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References
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Acknowledgements
We are grateful to M. Sauer for support and D. Helmerich for technical assistance. We also thank R. Thorsen, S. Stallinga and B. Rieger for discussion.
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The authors filed a patent with Carl Zeiss Microscopy GmbH (No. 10 2016 014 133.6).
Integrated supplementary information
Supplementary Figure 1 Theoretical emission profile and experimental spot.
a) Simulated areal Airy-PSF emission pattern of 2,500 photon counts distributed over 10 × 10 µm² without background (FWHM = 340 nm). Inverted image with ≥2 photon count per pixel (N px) saturation. Inset shows unsaturated partial image of the boxed region at the same scale, indicated in red. b) Top: representative experimental single-molecule image of Cy5 molecules under dSTORM conditions; middle: same region in saturation, experimental TRABI radius is shown as solid circle (0.865 µm), 1.86 × FWHM radius is shown as dashed circle (0.632 µm); bottom: intensity profile in black as indicated in the top panel, the background mean and s.d. from five consecutive frames is shown in gray and light gray, respectively. Scale bars, a) 5 µm, b) 2 µm.
Supplementary Figure 2 3D intensity-based biplane imaging of the DNA origami sample.
a) Representative part of a DNA-PAINT image. The numbers in nanometers reflect the calculated length of the selected nanoruler. b) Axial localization precision of BP-TRABI, s.d. of Gaussian fit = 17.2 nm (FWHM = 40.5 nm). c) Axial distance of the two spots, Gaussian fit yields 52.3 ± 16.5 nm (mean ± s.d.), median = 51.4 nm. d) Distribution of the calculated nanoruler length, Gaussian fit yields 74.8 ± 13.2 nm (mean ± s.d.), median = 75.5 nm. e) The absolute z-position of all localizations (black) and the nanoruler length (blue) as function of the distance from the center of the field of view (mean and s.d.). First and last data points are affected by a reduced sample size (Supplementary Table 1). Three independent fields of view (34 µm × 34 µm) were conflated for data analysis. n = 383 single spots comprising 40,091 individual localizations for b)- e) Scale bar, a) 2 µm.
Supplementary Figure 3 3D classical biplane imaging of the DNA origami sample.
a) Axial localization precision of BP-FWHM, s.d. of Gaussian fit = 26.8 nm (FWHM = 63.1 nm). b) Axial distance of the two spots, Gaussian fit yields 53.1 ± 15.6 nm (mean ± s.d.), median = 52.8 nm. d) Distribution of the calculated nanoruler length, Gaussian fit yields 75.8 ± 11.8 nm (mean ± s.d.), median = 75.5 nm. d) The absolute z-position of all localizations (black) and the nanoruler length (blue) as function of the distance from the center of the field of view (mean and s.d.). First and last data points are affected by a reduced sample size (Supplementary Table 1). Three independent fields of view (34 µm × 34 µm) were conflated for data analysis. n = 353 single spots comprising 41,928 individual localizations.
Supplementary Figure 4 Field-dependent nanoruler length of different 3D methods in comparison.
a) BP-TRABI (black, n = 383 single spots comprising 40,091 individual localizations) versus TRABI (blue, n = 222 single spots comprising 18,232 individual localizations). b) Classical biplane imaging (BP-FWHM, black, n = 353 single spots comprising 41,928 individual localizations) versus TRABI (blue). Displayed are mean value and s.d. (Supplementary Table 1).
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Supplementary Figs. 1–4, Supplementary Table 1 and Supplementary Methods
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Franke, C., van de Linde, S. Reply to ‘Impact of optical aberrations on axial position determination by photometry’. Nat Methods 15, 990–992 (2018). https://doi.org/10.1038/s41592-018-0228-3
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DOI: https://doi.org/10.1038/s41592-018-0228-3
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