Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy

Hess, Samuel T.; Girirajan, Thanu Prabha Kalambur; Mason, Michael D.

doi:10.1529/biophysj.106.091116

Cited by 3,290 publications

(2,581 citation statements)

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“…The diffraction limit has ceased to be a practical limit to resolution in far-field microscopy, following the demonstration of STED, 1,2,3 RESOLFT 4 and localisation microscopies 5,6,7 and the subsequent development of a plethora of super-resolved nanoscopy techniques. 8 In particular, stimulated emission depletion (STED) nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for super-resolved imaging in complex biological samples including live organisms.…”

Section: Key-words: Sted Nanoscopy Nanorods Super-resolution Plasmmentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

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Plasmonic nanoparticles influence the absorption and emission processes of nearby emitters due to local enhancements of the illuminating radiation and the photonic density of states. Here, we use the plasmon resonance of metal nanoparticles in order to enhance the stimulated depletion of excited molecules for super-resolved nanoscopy. We demonstrate stimulated emission depletion (STED) nanoscopy with gold nanorods with a long axis of only 26 nm and a width of 8 nm that provide an enhancement of up to 50% of the resolution compared to fluorescent-only probes without plasmonic components irradiated with the same depletion power. The nanoparticle-assisted STED probes reported here represent a ~2x10 3 reduction in probe volume compared to previously used nanoparticles. Finally, we demonstrate their application toward plasmon-assisted STED cellular imaging at low-depletion powers and we also discuss their current limitations. Key-words: STED nanoscopy, nanorods, super-resolution, plasmonic nanoparticles, bio-imagingThe diffraction limit has ceased to be a practical limit to resolution in far-field microscopy, following the demonstration of STED, 1,2,3 RESOLFT 4 and localisation microscopies 5,6,7 and the subsequent development of a plethora of super-resolved nanoscopy techniques. 8 In particular, stimulated emission depletion (STED) nanoscopy, which builds on the advantages of laser scanning confocal microscopy, is a powerful technique for super-resolved imaging in complex biological samples including live organisms. 9,10 STED nanoscopy uses stimulated emission to turn off the spontaneous fluorescence emission of dye molecules, typically overlapping a focused excitation beam with a "doughnut" shaped beam that deexcites emitters to the ground state everywhere except for the area within the centre of the doughnut, thus providing theoretically diffraction-unlimited resolution in the transverse plane by reducing the fullwidth half-maximum (FWHM) of the point spread function. By increasing the power of the depletion beam the emission region can be can be drastically reduced -theoretically allowing for sub-nanometre resolution -with resolutions of less than 10 nm being demonstrated. 11 The scaling of resolution with the square root of the depletion beam power means that relatively high-power lasers are typically used for STED nanoscopy. In practice, however, the use of high-power irradiation can result in problems such as photobleaching of the fluorophores and phototoxicity, and so the achievable resolution is compromised by the need to limit the intensity of the depletion laser radiation. Furthermore, high power lasers can add cost and complexity to STED microscopes and so the requirement for high power depletion beams presents challenges for parallelizing STED measurements 12,13 in terms of the lasers required, thus, limiting the potential for faster super-resolved imaging.To some extent the issue of photobleaching can be addressed with the development of more robust fluorescent labels such as quantum dots 14 as ...

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Section: Key-words: Sted Nanoscopy Nanorods Super-resolution Plasmmentioning

confidence: 99%

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Cortés¹,

Huidobro²,

Sinclair³

et al. 2016

ACS Nano

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“…SMLM was demonstrated in 2006, independently by three research groups [1][2][3] , and has enabled subsequent breakthroughs in diverse fields 4,5 . SMLM can resolve biological structures at the nanometer scale (typically 20 nm lateral resolution), circumventing Abbe's diffraction limit.…”

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confidence: 99%

Quantitative evaluation of software packages for single-molecule localization microscopy

et al. 2015

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the quality of super-resolution images obtained by singlemolecule localization microscopy (smlm) depends largely on the software used to detect and accurately localize point sources. in this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. these metrics reflect the various tradeoffs of smlm software packages and help users to choose the software that fits their needs.We have conducted a large-scale comparative study of software packages developed in the context of SMLM, including recently developed algorithms. We designed realistic data that are generic and cover a broad range of experimental conditions and compared the software packages using a multiple-criterion quantitative assessment that is based on a known ground truth.Our study is based on the active participation of developers of SMLM software. More than 30 groups have participated so far, and the study is still under way. We provide participants access to our benchmark data as an ongoing public challenge. Participants run their own software on our data and report their list of localized particles for evaluation. The results of the challenge are accessible online and updated regularly.SMLM was demonstrated in 2006, independently by three research groups 1-3 , and has enabled subsequent breakthroughs in diverse fields 4,5 . SMLM can resolve biological structures at the nanometer scale (typically 20 nm lateral resolution), circumventing Abbe's diffraction limit. At the cost of a relatively simple setup 6,7 , it has opened exciting new opportunities in life science research 8,9 .The underlying principle of SMLM is the sequential imaging of sparse subsets of fluorophores distributed over thousands of frames, to populate a high-density map of fluorophore positions. Such large data sets require automated image-analysis algorithms to detect and precisely infer the position of individual fluorophore, taking advantage of their separation in space and time.The acquired data cannot be visualized directly; further computerized image-reconstruction methods are required. These typically comprise four steps: preprocessing, detection, localization and rendering. Preprocessing reduces the effects of the background and noise; detection identifies potential molecule candidates in each frame; localization performs a subpixel refine...

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“…Despite the increased resolution, these produce conventional fluorescence images, i.e., arrays of pixels with values representing the fluorescence intensity at those locations. Quantification can be performed in the same way as for conventional microscopes.Another strategy is based on Single-Molecule Localisation Microscopy (SMLM) [4][5][6][7] . This relies on the temporal separation of the excitation of fluorophores in the sample whose PSFs would otherwise overlap at the detector.…”

mentioning

confidence: 99%

“…Another strategy is based on Single-Molecule Localisation Microscopy (SMLM) [4][5][6][7] . This relies on the temporal separation of the excitation of fluorophores in the sample whose PSFs would otherwise overlap at the detector.…”

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confidence: 99%

Bayesian cluster identification in single-molecule localization microscopy data

et al. 2015

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Correspondence should be addressed to patrick. rubin-delanchy@bristol.ac.uk and dylan.owen@kcl.ac.uk Single-molecule identification-based super-resolution microscopy techniques such as photo-activated localisation microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) produce pointillist data sets of molecular coordinates. While many algorithms exist for the identification and localisation of molecules from the raw image data, methods for analysing the resulting point patterns for properties such as clustering have remained relatively under-studied. Here, we present the first model-based Bayesian approach to evaluate molecular cluster assignment proposals which, in this article, are generated by analysis based on Ripley's Kfunction. The method is also the first to take full account of the individual localisation precisions calculated for each emitter. The technique is validated using simulated and experimental data from which we characterise the clustering behaviour of CD3ζ, an important subunit of the CD3-T cell receptor complex required for T cell function, in resting and activated primary human T cells.Conventional fluorescence microscopes produce images of the distribution of fluorophores in the sample convolved with the microscope Point Spread Function (PSF). Due to diffraction, this PSF typically has a width of hundreds of nanometres meaning the resulting image has a resolution, as assessed by the Rayleigh criterion, of ~200 nm. Several strategies now exist to circumvent this resolution limit 1 . Some of these, such as Stimulated Emission Depletion (STED) microscopy, rely on narrowing the excitation spot of a confocal microscope by means of a toroidal depletion beam and the process of stimulated emission 2,3 . Despite the increased resolution, these produce conventional fluorescence images, i.e., arrays of pixels with values representing the fluorescence intensity at those locations. Quantification can be performed in the same way as for conventional microscopes.Another strategy is based on Single-Molecule Localisation Microscopy (SMLM) [4][5][6][7] . This relies on the temporal separation of the excitation of fluorophores in the sample whose PSFs would otherwise overlap at the detector.The position of each fluorophore can then be estimated from the centres of the PSFs. Many algorithms are available to extract the x-y coordinates of the molecules [8][9][10] . Each emitter can be localised to a precision between 10 and 30 nm. Common strategies for the temporal separation of molecules involve intra-molecular rearrangements to switch from dark to fluorescent states or the exploitation of non-emitting molecular radicals 11,12 . These strategies are typically pursued using photoactivatable or photoconvertible fluorescent proteins or small molecule probes coupled with a reducing buffer and immunostaining protocols 13 . We refer to all such strategies as SMLM.Unlike non-pointillist microscopy methods, SMLM imaging does not produce a conventional image. Instead, the raw data is a list of the...

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Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy

Cited by 3,290 publications

References 52 publications

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Plasmonic Nanoprobes for Stimulated Emission Depletion Nanoscopy

Quantitative evaluation of software packages for single-molecule localization microscopy

Bayesian cluster identification in single-molecule localization microscopy data

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