Yiran Wang, Gangshan Liu, Ziyang Li, Zhengjun Liu*
School of Physics, Harbin Institute of Technology, Harbin 150001, China
Structured illumination microscopy (SIM) is a mainstream super-resolution imaging technique that is applied in fluorescence microscopy, effectively doubling the lateral resolution compared to conventional methods. Compared to other popular fluorescence super-resolution techniques such as STORM and STED, SIM offers advantages through its reduced light exposure, faster imaging speeds, and, consequently, lower phototoxicity and photobleaching, making it particularly suitable for live cell imaging. However, traditional 2D-SIM requires nine sequentially captured raw structured illumination images to reconstruct a single super-resolution image. This results in a ninefold increase in acquisition time compared to standard wide-field imaging. Reducing the exposure time can mitigate motion artifacts but at the cost of image quality; conversely, increasing the exposure time can heighten phototoxicity, impacting the overall observation time and imaging efficiency.
To address these challenges, we propose a novel method called 4CSIM, which replaces sinusoidal illumination with a checkerboard-patterned structure, requiring only four raw images to achieve super-resolution imaging. In 4CSIM, instead of treating the entire image as a whole, we establish a point-wise relationship between the pixels in the reconstructed super-resolution image and the corresponding pixels in the original SIM images, reconstructing the Fourier spectrum of the super-resolution image pixel by pixel. The checkerboard illumination pattern enables this "point-wise reconstruction" method, with the reconstruction process involving only three-dimensional matrix operations. Compared to the existing 4SIM method, 4CSIM significantly accelerates image reconstruction due to its non-iterative nature and low memory usage, avoiding the trade-off between a high frame rate acquisition and reconstruction speed observed in 4SIM methods.
In summary, 4CSIM enhances both the acquisition frame rate and reconstruction speed of SIM technology, making real-time video imaging feasible.
