Successful optimization of reconstruction parameters in structured illumination microscopy – a practical guide

Abstract

The impact of the different reconstruction parameters in super-resolution structured illumination microscopy (SIM) onto artifacts is carefully analyzed. They comprise the Wiener filter parameter, an apodization function, zero-frequency suppression and modifications of the optical transfer function. A detailed investigation of the reconstructed image spectrum is concluded to be suitable for identifying artifacts. For this purpose, two samples, an artificial test slide and a more realistic biological system, were used to characterize the artifact classes and their correlation with the image spectra as well as the reconstruction parameters. In addition, a guideline for efficient parameter optimization is suggested and the implementation of the parameters in selected up-to-date processing packages (proprietary and open-source) is depicted.

Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

“…Reconstruction of SIM images was done with the ZENBlack software structured illumination processing module. Wiener deconvolution was used as implemented in the Zeiss SIM module [ 46 ]. In a first step, the Wiener filter was set free and automatically determined, followed by a systematic alteration of its value.…”

High-fidelity structured illumination microscopy by point-spread-function engineering

“…By inverse Fourier transformation, the downward kinks in the OTF are transformed into sidelobes in the PSF. The abnormal features of OTF could be corrected by Wiener deconvolution, but this requires that the OTF used and estimated reconstruction parameters accurately match the actual imaging conditions 8 , 15 , 18 , 27 ; otherwise, the final SR images often exhibit reconstruction artifacts, such as sidelobe artifacts. Moreover, the residual background signals are usually concentrated at the raised peaks of , causing periodic honeycomb artifacts 7 , 12 , 18 and limiting the OS capability 35 – 38 .…”

“…Moreover, the residual background signals are usually concentrated at the raised peaks of , causing periodic honeycomb artifacts 7 , 12 , 18 and limiting the OS capability 35 – 38 . This defect is usually remedied by OTF attenuation 20 , 27 , 31 , 36 , but this solution cannot achieve a good balance between rejecting background fluorescence and protecting fine and weak structures. For a more detailed discussion of the thorny issues in Wiener-SIM reconstruction, see Supplementary Note 1 .…”

“…2b ). With an active compensation for the theoretical OTF (“dampening” factor = 0.3) 27 , 31 , the patchy features became weaker but still existed in the spectrum of fairSIM; hence, the SR image showed similar sidelobe artifacts. By contrast, the spectrum reconstructed by HiFi-SIM using the same OTF as in traditional Wiener-SIM showed homogenous features, yielding clean microspheres.…”

“…To acquire SIM images with minimal artifacts, dedicated practical guidelines were recommended for instrument refinement 16 , data acquisition 17,18 , and sample preparation 18,19 . Several studies have been conducted on reconstruction algorithms, including accurate illumination parameter estimation [20][21][22][23][24] , iterative deconvolution 8,25,26 , and fine tuning of reconstruction parameters 27 . Despite all these efforts, artifacts that limit the implementation of SIM as a daily imaging tool still frequently appear in SIM images.…”