A significant improvement in acquisition speed of structured illumination microscopy (SIM) opens a new field of applications to this already well-established super-resolution method towards 3D scanning real-time imaging of living cells. We demonstrate a method of increased acquisition speed on a two-beam SIM fluorescence microscope with a lateral resolution of ~100 nm at a maximum raw data acquisition rate of 162 frames per second (fps) with a region of interest of 16.5  ×  16.5 µm, free of mechanically moving components. We use a programmable spatial light modulator (ferroelectric LCOS) which promises precise and rapid control of the excitation pattern in the sample plane. A passive Fourier filter and a segmented azimuthally patterned polarizer are used to perform structured illumination with maximum contrast. Furthermore, the free running mode in a modern sCMOS camera helps to achieve faster data acquisition.

Abstract:We describe a two-beam interference structured illumination fluorescence microscope. The novelty of the presented system lies in its simplicity. A programmable electro-optical spatial light modulator in an intermediate image plane enables precise and rapid control of the excitation pattern in the specimen. The contrast of the projected light pattern is strongly influenced by the polarization state of the light entering the high NA objective. To achieve high contrast, we use a segmented polarizer. Furthermore, a mask with six holes blocks unwanted components in the spatial frequency spectrum of the illumination grating. Both these passive components serve their purpose in a simpler and almost as efficient way as active components. We demonstrate a lateral resolution of 114.2 ± 9.5 nm at a frame rate of 7.6 fps per reconstructed 2D slice.

Structured illumination microscopy (SIM) is a powerful technique for obtaining super-resolved fluorescence maps of samples, but it is very sensitive to aberrations or misalignments affecting the excitation patterns. Here, we present a reconstruction algorithm that is able to process SIM data even if the illuminations are strongly distorted. The approach is an extension of the recent blind-SIM technique, which reconstructs simultaneously the sample and the excitation patterns without a priori information on the latter. Our algorithm was checked on synthetic and experimental data using distorted and nondistorted illuminations. The reconstructions were similar to that obtained by up-to-date SIM methods when the illuminations were periodic and remained artifact-free when the illuminations were strongly distorted.

The microscope image of a thick fluorescent sample taken at a given focal plane is plagued by out-of-focus fluorescence and diffraction limited resolution. In this work, we show that a single slice of Structured Illumination Microscopy (two or three beam SIM) data can be processed to provide an image exhibiting tight sectioning and high transverse resolution. Our reconstruction algorithm is adapted from the blind-SIM technique which requires very little knowledge of the illumination patterns. It is thus able to deal with illumination distortions induced by the sample or illumination optics. We named this new algorithm thick slice blind-SIM because it models a three-dimensional sample even though only a single two-dimensional plane of focus was measured.