both for the fluorescent beads as well as biological samples. The tSIM geometry decouples the illumination and the collection light paths consequently enabling free change of the imaging objective lens without influencing the spatial frequency of the illumination pattern that are defined by the tilt-mirrors. The large and scalable FOV supported by tSIM will find usage for applications where scanning large areas are necessary as in pathology and applications where images must be correlated both in space and time.