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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

Quantitative intensity and phase imaging to localize nanoparticles.(a) Schematic of the optical set-up and sample. The microscope port highlighted in yellow is dedicated to intensity and phase imaging of nanoparticles in transillumination using the microscope lamp, with MHM the acronym for Modified Hartman Mask. The port highlighted in red corresponds to dSTORM imaging. (b) Intensity and phase images of two 100 nm gold nanoparticles, in-focus (z=0) or slightly defocused (z=±250 nm). Note the contrast inversion in the phase images on defocusing and the weak variations of the intensity signal. The scale bar, 2 μm. (c) Intensity (black squares) and phase (red dots) response of a single 100 nm gold nanoparticle recorded versus the mechanical sample displacement. Lines are the results of the numerical propagation computed using the z=0 plane data only; they are not numerical fits to the experimental data.
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f1: Quantitative intensity and phase imaging to localize nanoparticles.(a) Schematic of the optical set-up and sample. The microscope port highlighted in yellow is dedicated to intensity and phase imaging of nanoparticles in transillumination using the microscope lamp, with MHM the acronym for Modified Hartman Mask. The port highlighted in red corresponds to dSTORM imaging. (b) Intensity and phase images of two 100 nm gold nanoparticles, in-focus (z=0) or slightly defocused (z=±250 nm). Note the contrast inversion in the phase images on defocusing and the weak variations of the intensity signal. The scale bar, 2 μm. (c) Intensity (black squares) and phase (red dots) response of a single 100 nm gold nanoparticle recorded versus the mechanical sample displacement. Lines are the results of the numerical propagation computed using the z=0 plane data only; they are not numerical fits to the experimental data.

Mentions: Our stabilization method relies on monitoring the position of a gold nanoparticle using combined intensity and phase imaging with a commercial wavefront-sensing device (Fig. 1a). In the visible and near infrared spectral range, the imaginary part of the complex refractive index of gold dominates over the real part28. Therefore, when a gold nanoparticle is perfectly set at the microscope focus (Fig. 1b; z=0), the intensity drop is large as light is lost due to absorption and scattering2930, and the phase response is weak as the optical retardation is almost negligible for a subwavelength nanoparticle. The intensity image at the focus provides 2D super-localization of the nanoparticle in the transverse plane using a conventional 2D Gaussian fit of the diffraction-limited spot3132, as reported for fluorescence emitters in earlier works33. However, in the case of a nanometre axial defocus (Fig. 1b,c), the intensity barely changes. Moreover, the intensity dependence on the axial position is symmetric respective to the optimal focus position. Axial super-localization is therefore difficult to achieve using only the intensity images without time-consuming physical displacement of the sample stage and acquisition of multiple intensity images.


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)

Quantitative intensity and phase imaging to localize nanoparticles.(a) Schematic of the optical set-up and sample. The microscope port highlighted in yellow is dedicated to intensity and phase imaging of nanoparticles in transillumination using the microscope lamp, with MHM the acronym for Modified Hartman Mask. The port highlighted in red corresponds to dSTORM imaging. (b) Intensity and phase images of two 100 nm gold nanoparticles, in-focus (z=0) or slightly defocused (z=±250 nm). Note the contrast inversion in the phase images on defocusing and the weak variations of the intensity signal. The scale bar, 2 μm. (c) Intensity (black squares) and phase (red dots) response of a single 100 nm gold nanoparticle recorded versus the mechanical sample displacement. Lines are the results of the numerical propagation computed using the z=0 plane data only; they are not numerical fits to the experimental data.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Quantitative intensity and phase imaging to localize nanoparticles.(a) Schematic of the optical set-up and sample. The microscope port highlighted in yellow is dedicated to intensity and phase imaging of nanoparticles in transillumination using the microscope lamp, with MHM the acronym for Modified Hartman Mask. The port highlighted in red corresponds to dSTORM imaging. (b) Intensity and phase images of two 100 nm gold nanoparticles, in-focus (z=0) or slightly defocused (z=±250 nm). Note the contrast inversion in the phase images on defocusing and the weak variations of the intensity signal. The scale bar, 2 μm. (c) Intensity (black squares) and phase (red dots) response of a single 100 nm gold nanoparticle recorded versus the mechanical sample displacement. Lines are the results of the numerical propagation computed using the z=0 plane data only; they are not numerical fits to the experimental data.
Mentions: Our stabilization method relies on monitoring the position of a gold nanoparticle using combined intensity and phase imaging with a commercial wavefront-sensing device (Fig. 1a). In the visible and near infrared spectral range, the imaginary part of the complex refractive index of gold dominates over the real part28. Therefore, when a gold nanoparticle is perfectly set at the microscope focus (Fig. 1b; z=0), the intensity drop is large as light is lost due to absorption and scattering2930, and the phase response is weak as the optical retardation is almost negligible for a subwavelength nanoparticle. The intensity image at the focus provides 2D super-localization of the nanoparticle in the transverse plane using a conventional 2D Gaussian fit of the diffraction-limited spot3132, as reported for fluorescence emitters in earlier works33. However, in the case of a nanometre axial defocus (Fig. 1b,c), the intensity barely changes. Moreover, the intensity dependence on the axial position is symmetric respective to the optimal focus position. Axial super-localization is therefore difficult to achieve using only the intensity images without time-consuming physical displacement of the sample stage and acquisition of multiple intensity images.

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