Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples

Juette, Manuel F.; Gould, Travis J.; Lessard, Mark D.; Mlodzianoski, Michael J.; Nagpure, Bhupendra S.; Bennett, Brian; Hess, Samuel T.; Bewersdorf, Joerg

doi:10.1038/nmeth.1211

Cited by 769 publications

(638 citation statements)

References 15 publications

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“…In the future, our reference data will include more features, such as drift, 3D localization (PSF engineering 57,58 and multiple planes 59,60 , additional levels of molecule density, multiple fluorescent channels, asymmetrical PSF due to dipole effect 61 , scattering effects, and a richer variety of noise models associated with various types of cameras, EMCCD, sCMOS 62,63 . It will also be interesting to generate benchmarking data to test the impact of clustering (spatial aggregation) and diffusion for single-particle tracking.…”

Section: Competing Financial Interestsmentioning

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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Section: Competing Financial Interestsmentioning

confidence: 99%

Quantitative evaluation of software packages for single-molecule localization microscopy

et al. 2015

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“…20,21 We have recently reported a new family of switchable rhodamine dyes whose fluorescence ability can be selectively switched on in a single layer of the sample by two-photon absorption, thus conferring noninvasive optical sectioning to single-molecule-switching-based nanoscopy. 22 In combination with localization along the third dimension, 3D nanoscopy of extended sample regions is feasible, 23,24 thus complementing 3D-approaches based on STED. [25][26][27][28] Fortunately, switching emitters to reach nanoscopical resolution largely maintains the versatility of the fluorescence readout.…”

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confidence: 99%

Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

et al. 2008

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By combining the photoswitching and localization of individual fluorophores with spectroscopy on the single molecule level, we demonstrate simultaneous multicolor imaging with low crosstalk and down to 15 nm spatial resolution using only two detection color channels. The applicability of the method to biological specimens is demonstrated on mammalian cells. The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques.

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“…Highly uniform gold NPs could be specially synthesized to minimize such axial uncertainty. We believe that other well-known strategies of axial localization such as highly axially dependent illumination, bi-plane detection or PSF engineering [24][25][26] will provide higher precision as these methods do not rely on the scattering intensity but the shape of PSFs over height. Thus, the illumination profile and the uniformity of NPs can become no longer critical.…”

Section: Resultsmentioning

confidence: 99%

“…Our 3D imaging method would have an imaging depth of less than 1 μm mainly due to axial localization enabled by TIR illumination. However, thicker samples could also in principle be imaged in PBM by employing the aforementioned strategies [24][25][26]. Also, fluorescent particles rather than gold NPs could be considered in certain circumstances.…”

Section: Resultsmentioning

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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Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples

Cited by 769 publications

References 15 publications

Quantitative evaluation of software packages for single-molecule localization microscopy

Quantitative evaluation of software packages for single-molecule localization microscopy

Multicolor Far-Field Fluorescence Nanoscopy through Isolated Detection of Distinct Molecular Species

Three-dimensional nanoscale imaging by plasmonic Brownian microscopy

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