Two‐dimensional structured illumination microscopy

In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms 'quasi-confocal images' can be derived from a given number of such phase-images. Here, we present an alternative structured illumination microscopy approach, which employs two-dimensional patterns instead of a one-dimensional one. While in one-dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two-dimensional approach it is shifted at a single, pattern-dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern-shift and -rotation. Moreover, our two-dimensional approach also yields a better signal-to-noise ratio in the evaluated image.