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A super-resolution platform for correlative live single-molecule imaging and STED microscopy


Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.

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Fig. 1: Correlative STED, PALM and uPAINT enables super-resolution imaging of spine morphology and synaptic proteins.
Fig. 2: Spatial organization and mobility of PSD95 and GluA1 in different dendritic/spine compartments.
Fig. 3: Correlative imaging of pre- and postsynaptic contacts and growth cones.

Data availability

The data that support the findings of this study are available from the corresponding author upon responsible request.

Code availability

The custom-made SML-Transformer software to overlay STED and SMLM images is available as Supplementary Software with training data.


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We thank E. Gouaux for the anti-GluA2 antibody and the IINS Cell Biology Core Facility for cell culture and plasmid production. This work was supported by funding from the Conseil Régional d’Aquitaine, Labex BRAIN, France-BioImaging (grant no. ANR-10-INBS-04), Fondation pour la Recherche Médicale (grant no. DEQ20160334901 to U.V.N.) and European Research Council (ERC grant no. 787340 Dyn-Syn-Mem to D.C.). We thank members of the Nägerl team for comments on the manuscript.

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Authors and Affiliations



J.B.S. and U.V.N. conceived and supervised the study. V.V.G.K.I. and M.O.L. built the microscope. V.V.G.K.I. optimized the two modalities and performed all the experiments and data analysis. C.B. developed the coordinate transformation plugin. J.A. and B.C. performed electroporations and transfections. F.L. developed the ImageJ plugin for spine analysis. J.T. contributed to the design of the STED microscope. O.R. and G.G. designed and performed the fibroblast experiments with V.V.G.K.I. E.H. and B.C. advised on uPAINT experiments. O.T. advised on growth cone experiments. D.C. provided intellectual input. V.V.G.K.I., J.B.S. and U.V.N. wrote the manuscript with comments from all authors.

Corresponding authors

Correspondence to Jean-Baptiste Sibarita or U. Valentin Nägerl.

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The authors declare no competing interests.

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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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Integrated supplementary information

Supplementary Figure 1 Point spread function and spatial resolutions.

a) Lateral (XY) and axial (XZ) images of the excitation (cyan hot) and STED (doughnut and bottle beams, orange hot), measured from 150 nm gold beads adsorbed on glass coverslip, scale bar 500 nm. b) Confocal and STED images of 40 nm fluorescent beads adsorbed on glass coverslip, Scale bar 500 nm. c) Confocal and STED image cross-sections of 40 nm fluorescent beads, scale bar 500 nm. One representative image (a, b and c) of everyday alignment. d) Lateral and axial FWHM measured on 40 nm fluorescent beads measured by Gaussian (confocal) and Lorentzian (STED) fitting with ImageJ and Prism software. N = 25 beads, box plots represent the mean with range. e) Fourier Ring Correlation (FRC) measurements on 40 nm fluorescent beads, shown in b). f) Dissociated neurons imaged in confocal (left) and STED (right), scale bar 1 µm. Inset shows the resolution improvement with STED, scale bar 500 nm. One representative image out of 3 independent experiments. g) Quantification of spine neck width and spine head area on confocal and STED images (two-tailed paired Wilcoxon, p<0.0001, N = 90 spines, 5 cells, 3 cultures). h) PSD95_mEos3.2 imaged in widefield (left) and PALM (right), scale bar 2 µm. Inset shows the resolution improvement with PALM, scale bar 1 µm. One representative image out of 3 independent experiments. i) Quantification of PSD95 area measured on widefield and PALM images (two-tailed paired Wilcoxon, p<0.0001, N = 90 spines, 5 cells, 3 cultures). j) Summary table of STED vs confocal and widefield vs PALM measurements made on beads (N = 25), spine neck, spine width and spine head area of dissociated neurons and PSD95 clusters, N = 90 spines, the values shown in the table are mean with SEM. k) Summary table of fluorophores and spectral ranges used for the STED/SMLM experiments. Data points (red color) in g and i represents the mean with SEM.

Supplementary Figure 2 Sub-pixel alignment of STED and SMLM data.

a) Images of 100 nm multicolor bead acquired in STED and SMLM after optical alignment and before sub-pixel alignment, scale bar 5 µm, 160 nm/pixel. b) Overlay between bead localizations and STED image before (green cross) and after (magenta cross) coordinate transformation. c) Histogram of pair distances before and after transformation.

Supplementary Figure 3 STED/sptPALM time-lapse imaging and STED laser effect on mEos3.2.

a) STED/sptPALM time-lapse imaging showing GluA1_mEos3.2 trajectories along the dendritic spines with inset showing zoom-in on spine, scale bar 1 µm, inset scale bar 500 nm, two representative images out of 6 independent experiments. b) STED/sptPALM time-lapse imaging showing PSD95_mEos3.2 trajectories along the dendritic spines with inset showing zoom-in on spine, scale bar 1 µm, inset scale bar 500 nm, two representative images out of 6 independent experiments. c) Decay of mEos3.2 during sptPALM/STED time-lapse experiments. Left: at constant 405 nm power without STED images in between (PALM bleaching). Middle: at constant 405 nm power, with STED images in between (PALM + STED bleaching). Right: modulating the 405 nm excitation power from 3 to 80 µW (measured at the sample plane) to compensate for mEos3.2 bleaching. Data points represent mean with SD, N = 5 cells, 2 different preparations. d) Fraction of mobile trajectories for GluA1_mEos3.2 and PSD95_mEos3.2 during three cycles, one-way ANOVA test, N = 12 cells, 6 cultures, N.S = not significant, * p = 0.01, p value), indicating that STED laser is not significantly affecting the dynamics of GluA1 and PSD95 molecules. e) Cumulative and normal distributions of GluA1_mEos3.2 during three cycles, N = 12 cells, 6 cultures. f) Cumulative and normal distributions of PSD95_mEos3.2 during three cycles, N = 12 cells, 6 cultures. g) Cumulative and normal distributions of PSD95_mEos3.2 diffusion coefficients without STED laser, N = 8 cells, 5 cultures. Data points in d, e, f and g represent mean with SEM.

Supplementary Figure 4 STED/sptPALM correlative nanoscale imaging of focal adhesion sites and adhesion molecules in fibroblasts.

a) From left to right: STED image of paxillin, sptPALM trajectories of integrins in fibroblasts and overlay of both, scale bar 2 µm. b) Zoom of focal adhesion sites which are better resolved by STED (right) compared to widefield (left) and confocal (middle), scale bar 1 µm. c) STED/sptPALM overlay illustrates that immobilized molecules (cyan) colocalize with adhesion sites resolved by STED microscopy, while freely moving molecules (green) are more localized outside the adhesion sites. One representative image (a, b and c) out of 4 independent experiments. d) In widefield and confocal microscopy, immobile trajectories are 10% and 4.5 % underestimated in FAs, respectively, compared to STED (One-way ANOVA, p<0.0001, N = 22 FAs, from 4 cells, 2 cultures), box plots represents the average with range.

Supplementary Figure 5 STED/PALM/uPAINT correlative nanoscale imaging of spine morphology with GluA2 and PSD95 membrane proteins.

a) Representative image of dissociated neuron imaged by correlative STED, PALM and uPAINT, scale bar 1 µm. b) Overlay of the 3 channels with a zoom on a spine, showing GluA2 AMPA receptor moving inside and outside the PSD95, scale bar 500 nm. One representative image (a, b) out of 3 independent experiments. c) Cumulative distribution of PSD95 and GluA2 AMPA receptor diffusion coefficients.

Supplementary Figure 6 Statistical analysis and correlation between GluA1 mobility and spine morphology.

a) One-way ANOVA test results of GluA1 mobility in different compartments. 8 cells, 7 cultures (b-d) Correlation between mobile/immobile ratio of GluA1_mEos3.2 and b) spine neck width, c) spine neck length, and d) spine head area. N= 67 spines, 6 cells, 6 cultures e) Distribution of PSD95_mEos3.2 (blue) and GluA1_mEos3.2 (magenta) diffusion coefficients inside the whole dendrite, N = 8 cells, 7 cultures. f) Distribution of PSD95_mEos3.2 (blue) and GluA1_SEP (magenta) diffusion coefficient, N = 6 cells, 3 cultures. g) Distribution of PSD95_mEos3.2 (blue) and GluA1_SEP (magenta) diffusion coefficient, N = 5 cells, 3 cultures. The vertical dashed line separates immobile (left) from mobile (right) molecules. Data points in e, f and g represents the mean with SEM.

Supplementary Figure 7 STED/uPAINT correlative time-lapse imaging and statistics.

a) Time-lapse imaging of GluA1_SEP molecules (uPAINT) along the dendritic spines (STED) during 6 cycles, scale bar 1 µm, One representative image out of 3 independent experiments. b) Distribution of the diffusion coefficients of GluA1_SEP for the 6 cycles. c) Cumulative distribution of the diffusion coefficients of GluA1_SEP for 6 cycles. N = 6 cells, 3 cultures. Data points in b and c represents the mean with SEM. d) Spine neck width during the 6 cycles. e) Spine head area during the 6 cycles. f) One-way ANOVA test results of neck width, and head area for the different cycles. (N = 44 spines; N = 6 cells; 3 cultures). Data points (red color) in d and e represents the mean with SD.

Supplementary Figure 8 Visualization of synaptic contacts with correlative SUSHI/PALM.

a) Examples of synaptic contacts resolved by correlative SUSHI (gray scale, inverted colors) and PALM (magenta) with FWHM measurements of gap between pre- and postsynaptic contacts (green line), b stands for presynaptic bouton, scale bar 1 µm, six representative images out of 9 independent experiments. b) FWHM measurements on SUSHI image shown in Fig. 3c.

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Inavalli, V.V.G.K., Lenz, M.O., Butler, C. et al. A super-resolution platform for correlative live single-molecule imaging and STED microscopy. Nat Methods 16, 1263–1268 (2019).

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