New directions in single-molecule imaging and analysis

Moerner, W. E.

doi:10.1073/pnas.0610081104

Cited by 418 publications

(343 citation statements)

References 128 publications

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“…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.…”

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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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Quantitative evaluation of software packages for single-molecule localization microscopy

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“…Apart from biological applications, single-molecule spectroscopy has provided exceptional insights into the properties of soft and complex materials, and holds great promise for further development in this fi eld. For recent reviews, see Refs [3][4][5][6][7][8]. Th ere will, no doubt, be further qualitative progress related to recently developed super-resolution microscopic techniques [9][10], which overcome the diff raction limit in optical imaging and bring the resolution closer to the molecular scale.…”

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Conformation and physics of polymer chains: a single-molecule perspective

Vácha

Habuchi

2010

NPG Asia Mater

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N anotechnology has been one of the driving forces of science and technology over the past 20 years. Scaling down the size of an object to nanometer dimensions can result in dramatic changes to its physical properties. With recent progress in the nanoscience and nanotechnology of polymeric materials, new methods are now required for the characterization of polymer structures and properties. Despite the great success of electron microscopy and scanning probe microscope techniques, there remains a need for a non-invasive method that could provide information not only on the structure but also on the physical phenomena occurring on the nanoscale level. One method that has recently emerged as an excellent tool for nanoscale characterization is single-molecule optical detection and spectroscopy. Single-molecule spectroscopy relies on the detection and measurement of the fl uorescence signal and spectra from single isolated molecules or polymer chains (Figure 1). Th ese can either be immobilized in a solid matrix or be diff using in a solution or melt. In all cases, the concentration of the emitting molecules must be suffi ciently low to ensure that the spatial separation between individual molecules is larger than the optical resolution of the setup, typically on the order of micrometers. As the fl uorescence signal from a molecule is strongly infl uenced by the nanoscale environment, measuring many molecules one by one provides the distribution of physical properties of the respective nano-environments. Moreover, monitoring a molecule over a period of time reveals dynamic changes in its environment. Th ese static and dynamic heterogeneities of nanoscale properties are averaged and hidden in the usual ensemble experiments. Th e strength of fl uorescence detection is in its sensitivity and low background level -emissions of just a few hundred photons per second can be readily detected. Th e technique also off ers remarkable dynamic range, from sub-nanosecond timescales to seconds and longer, and is non-invasive, making it possible to detect emissions from molecules buried deep in a sample.Single-molecule spectroscopy has evolved since the beginning of the 1990s from low-temperature, high-resolution optical spectroscopic

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“…Steps toward super-resolution with single-molecule emitters. For acronyms, see (139) In 1995, after spending years developing near-field optical imaging at Bell Labs, Eric Betzig wrote a seminal paper noting that a control variable that distinguishes molecules along another dimension could be used for superresolution microscopy, and he suggested the use of many molecules with different colors, as in the low temperature studies (131). Subsequently, in 1998 Antoine van Oijen et al experimentally demonstrated this idea directly: they used spectral tunability at low temperatures to spatially resolve a set of single molecules in three dimensions, with 40 nm lateral and 100 nm axial resolution, far below the optical diffraction limit (132,133).…”

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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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New directions in single-molecule imaging and analysis

Cited by 418 publications

References 128 publications

Quantitative evaluation of software packages for single-molecule localization microscopy

Quantitative evaluation of software packages for single-molecule localization microscopy

Conformation and physics of polymer chains: a single-molecule perspective

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

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