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Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy.

Bon P, Bourg N, Lécart S, Monneret S, Fort E, Wenger J, Lévêque-Fort S - Nat Commun (2015)

Bottom Line: Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry.We show how to combine the intensity and phase information to provide the key to the third axial dimension.We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratoire Photonique Numérique et Nanosciences (LP2N), CNRS UMR5298, Institut d'Optique Graduate School, Bordeaux University, Rue Francois Mitterand, 33400 Talence, France [2] Institut Langevin, ESPCI ParisTech, CNRS UMR 7587, PSL Research University, 1 rue Jussieu, Paris 75238, France [3] Institut des Sciences Moléculaires d'Orsay (ISMO), University Paris-Sud, CNRS UMR 8214, Orsay 91405, France.

ABSTRACT
Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

No MeSH data available.


Related in: MedlinePlus

Super-localization enhanced dSTORM imaging on actin phalloidin-A647 labelling.(a) Epi-fluorescence image. (b) dSTORM image without drift correction. (c) dSTORM reconstruction with lateral (x, y) drifts stabilization. The detection threshold is lowered to maximize the number of detected molecules and thus reduce the negative effects of axial drifts. (d) dSTORM reconstruction with full 3D drift correction. (e) Intensity cuts along the lines in the sub-images (a–d). The total acquisition time is 25 min.
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f3: Super-localization enhanced dSTORM imaging on actin phalloidin-A647 labelling.(a) Epi-fluorescence image. (b) dSTORM image without drift correction. (c) dSTORM reconstruction with lateral (x, y) drifts stabilization. The detection threshold is lowered to maximize the number of detected molecules and thus reduce the negative effects of axial drifts. (d) dSTORM reconstruction with full 3D drift correction. (e) Intensity cuts along the lines in the sub-images (a–d). The total acquisition time is 25 min.

Mentions: Figure 3 shows dSTORM images of F-actin networks in fixed CHO cells labelled by Alexa Fluor 647 phalloidin (more dSTORM images on tubulin network are shown in the Supplementary Fig. 1). When no stabilization is active to compensate for spatial drifts (Fig. 3b), very few fluorescent molecules are detected and the spatial resolution is poor. A conventional approach to recover the detection events in the case of defocus is to lower the fluorescence detection threshold. The lateral (x, y) drifts can be compensated using the intensity images from the gold nanoparticles fiducials. However, in that case (Fig. 3c), the localization signal-to-noise ratio for each fluorophore is low, hence the reconstruction uncertainty is relatively large and the dSTORM resolution is limited. In the case of the full 3D stabilization using our method (Fig. 3d), the resolution gain is clearly visible as sub-networks are appearing on the reconstructed images. We estimate the dSTORM spatial resolution from localization histograms (Fig. 3e and Supplementary Fig. 2). The lateral resolution for the F-actin experiments with 3D stabilization is 11 nm for the smallest fibres, realizing a threefold improvement of the resolution obtained without lateral drift compensation (see Supplementary Fig. 2 and Supplementary Table 2).


Three-dimensional nanometre localization of nanoparticles to enhance super-resolution microscopy.

Bon P, Bourg N, Lécart S, Monneret S, Fort E, Wenger J, Lévêque-Fort S - Nat Commun (2015)

Super-localization enhanced dSTORM imaging on actin phalloidin-A647 labelling.(a) Epi-fluorescence image. (b) dSTORM image without drift correction. (c) dSTORM reconstruction with lateral (x, y) drifts stabilization. The detection threshold is lowered to maximize the number of detected molecules and thus reduce the negative effects of axial drifts. (d) dSTORM reconstruction with full 3D drift correction. (e) Intensity cuts along the lines in the sub-images (a–d). The total acquisition time is 25 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525210&req=5

f3: Super-localization enhanced dSTORM imaging on actin phalloidin-A647 labelling.(a) Epi-fluorescence image. (b) dSTORM image without drift correction. (c) dSTORM reconstruction with lateral (x, y) drifts stabilization. The detection threshold is lowered to maximize the number of detected molecules and thus reduce the negative effects of axial drifts. (d) dSTORM reconstruction with full 3D drift correction. (e) Intensity cuts along the lines in the sub-images (a–d). The total acquisition time is 25 min.
Mentions: Figure 3 shows dSTORM images of F-actin networks in fixed CHO cells labelled by Alexa Fluor 647 phalloidin (more dSTORM images on tubulin network are shown in the Supplementary Fig. 1). When no stabilization is active to compensate for spatial drifts (Fig. 3b), very few fluorescent molecules are detected and the spatial resolution is poor. A conventional approach to recover the detection events in the case of defocus is to lower the fluorescence detection threshold. The lateral (x, y) drifts can be compensated using the intensity images from the gold nanoparticles fiducials. However, in that case (Fig. 3c), the localization signal-to-noise ratio for each fluorophore is low, hence the reconstruction uncertainty is relatively large and the dSTORM resolution is limited. In the case of the full 3D stabilization using our method (Fig. 3d), the resolution gain is clearly visible as sub-networks are appearing on the reconstructed images. We estimate the dSTORM spatial resolution from localization histograms (Fig. 3e and Supplementary Fig. 2). The lateral resolution for the F-actin experiments with 3D stabilization is 11 nm for the smallest fibres, realizing a threefold improvement of the resolution obtained without lateral drift compensation (see Supplementary Fig. 2 and Supplementary Table 2).

Bottom Line: Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry.We show how to combine the intensity and phase information to provide the key to the third axial dimension.We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratoire Photonique Numérique et Nanosciences (LP2N), CNRS UMR5298, Institut d'Optique Graduate School, Bordeaux University, Rue Francois Mitterand, 33400 Talence, France [2] Institut Langevin, ESPCI ParisTech, CNRS UMR 7587, PSL Research University, 1 rue Jussieu, Paris 75238, France [3] Institut des Sciences Moléculaires d'Orsay (ISMO), University Paris-Sud, CNRS UMR 8214, Orsay 91405, France.

ABSTRACT
Meeting the nanometre resolution promised by super-resolution microscopy techniques (pointillist: PALM, STORM, scanning: STED) requires stabilizing the sample drifts in real time during the whole acquisition process. Metal nanoparticles are excellent probes to track the lateral drifts as they provide crisp and photostable information. However, achieving nanometre axial super-localization is still a major challenge, as diffraction imposes large depths-of-fields. Here we demonstrate fast full three-dimensional nanometre super-localization of gold nanoparticles through simultaneous intensity and phase imaging with a wavefront-sensing camera based on quadriwave lateral shearing interferometry. We show how to combine the intensity and phase information to provide the key to the third axial dimension. Presently, we demonstrate even in the occurrence of large three-dimensional fluctuations of several microns, unprecedented sub-nanometre localization accuracies down to 0.7 nm in lateral and 2.7 nm in axial directions at 50 frames per second. We demonstrate that nanoscale stabilization greatly enhances the image quality and resolution in direct stochastic optical reconstruction microscopy imaging.

No MeSH data available.


Related in: MedlinePlus