Precise Nanometer Localization Analysis for Individual Fluorescent Probes

Thompson, Russell E.; Larson, Daniel R.; Webb, Watt W.

doi:10.1016/s0006-3495(02)75618-x

Cited by 2,121 publications

(2,262 citation statements)

References 32 publications

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“…We confirmed in this study of SMLM software packages that the experimental accuracy is one order of magnitude better than the classical diffraction limit, which supports theoretical findings 24,25 . This is the best one can hope for; indeed, a few software packages nearly achieve the Cramér-Rao lower bound.…”

Section: Discussionsupporting

confidence: 88%

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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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Section: Discussionsupporting

confidence: 88%

“…This Cramér-Rao lower bound (CRLB) was initially introduced as a fundamental limit of accuracy 23,24,37 . There also exist refined CRLBs that take pixelation, various sources of noise, and fluorescence background into account.…”

Section: Evaluation and Scoring Calculation Theoretical Accuracymentioning

confidence: 99%

Quantitative evaluation of software packages for single-molecule localization microscopy

et al. 2015

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“…Thompson et al [34] analyzed the sources contributing to localization precision in light microscopy images. They determined that the localization precision (or error), Δx, can be approximated as:…”

Section: Localization Precisionmentioning

confidence: 99%

“…The primary complicating factor is the fact that the noise in the presence (σ o ) and absence (σ b ) of signal is different, due to the presence of photon shot noise only when a fluorescent object is present [34]. One approach is to propagate the noise error as [35].…”

Section: Localization Precisionmentioning

confidence: 99%

“…One approach is to propagate the noise error as [35]. This error propagation formula assumes that the errors are independent, which they are not since σ o contains contributions from both shot noise and background noise errors [34]. Furthermore, for low signal levels, such as those typically observed in SMF experiments, this expression reduces to , which is a conservative (over)estimate of the true noise.…”

Section: Localization Precisionmentioning

confidence: 99%

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Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Yang

Musser

2006

Methods

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The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and ∼15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.

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Single‐Molecule Tracking Photoactivated Localization Microscopy to Map Nano‐Scale Structure and Dynamics in Living Spines

MacGillavry

Blanpied

2013

CP Neuroscience

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Super-resolution microscopy has rapidly become an indispensable tool in cell biology and neuroscience by enabling measurement in live cells of structures smaller than the classical limit imposed by diffraction. The most widely applied super-resolution method currently is localization microscopy, which takes advantage of the ability to determine the position of individual fluorescent molecules with nanometer accuracy even in cells. By iteratively measuring sparse subsets of photoactivatable fluorescent proteins, protein distribution in macromolecular structures can be accurately reconstructed. Moreover, the motion trajectories of individual molecules within cells can be measured, providing unique ability to measure transport kinetics, exchange rates, and binding affinities of even small subsets of molecules with high temporal resolution and great spatial specificity. This unit describes protocols to measure and quantify the distribution of scaffold proteins within single synapses of cultured hippocampal neurons, and to track and measure the diffusion of intracellular constituents of the neuronal plasma membrane.

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Precise Nanometer Localization Analysis for Individual Fluorescent Probes

Cited by 2,121 publications

References 32 publications

Quantitative evaluation of software packages for single-molecule localization microscopy

Quantitative evaluation of software packages for single-molecule localization microscopy

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

Single‐Molecule Tracking Photoactivated Localization Microscopy to Map Nano‐Scale Structure and Dynamics in Living Spines

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