A protocol for structured illumination microscopy with minimal reconstruction artifacts

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, reconstruction 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 reconstruction. 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 conditions. This procedure effectively suppressed the artifacts in the super-resolution images reconstructed from raw data of low signal-to-noise ratio.