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Polarization modulation adds little additional information to super-resolution fluorescence microscopy

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Figure 1: Images from hippocampal neurons labeled with a farnesylated form of EGFP, reconstructed using the SPEED algorithm with and without polarization information.

References

  1. Hell, S.W. Nat. Methods 6, 24–32 (2009).

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  2. Hafi, N. et al. Nat. Methods 11, 579–584 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Bates for critical reading of the manuscript.

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Correspondence to Jan Keller.

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Integrated supplementary information

Supplementary Figure 1 Analysis of the polarization modulation of the raw SPoD data in Figure 4b of ref. 2.

(a) Average of the raw modulation data of the region shown in Fig. 1a (Diffraction limited resolution). (b) Modulation amplitude along the polarization direction determined from a simple sinusoidal fit. (c) Raw SPoD data along the line profiles shown in (a) and (b). Scale bars, 300nm.

Supplementary Figure 2 Analysis of ExPAN data from Figure 2d of ref. 2.

(a) Average of the raw modulation data of data in Fig. 2d in Hafi et al.2 (Diffraction-limited resolution). (b) Deconvolved images using the SPEED algorithm with (top) and without (bottom) taking modulation information into account, respectively. (c) Raw ExPAN modulation data along the line profile in (a). (d) Intensity profile plots along the dotted line of the diffraction-limited data in (a) (black), the SPEED deconvolution with modulation (b, top) (blue) and the SPEED deconvolution without modulation (b, bottom) (orange), respectively. Scale bars, 200 nm.

Supplementary Figure 3 Central cutout of the neuron in Figure 4b of ref. 2 after processing with various deconvolution algorithms.

Richardson-Lucy (RL) deconvolution (left column), deconvolution by the SPEED algorithm of Hafi et al. for different values of the λ1 sparsity parameter (second to fourth columns) after 200 iterations (a) or 520 iterations (b). All other parameters as specified in the Supplementary Software package of Hafi et al.2. (c) FWHMs (after linear interpolation) of the spine neck (dotted lines in (a, b)) of the deconvolved data as function of the number of iterations performed in each deconvolution algorithm. Scale bars, 1 μm.

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Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 3376 kb)

Supplementary Software

Modified algorithm to analyse SPoD data and raw data of original Figs. 4b and 2d. (ZIP 2077 kb)

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Frahm, L., Keller, J. Polarization modulation adds little additional information to super-resolution fluorescence microscopy. Nat Methods 13, 7–8 (2016). https://doi.org/10.1038/nmeth.3687

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