Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.
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High-fidelity 3D live-cell nanoscopy through data-driven enhanced super-resolution radial fluctuation
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J.V.T. and P.H. are candidates for joint PhD degrees from EMBL and Heidelberg University. PA-JF549 and Halo-Cy5 were a kind gift of L. Lavis, HHMI Janelia Research Campus. We thank the EMBL advanced light microscopy facility for their help. This work was supported by the European Research Council (grant no. ERC CoG-724489 to J.R., M.M., P.H. and J.V.T.), the National Institutes of Health Common Fund 4D Nucleome Program (grant no. U01 EB021223/U01 DA047728 to J.E. and J.R.), the Allen Distinguished Investigator Program through The Paul G. Allen Frontiers Group (J.E.), the UK Biotechnology and Biological Sciences Research Council (grant nos. BB/M022374/1, BB/P027431/1, BB/R000697/1 and BB/S507532/1 to R.H. and P.M.P.), the Wellcome Trust (203276/Z/16/Z, R.H. and P.M.P.), the EMBL Interdisciplinary Postdoc Programme (EIPOD) under Marie Curie Actions COFUND (Y.L.), the Human Frontier Science Program (RGY0065/2017 to J.R.) and the European Molecular Biology Laboratory (J.V.T., K.C., P.H., S.K.P., K.C.K., Y.L., Y.W., M.M., U.M., B.N., M.K., V.J.S., J.E. and J.R.). V.J.S. acknowledges support by the Boehringer Ingelheim Fonds.
The authors declare no competing interests.
Peer review information 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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(a-d) Southern blots of (a) Nup96-SNAP, (b) Nup96-Halo, (c) Nup96-mMaple, (d) Nup96-mEGFP. Blots on the left were generated from probes against Nup96-C-term and blots on the right were generated from probes against respective tags. The presence of a single band was indicative of a homozygous knock-in (in red). (e) Western blot of the homozygous cell lines probed with an anti-Nup98 antibody. Reduction of band intensity in Nup96-WT indicates the specificity of the antibody to Nup96. siRNA concentrations used: 0.6 µg, 1.2 µg and 1.8 µg. Representative images of one (a-d) or two (e) independent experiments are shown. Full scans of all blots are shown in Supplementary Fig. 11.
Supplementary Figure 2 SMLM with total internal reflection fluorescence (TIRF) excitation on Nup96-SNAP-AF647.
In the majority of cells, the lower nuclear envelope is sufficiently close to the coverslip to be efficiently excited with TIRF. Representative images from two independent experiments are shown. Scale bars 1 µm.
(a) Logarithmic power spectrum for Fig. 1f–i. (b) Fourier Ring Correlation for Fig. 1m–q including resolution estimates. To calculate these curves, the localizations were divided into ten time windows, and the correlation was computed between even and odd time windows. A correction for spurious correlation due to re-activations was not applied. (c) Histogram of radii resulting from a ring fit to Fig. 1p. From the average measured radius <R> = 174 ± 25 nm and the known radius (Fig. 2) the expansion factor was estimated to be 3.2. Values depict mean ± SD.
Representative images from three independent experiments are shown. Scale bars 10 µm (upper panel) and 1 µm (lower panel).
(a) Nup96-SNAP-AF647. R = 53.7 ± 2.1 nm (N = 3, nC = 6 nNPC = 1856). (b) Nup96-Halo-Cy5. R = 54.5 ± 2.6 nm (N = 2, nC = 6 nNPC = 4959). (c) Nup96-mMaple. R = 55.4 ± 3.5 nm (N = 3, nC = 6 nNPC = 4276). (d) Nup96-GFP-Nanobody-Q-AF647. R = 55.0 ± 1.9 nm (N = 2, nC = 6 nNPC = 2913). (e) Nup96-GFP-Antibody-AF647. R = 64.3 ± 2.6 nm N = 2, nC = 6 nNPC = 3158). (f) Box plots of the radii for the different labels. Each data point corresponds to one cell. The center of the box plot shows the median, while lower and upper boundaries indicate the 25th and 75th percentiles, respectively. N denotes the number of biologically independent experiments, nC the number of imaged cells and nNPC the number of analyzed NPCs. Values show weighted mean ± SD, based on nNPC. Measurements were performed in GLOX/MEA blinking buffer (a,b,d,e) or 50 mM Tris in D2O (c).
Supplementary Figure 6 Depth induced aberrations lead to local deformations in z (related to Fig. 2j).
Distance between rings plotted vs the z-position a) before correction and b) after correction (N = 2, nC = 8, nNPC = 7234). Straight lines are the linear fit of the ring distance for each cell. Before correction these values are highly correlated (Pearson coefficient −0.27 ± 0.10), after correcting for the localization errors, the correlation is reduced (Pearson coefficient 0.04 ± 0.15). N denotes the number of biologically independent experiments, nC the number of imaged cells and nNPC the number of analyzed NPCs. Values depict mean ± SD.
Error in determining ELE (inferred ELE – true ELE) in dependence on (a) the brightness of the fluorophores, (b) the labeling efficiency, (c) the number of re-activations for bright (5000 photons) fluorophores and for (d) dim (500 photons) fluorophores and (e) in dependence on the number of nuclear pores analyzed. (f) Statistical accuracy (SEM) in determining the ELE in dependence on the number of nuclear pores. Unless otherwise indicated the simulation parameters were: labeling efficiency 0.5 (a,e,f) and 0.3 (c,d), number of photons = 5000, on average 1 re-activation, background 20 photons, 900 nuclear pores. Error bars denote mean ± SD.
ELE of Nup107-SNAP labeled with BG-AF647 and stored at 4 °C imaged (a) on the day of sample preparation, (b) two months after sample preparation and (c) two years after sample preparation. Representative images from one independent experiment are shown. Scale bars 1 µm.
Representative images of two (a-f,I,k,m,n,p), three (g,j,o,r), four (h), five (l) or six (q) independent experiments are shown. Scale bars 100 nm.
(a) Lanes 1 & 2: 2 different cell line clones under tetracycline (tet) induction. Lanes 3–7: clone no. 10 under tet induction for 4-0 days. Lane 8: blank. Lane 9: HEK293T wildtype. Lane 10: ladder. GAPDH is used as loading control. Clone 10 was used for all subsequent experiments and 2 days of tet induction was sufficient to knock down endogenous Nup107 and induce expressions of mMaple-Nup107. (b) Dose titration of tet concentration to induce mMaple-Nup107 expression. 10 ng/mL was sufficient to knock down endogenous Nup107 and induce expressions of mMaple-Nup107. Increasing tet concentration resulted in increasing expression of mMaple-Nup107. A tet concentration of 1 mg/mL was used for all subsequent experiments. (c) Recovery of endogenous expression of Nup107 after removal of tet from media. Expression of endogenous Nup107 can be observed after 1 day off tet induction with an increasing trend the longer tet is removed. The opposite trend is observed for expression levels of mMaple-Nup107. Representative images of one independent experiment are shown. Full scans of all blots are shown in Supplementary Fig. 12.
(a-e) Original Southern blots for U2OS cell line validation with the probes indicated above each blot. The clones chosen for all subsequent experiments are marked with a red asterisk. (f) Original Western blots for U2OS cell line validation decorated for Nup96 and GAPDH, respectively.
(a-c) Original Western blots for HEK293T cell validation and tetracycline titration decorated for Nup107 and GAPDH, respectively.
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Thevathasan, J.V., Kahnwald, M., Cieśliński, K. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat Methods 16, 1045–1053 (2019). https://doi.org/10.1038/s41592-019-0574-9
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