Structured illumination microscopy (SIM) is a powerful super-resolution method in bioscience, featuring full-field imaging and high photon efficiency. However, artifact-free super-resolution image reconstruction requires precise knowledge about the illumination parameters. In this work, we propose an efficient and robust SIM algorithm based on principal component analysis (PCA-SIM) combines iteration-free reconstruction, noise robustness, and limited computational complexity. These characteristics make PCA-SIM a promising method for high-speed, long-term, artifact-free super-resolution imaging of live cells.
Structured Illumination Microscopy(SIM) is a suitable instrument for fluorescence imaging, especially dynamic imaging of live cells. It has two significant advantages of fast imaging speed and low excitation light energy density, and can reach the resolution of 100nm. In this article, we focus on the traditional reconstruction algorithm of SIM, which separates the spectrum with 9 raw images of 3-step phase-shifted picture in 3 orientations and operates the reconstruction with the correct displacement in Fourier space. However, without considering the initial phase error and the possibility that the displacement is a sub-pixel, there are artifacts in the results of the traditional reconstruction algorithm of SIM. To eliminate the artifacts and improve the imaging quality, we analyze the causes of various artifacts, and study the cross-correlation-based reconstruction algorithm of SIM, using the maximum cross-correlation value between the spectrum to get the correct displacement. Then, we simulate the illumination experimental parameters on the images of the reconstructed results. From the perspective of both hardware and software, we respectively consider the construction of the home-built SIM setup and the reconstruction software design, and finally realize the tri-color SIM system.
KEYWORDS: Phase shifts, Microscopy, Real time imaging, Image processing, Super resolution, Optical transfer functions, Modulation, Microscopes, Luminescence, Double patterning technology
With the development of various fluorescence technologies and optical control, fluorescence super-resolution microscopy has broken the limit of optical diffraction. Among them, the structural illumination microscopy (SIM), which combines structured light illumination and wide-field fluorescence imaging, uses structural illumination to mix in Fourier space to bring high-frequency information into the passband of the optical transfer function (OTF) to achieve super-resolution imaging. And having the advantages of weak phototoxicity and photobleaching, and fast imaging speed, SIM is currently one of the most mainstream techniques for super-resolution microscopy imaging of living cells. In this work, we have completed the theoretical simulation of SIM and the experimental operation. Firstly, we review the development of SIM, and systematically introduces its super-resolution imaging principle. Then, we discuss the technical difficulties of the hardware part, and builds a set of dual-beam interferometric SIM based on ferroelectric liquid crystal spatial light modulator, achieving that it only takes 270ms to collect 9 original images, and modulates the polarization characteristics of the illumination light to improve the interference fringe contrast and energy utilization. Finally, by using the open-source plugin Hifi-SIM to achieve image reconstruction, we obtain some ideal results.
Among the popular fluorescence super-resolution microscopy imaging technologies that had broken the optical diffraction limit, structured illumination microscopy (SIM) holds the advantages of low phototoxicity, weak photobleaching, and fast imaging speed, and it is currently one of the mainstream technologies for super-resolution microscopy imaging of living cells. SIM uses the modulation of the structured illumination patterns to encode highfrequency information in the raw images into the low-frequency region, allowing it to pass through the optical transfer function (OTF), and then obtains super-resolution images through demodulation and reconstruction. The reconstructed image is affected by some important parameters of the illumination light field, so it is necessary to accurately estimate the unknown parameters of the illumination light field, especially the initial phase, to minimize artifacts in the reconstructed image. In this work, we have completed the experimental operation of SIM, and image reconstruction based on different phase reconstruction algorithms. Firstly, we reviewed the development history of SIM, and systematically introduced the principle of SIM to achieve super-resolution imaging and the phase estimation algorithms. Then, we discussed the technical difficulties of the hardware setup, and built a dual-beam interference SIM system based on the ferroelectric liquid crystal spatial light modulator (FLC-SLM). Finally, we used different phase estimation algorithms to extract the initial phases of the collected images, and some comparable results are obtained.
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