Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms

Huang, Fang; Hartwich, Tobias M.P.; Rivera‐Molina, Felix; Lin, Yu; Duim, Whitney C.; Long, Jane J.; Uchil, Pradeep D.; Myers, Jordan; Baird, Michelle A.; Mothes, Walther; Davidson, Michael W.; Toomre, Derek; Bewersdorf, Joerg

doi:10.1038/nmeth.2488

Cited by 497 publications

(552 citation statements)

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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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“…Thus, each localized singlemolecule emitter contains spatial and temporal information that provides quantitative data that surpass those obtained from other super-resolution techniques. We took advantage of recent developments in instrumentation and data analysis methods (26)(27)(28)(29)(30)(31)(32) to perform FPALM super-resolution microscopy at ∼35-nm resolution (localization precision, σ loc , ∼14 nm). These methods enabled us to observe that nodes are uniform units with stoichiometric ratios and distinct distributions of constituent proteins, and provided the quantitative data necessary to build a molecular model of the node.…”

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Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Laplante

Huang

Tebbs

et al. 2016

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

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Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/ Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring.T he mechanism of cell division by a contractile ring of actin and myosin II appeared in the common ancestor of amoebas, fungi, and animals (1). Although much is known about the protein composition of contractile rings, relatively little is known about the 3D organization of these proteins. This information is required to formulate computer models and simulations to test ideas regarding the mechanisms of contractile ring function.Electron microscopy has shown actin filaments in contractile rings of animal cells (2, 3) and yeast cells (4,5). Myosin II concentrates in cleavage furrow (6) and is the main motor for constricting contractile rings (7,8). In animal cells, electron microscopy has revealed rods the size of myosin II minifilaments in contractile rings (2, 9), and structured-illumination fluorescence microscopy has shown that this myosin II is organized in bipolar assemblies (10). Contractile rings contain other structural and regulatory proteins, including anillin (11), IQ motif containing GTPase-activating proteins (IQGAP) (12), formins (13), alphaactinin (14), and Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) proteins (15), but how these proteins are anchored to the plasma membrane or organized into functional complexes in animal cells is unknown.Our understanding of the cytokinetic apparatus is most advanced in fission yeast (16). Only in fission yeast do we know the concentrations of the major cytokinesis proteins (17) and the time course of events leading to cellular division (18,19). Molecularly explicit computer models can account for both the ...

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“…The experimenter must choose actively use some method to control the emitting concentration. Of course, the imaging is still timesequential, thus this approach is best for quasi-static structures or fixed cells, but significant progress has been made in increasing the imaging speed (152). Selected reviews may be consulted for additional detail of modern challenges and progress (153)(154)(155)(156) (157)(158-161).…”

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confidence: 99%

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

Moerner

2015

Rev. Mod. Phys.

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The initial steps toward optical detection and spectroscopy of single molecules in condensed matter arose out of the study of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral signatures relating to the fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 90's, many fascinating physical effects were observed for individual molecules, and the imaging of single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency provided important forerunners of the later superresolution microscopy with single molecules. In the room temperature regime, imaging of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic observation and localization of individual fluorophores is a key ingredient to imaging beyond the optical diffraction limit. Combining this with active control of the number of emitting molecules in the pumped volume led to the super-resolution imaging of Eric Betzig and others, a new frontier for optical microscopy beyond the diffraction limit. The background leading up to these observations is described and current developments are summarized. The early days Introduction and early inspirationsI want to thank the Nobel Committee for Chemistry, the Royal Swedish Academy of Sciences, and the Nobel Foundation for selecting me for this prize recognizing the development of super-resolved fluorescence microscopy. I am truly honored to share the prize with my two esteemed colleagues, Stefan Hell and Eric Betzig. My primary contributions center on the first optical detection and spectroscopy of single molecules in the condensed phase(1), and on the observations of imaging, blinking and photocontrol not only for single molecules at low temperatures in solids, but also for useful variants of the green fluorescent protein at room temperature (2). This lecture describes the context of the events leading up to these advances as well as a portion of the subsequent developments both around the world and in my laboratory.In the mid-1980's, I derived much early inspiration from amazing advances that were occurring around the world where single nanoscale quantum systems were detected and explored for both scientific and technological reasons. Some of these were (i) the spectroscopy of single electrons or ions confined in vacuum electromagnetic traps (3-5), (ii) scanning tunneling microscopy (STM) (6) and atomic force microscopy (AFM) (7), and (iii) the study of ion currents in single membrane-embedded ion channels (8). But why not optical detection and ...

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Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms

Cited by 497 publications

References 32 publications

Quantitative evaluation of software packages for single-molecule localization microscopy

Quantitative evaluation of software packages for single-molecule localization microscopy

Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

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