Superresolution by localization of quantum dots using blinking statistics

Lidke, Keith A.; Rieger, Bernd; Jovin, Thomas M.; Heintzmann, Rainer

doi:10.1364/opex.13.007052

Cited by 332 publications

(259 citation statements)

References 8 publications

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“…A final image is formed by summing the locations of all single molecules derived from imaging the separate randomly generated sparse collections. Variations on this idea have also appeared, for example, by using accumulated binding of diffusible probes (17) or quantum dot blinking (18). Importantly, several of these techniques have recently been pushed to 3 dimensions by using astigmatism (19), multiplane methods (20), and 2-photon activation by temporal focusing (21) to quantify the z position of the emitters.…”

mentioning

confidence: 99%

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Pavani

Thompson

Biteen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

928

787

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We demonstrate single-molecule fluorescence imaging beyond the optical diffraction limit in 3 dimensions with a wide-field microscope that exhibits a double-helix point spread function (DH-PSF). The DH-PSF design features high and uniform Fisher information and has 2 dominant lobes in the image plane whose angular orientation rotates with the axial (z) position of the emitter. Single fluorescent molecules in a thick polymer sample are localized in single 500-ms acquisitions with 10-to 20-nm precision over a large depth of field (2 m) by finding the center of the 2 DH-PSF lobes. By using a photoactivatable fluorophore, repeated imaging of sparse subsets with a DH-PSF microscope provides superresolution imaging of high concentrations of molecules in all 3 dimensions. The combination of optical PSF design and digital postprocessing with photoactivatable fluorophores opens up avenues for improving 3D imaging resolution beyond the Rayleigh diffraction limit.microscopy ͉ photoactivation ͉ superresolution ͉ computational imaging ͉ PSF engineering F luorescence microscopy is ubiquitous in biological studies because light can noninvasively probe the interior of a cell with high signal-to-background and remarkable label specificity. Unfortunately, optical diffraction limits the transverse (x-y) resolution of a conventional fluorescence microscope to approximately /(2NA), where is the optical wavelength and NA is the numerical aperture of the objective lens (1). This limitation requires that point sources need to be Ͼ Ϸ200 nm apart in the visible wavelength region to be distinguished with modern high-quality fluorescence microscopes. Diffraction causes the image of a single-point emitter to appear as a blob (i.e., the point-spread function or PSF) with a width given by the diffraction limit. However, if the shape of the PSF is measured, then the center position of the blob can be determined with a far greater precision (termed superlocalization) that scales approximately as the diffraction limit divided by the square root of the number of photons collected, a fact noted as early as Heisenberg in the context of electron localization with photons (2) and later extended to point objects (3, 4) and single-molecule emitters (5-8). Because single-molecule emitters are only a few nanometers in size, they represent particularly useful point sources for imaging, and superlocalization of single molecules at room temperature has been pushed to the 1-nm regime (9) in transverse (2-dimensional) imaging. In the third (z) dimension, diffraction also limits resolution to Ϸ2n /NA 2 with n the index of refraction, corresponding to a depth of field of Ϸ500 nm in the visible wavelength region with modern microscopes. Improvements in 3D localization beyond this limit are also possible by using astigmatism (10, 11), defocusing (12), or simultaneous multiplane viewing (13).Until recently, superlocalization of individual molecules was unable to provide true resolution beyond the diffraction limit (superresolution) because the concentration of emi...

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mentioning

confidence: 99%

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Pavani

Thompson

Biteen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

928

787

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“…When combined with a PSF measurement, this approach has been able to distinguish pairs (4) and quartets (5) of molecules with nanometer precision. Another approach has been to analyze the blinking trajectory of the emission of quantum dots to distinguish between a single dot and groups of them clustered on the nanometer scale (6). The intermittency in the fluorescence signal caused by diffusioncontrolled collisions of a fluorescent probe with objects of interest was recently introduced as a method of subdiffraction imaging (7).…”

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confidence: 99%

Wide-field subdiffraction imaging by accumulated binding of diffusing probes

Sharonov

Hochstrasser

2006

Proc. Natl. Acad. Sci. U.S.A.

959

907

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A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the solution. Because the flux of probes at the object is essentially constant over long time periods, the examination of an almost unlimited number of individual probe molecules becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resolution of Ϸ25 nm is readily achieved. The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.diffusion controlled ͉ fluorescence ͉ single molecule F or the purpose of visualizing biological processes and structures, it is essential to develop new methods that can determine the spatial locations of molecules with the highest possible precision. The submolecular methods of spatial resolution such as x-ray and electron topography, atomic force microscopy, and near-field optical microscopy have contributed enormously toward this goal. However, new approaches that are not invasive to biological objects and are accessible to bulk, as well as surface structures, could have a significant impact and could address many new questions in the life sciences (1). Optical methods based on fluorescence detection do have single-molecule sensitivity and can be arranged to be nondestructive even when performed in natural environments, incorporating such complex and sensitive structures as living cells. In principle, optical responses can probe the entire 3D space. Although diffraction limits the spatial resolution of optical methods, many approaches have been developed recently that allow distance measurements on scales that are much shorter than the wavelength of the light.It is possible to specify the location of a single molecule with very high precision from measurements of its fluorescence by fitting the emitted intensity distribution to the 2D spatial parameters of the point-spread function (PSF). This approach has been shown to locate emitters with Ϸ1-nm precision (2). The distance between two optically different molecules can be estimated by FRET methods that can serve as molecular rulers at nanometer accuracy (3). Two molecules emitting light at different frequencies have also been resolved by means of PSF measurements. Even for molecules having the same spectra, the process of photobleaching causes the fluorescence source to be switched between molecules. When combined with a PSF measurement, this approach has been able to distinguish pairs (4) and quartets (5) of molecules with nanometer precision. Another approach has been to analyze the blinking trajectory of the emission of quantum dots to distinguish between a single dot and groups of them clustered on the nanom...

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“…The lateral resolution in PBM depends primarily on the localization precision of particles and their lateral diffusion lengths during single frame exposure time. The strong plasmon resonance of the metal NPs results in a couple of orders brighter PSFs (~25,000 photons) than when obtained with typical bright organic dye molecules employed in SMLM [18][19][20][21]. Thus, if they were stationary, localization precision [22,23] of their lateral positions could be down to 5 nm at a typical per-frame exposure time of 1-5 ms. More dominant influence on lateral resolution is attributed to Brownian diffusion of particles that induces around 20-30 nm positional uncertainty in our typical exposure time.…”

Section: Resultsmentioning

confidence: 99%

“…Plasmonic superlens [13][14][15][16] enables super-resolution optical imaging beyond the diffraction limit but only provides two-dimensional imaging at a smaller area [17]. Single molecule localization microscopy (SMLM) allows 3D imaging of complex objects with nanometer accuracy, but requires the samples labeled with emitter molecules such as fluorophores and quantum dots [18][19][20][21]. The staining requirement makes SMLM primarily applicable to biological objects with known molecular identities.…”

Section: Introductionmentioning

confidence: 99%

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

et al. 2017

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Three-dimensional (3D) imaging at the nanoscale is a key to understanding of nanomaterials and complex systems. While scanning probe microscopy (SPM) has been the workhorse of nanoscale metrology, its slow scanning speed by a single probe tip can limit the application of SPM to wide-field imaging of 3D complex nanostructures. Both electron microscopy and optical tomography allow 3D imaging, but are limited to the use in vacuum environment due to electron scattering and to optical resolution in micron scales, respectively. Here we demonstrate plasmonic Brownian microscopy (PBM) as a way to improve the imaging speed of SPM. Unlike photonic force microscopy where a single trapped particle is used for a serial scanning, PBM utilizes a massive number of plasmonic nanoparticles (NPs) under Brownian diffusion in solution to scan in parallel around the unlabeled sample object. The motion of NPs under an evanescent field is three-dimensionally localized to reconstruct the superresolution topology of 3D dielectric objects. Our method allows high throughput imaging of complex 3D structures over a large field of view, even with internal structures such as cavities that cannot be accessed by conventional mechanical tips in SPM.

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Superresolution by localization of quantum dots using blinking statistics

Cited by 332 publications

References 8 publications

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function

Wide-field subdiffraction imaging by accumulated binding of diffusing probes

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

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