A protocol for structured illumination microscopy with minimal reconstruction artifacts

• 作者: Junchao Fan Liangyi Chen Liuju Li Shan Tan Xiaoshuai Huang
• 来源期刊: 生物物理学报:英文版
• 学科: 生物学
• 关键词: ULTRAFAST SIM Low SIGNAL-TO-NOISE ratio RECONSTRUCTION
• 年，卷（期）: 2019,（2）
• 页码范围: 80-90
• 页数: 11页
• 分类号: Q

摘要

The imaging rate of structured illumination microscopy (SIM) reached 188 Hz recently. As the exposure time decreases, the camera detects fewer virtual photons, while the noise level remains the same. As a result, the signal-to-noise ratio (SNR) decreases sharply. Furthermore, the SNR decreases further because of photobleaching and phototoxicity. This decreased quality of SIM raw data may lead to surprising artifacts with various causes, which may confuse a new user of SIM microscopy. We summarize three significant possible sources of severe artifacts in reconstructed super-resolution (SR) images. Ultrafast motion of a biological sample or an uneven illumination pattern is the most difficult to be identified. The estimated parameter could also be incorrect, leading to artifact of regular patterns. Furthermore, rec on struction with the Wiener method generates stochastic artifacts due to the amplification of noise during the deconvolution process. To deal with these problems, we have established a protocol to reconstruct ultrafast SIM raw data obtained in low SNR conditions. First, we checked the quality of the raw data with the ImageJ plugin SIMcheck before rec on struction. Then, a modified parameter estimation method was used to improve the precision of the parameters. Finally, an iterative algorithm was used for SIM reconstruction under low signal-to-noise ratio cond让ions. This procedure effectively suppressed the artifacts in the super-resoluti on images rec on structed from raw data of low signal-to-noise ratio.