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Structured illumination microscopy with noise-controlled image reconstructions

Nature Methods volume 18, pages 821–828 (2021)Cite this article

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Abstract

Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artifacts that limit their applicability for lower signal-to-noise data. Here we present a physically realistic noise model that explains the structured noise artifact, which we then use to motivate new complementary reconstruction approaches. True-Wiener-filtered SIM optimizes contrast given the available signal-to-noise ratio, and flat-noise SIM fully overcomes the structured noise artifact while maintaining resolving power. Both methods eliminate ad hoc user-adjustable reconstruction parameters in favor of physical parameters, enhancing objectivity. The new reconstructions point to a trade-off between contrast and a natural noise appearance. This trade-off can be partly overcome by further notch filtering but at the expense of a decrease in signal-to-noise ratio. The benefits of the proposed approaches are demonstrated on focal adhesion and tubulin samples in two and three dimensions, and on nanofabricated fluorescent test patterns.

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Fig. 1: Noise propagation in SIM reconstructions.
Fig. 2: Noise-controlled SIM reconstructions.
Fig. 3: Trade-off between noise and contrast in SIM reconstructions.
Fig. 4: Noise-controlled 3D-SIM reconstructions.
Fig. 5: Resolution improvement and noise enhancement in deconvolution of GFP-zyxin dataset.

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Data availability

Data are available at https://doi.org/10.4121/12942932.

Code availability

MATLAB code is available at https://github.com/qnano/simnoise. ImageJ code for 2D-SIM is available at https://github.com/fairSIM.

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Acknowledgements

We thank M. Booth and B. Rieger for stimulating research advice, A. York for suggesting the binomial random splitting of Poisson-distributed variables and W. Baarends for kindly providing mCherry-SYCP3 samples. C.S. was supported by a Junior Research Fellowship through Merton College (Oxford, UK). L.S. acknowledges support by the Wellcome Trust Strategic Award 107457 and the European Research Council MSC ITN grant no. 766181. N.C. acknowledges European Research Council grant no. 648580. C.H. acknowledges support from the Netherlands Organization for Scientific Research (ZonMW-435002021). S.H. acknowledges support by NanoNextNL, a consortium of the Dutch government and 130 public and private partners.

Author information

Authors and Affiliations

  1. Department of Imaging Physics, Delft University of Technology, Delft, the Netherlands

    Carlas S. Smith, Nadya Chakrova, Sangeetha Hari, Yoram Vos, Cornelis W. Hagen, Jacob P. Hoogenboom & Sjoerd Stallinga

  2. Department of Physiology, Anatomy and Genetics, Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK

    Carlas S. Smith

  3. Department of Pathology, Erasmus Optical Imaging Centre, Erasmus Medical Center, Rotterdam, the Netherlands

    Johan A. Slotman, Wiggert van Cappellen & Adriaan B. Houtsmuller

  4. Micron Advanced Bioimaging Unit, Department of Biochemistry, University of Oxford, Oxford, UK

    Lothar Schermelleh

  5. Biochemistry, Molecular and Structural Biology Section, Leuven University, Leuven, Belgium

    Marcel Müller

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Contributions

Imaging experiments were done by C.S.S., J.A.S., L.S., N.C., W.v.C. and A.B.H. J.A.S., S.H., Y.V., C.W.H. and J.P.H. designed and manufactured nanofabricated test samples. C.S.S., N.C., M.M. and S.S. analyzed data. S.S. derived theory, wrote the paper and supervised the research. All authors read and approved the manuscript.

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Correspondence to Sjoerd Stallinga.

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The authors declare no competing financial interests.

Additional information

Peer review Information Nature Methods thanks Florian Ströhl and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Noise-controlled SIM reconstructions of GFP-zyxin protein in focal adhesions (green) and noise fraction map (magenta) over full FOV.

a–d, State-of-art SIM (w = 5 × 10−4), true-Wiener SIM, flat-noise SIM, and notch-filtered SIM reconstructions. Contours of the noise fraction map are added in white with contour level indicated. In all reconstructions the noise fraction is lowest in the foreground features and highest in the background region outside the cell. Overall, flat-noise SIM and true-Wiener SIM offer the lowest, and notch-filtered SIM the highest noise enhancement. Scale bar 5 μm.

Extended Data Fig. 2 Multi-color noise-controlled 2D-SIM reconstructions.

a, Combined widefield, true-Wiener SIM, flat-noise SIM, and notch-filtered SIM reconstructions of a fluorescent test slide of a bovine pulmonary artery endothelial cell (red channel: mitochondria labeled with MitoTracker Red, green channel: actin labeled with Alexa Fluor 488, blue channel: DNA labeled with DAPI). Note that due to embedding in hardening mounting medium, cells are flattened and 3D nuclear morphology is compromised. b-f, Insets of the DAPI channel comparing state-of-the-art SIM with clear noise amplification artifact to the noise-controlled SIM reconstructions. The SSNR in the DAPI channel is low in this example case, due to reduced signal intensity and compromised morphology. The low SSNR is properly taken into account by the noise-controlled SIM reconstructions, without introducing artifacts, but not by the state-of-the-art SIM reconstruction. Scale bar (a) 10 μm, scale bar (b-f) 5 μm.

Extended Data Fig. 3 Noise propagation in DMD-SIM.

a, Reconstructions of Alexa Fluor 488 labeled actin filaments in a bovine pulmonary artery endothelial cell with the iterative pattern-illuminated Fourier Ptychography (piFP) algorithm (see Supplementary Note 1) and with a band-pass regularization approach for flat-noise SIM. b, Comparison of flat-noise SIM to a widefield reconstruction obtained by summing the whole set of acquired images. c-e, Insets of the boxed region in (a) and (b). Both piFP and flat-noise SIM offer a resolution improvement, but piFP has better contrast than flat-noise SIM. The piFP reconstruction shows corrugated line structures and punctuated features (upper right of insets), similar to the structured noise artifact in state-of-the-art SIM with line illumination patterns, flat-noise SIM shows this to a lesser degree. Scale bar (a,b) 10 μm, scale bar (c-e) 4 μm.

Extended Data Fig. 4 Flat-noise SIM provides better visibility of high spatial frequency structures.

a–d, Widefield, true-Wiener, flat-noise, and notch-filtered SIM reconstructions of a nanofabricated test structure of lines with 140 nm pitch. The line pattern is just visible in flat-noise, and notch-filtered SIM but overshadowed by the noise pattern with uneven distribution of noise over spatial frequencies in true-Wiener SIM. Scale bar 1 μm.

Extended Data Fig. 5 Noise-controlled 2D-SIM of synaptonemal complex.

a–d, Widefield, and true-Wiener, flat-noise and notch-filtered SIM reconstructions of the mCherry-CSYCP3 protein in the synaptonemal complex. e–h, Line profiles along the lines indicated in (b). The SIM reconstructions reveal the two cable sub-structure with a line distance of around 200 nm, flat-noise SIM has less contrast but shows smoother lines and no background noise structure. Scale bar 3 μm.

Extended Data Fig. 6 Cross-sections in xy and yz-planes of 3D-reconstructions of tubulin (green) and noise fraction maps (magenta) for different camera exposure times.

a, Widefield, (b–d) state-of-the-art SIM for low, medium and high regularization, (e) true-Wiener SIM, (f) flat-noise SIM, and (g) notch-filtered SIM. The dashed lines in (a) indicate the location of the xz and xy slices. Scale bar 3 μm.

Extended Data Fig. 7 Widefield and noise-controlled 3D-SIM reconstructions of a 100 nm bead layer sample.

a,b, Widefield, (c,d) true-Wiener SIM, (e,f) flat-noise SIM, (g,h) notch-filtered SIM. The white box in (c) indicates the insets (b,d,f,h). i,j, SSNR of the SIM reconstructions without (i) and with (j) notch filtering. The data is averaged over rings in Fourier space and the plot is on a logarithmic scale according to log10(1+SSNR). The red line indicates the (ring averaged) support of the SIM-OTF, the white line indicates the SSNR = 5 region in Fourier space used for the extrapolation of the true-Wiener regularization filter. k, FRC curves for SIM obtained from 4 repeated acquisitions of the bead layer sample. The FRC resolution is 106.3 ± 0.5 nm, very close to the extended SIM diffraction limit 1/(2NA/λ + 2/p) = 99 nm for the estimated pattern pitch p = 416 nm, consistent with the relatively high signal level (peak pixel intensities above 104 detected photons) and the broad support of SSNR above one in spatial frequency space. Scale bar (a,c,e,g) 3 μm, scale bar (b,d,f,h) 1 μm.

Extended Data Fig. 8 Widefield and 3D noise-controlled SIM reconstructions of a bovine pulmonary artery endothelial cell.

a, Widefield, (b) true-Wiener SIM, (c) flat-noise SIM, (d) notch-filtered SIM (BPAEC, red channel: mitochondria labeled with Alexa Fluor 594, green channel: actin labeled with FITC, blue channel: DNA labeled with DAPI). Scale bar 5 μm.

Extended Data Fig. 9 Widefield and 3D noise-controlled SIM reconstructions of a mouse C127 cell.

a, Widefield, (b) true-Wiener SIM, (c) flat-noise SIM, (d) notch-filtered SIM (magenta channel: DNA labeled with DAPI, green channel: H3K4me3 labeled with Alexa Fluor 488, blue channel: DNA labeled with DAPI). Scale bar 5 μm.

Extended Data Fig. 10

Widefield, state-of-the-art SIM and noise-controlled 3D-SIM reconstructions for one timeframe of the 15 timeframe, 7-layer dataset of H2B-GFP histone in a live HeLa cell. Scale bar 6 μm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Note.

Reporting Summary

Supplementary Video 1

Impact of regularization parameter on state-of-the-art SIM.

Supplementary Video 2

Noise-controlled SIM of 10× repeated acquisition of GFP-zyxin.

Supplementary Video 3

Noise controlled 3D SIM of tubulin, signal level 1.

Supplementary Video 4

Noise controlled 3D SIM of tubulin, signal level 2.

Supplementary Video 5

Noise controlled 3D SIM of tubulin, signal level 3.

Supplementary Video6

Noise controlled 3D-SIM of tubulin, signal level 4.

Supplementary Video 7

Noise controlled 3D three-color SIM of BPAEC cell.

Supplementary Video 8

Noise controlled 3D two-color SIM of C127 cell.

Supplementary Video 9

Noise controlled 3D-SIM of live HeLa cell.

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Smith, C.S., Slotman, J.A., Schermelleh, L. et al. Structured illumination microscopy with noise-controlled image reconstructions. Nat Methods 18, 821–828 (2021). https://doi.org/10.1038/s41592-021-01167-7

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