The Lateral and Axial Localization Uncertainty in Super‐Resolution Light Microscopy

Rieger, Bernd; Stallinga, Sjoerd

doi:10.1002/cphc.201300711

Cited by 124 publications

(136 citation statements)

References 39 publications

(64 reference statements)

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“…Selecting the five best algorithms, we found that the accuracies are 21.05 nm and 32.13 nm for LS1 and LS2, respectively. They are worse than predicted by Thompson 20 .…”

Section: Evaluation and Scoring Calculation Theoretical Accuracymentioning

confidence: 59%

“…There also exist refined CRLBs that take pixelation, various sources of noise, and fluorescence background into account. A survey of localization accuracy and precision in the SMLM context can be found in Deschout et al 64 , while uncertainties in the lateral localization in super-resolution microscopy were also addressed in Rieger et al 20 .…”

Section: Evaluation and Scoring Calculation Theoretical Accuracymentioning

confidence: 99%

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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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“…Selecting the five best algorithms, we found that the accuracies are 21.05 nm and 32.13 nm for LS1 and LS2, respectively. They are worse than predicted by Thompson 20 .…”

Section: Evaluation and Scoring Calculation Theoretical Accuracymentioning

confidence: 59%

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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“…Here, N l is the total number of photons, ðx l ; y l Þ is the fluorophore position, ands l is the effective width of the PSF. The latter accounts for the finite pixel size a Â a and is related to the true Gaussian width s by the relations 2 ¼ s 2 þ a 2 =12 (15,16,19,20). Analytical CRLBs for parameter errors are calculated by inserting this PSF model into Eq.…”

Section: Resultsmentioning

confidence: 99%

Localization Precision in Stepwise Photobleaching Experiments

Schoen

2014

Biophysical Journal

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The precise determination of the position of fluorescent labels is essential for the quantitative study of biomolecular structures by various localization microscopy techniques. Localization by stepwise photobleaching is especially suited for measuring nanometer-scale distances between two labels; however, the precision of this method has remained elusive. Here, we show that shot noise from other emitters and error propagation compromise the localization precision in stepwise photobleaching. Incorporation of point spread function-shaped shot noise into the variance term in the Fisher matrix yielded fundamental Cràmer-Rao lower bounds (CRLBs) that were in general anisotropic and depended on emitter intensity and position. We performed simulations to benchmark the extent to which different analysis procedures reached these ideal CRLBs. The accumulation of noise from several images accounted for the worse localization precision in image subtraction. Propagation of fitting errors compromised the CRLBs in sequential fitting using fixed parameters. Global fitting of all images was also governed by error propagation, but made optimal use of the available information. The precision of individual distance measurements depended critically on the exact bleaching kinetics and was correctly quantified by the CRLBs. The methods presented here provide a consistent framework for quantitatively analyzing stepwise photobleaching experiments and shed light on the localization precision in some other bleaching- or blinking-assisted techniques.

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“…The increase in spot footprint is comparable to the different methods for 3D-localization [12]. These predictions are tested with simulations, using realistically simulated spot shapes including effects of vector diffraction [13] and of a finite spectral emission bandwidth (Gaussian shape, FWHM 35 nm).…”

mentioning

confidence: 90%

“…The precision for finding the center positions of the different orders is Δx j σ a ∕ N j 1 − I 1 τ j p , and the precision for finding the photon counts for each diffraction order is ΔN j N j ∕1 − I 0 τ j p , with the functions I n τ τ∕n! R ∞ 0 dtt n ∕1 τe t of the dimensionless background parameter τ j 2πσ 2 a b∕N j a 2 , where σ 2 a σ 2 a 2 ∕12 [11,12]. A Cramér-Rao lower bound (CRLB) analysis gives the best possible estimate for the emitter position and the wavelength as:…”

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confidence: 99%

Simultaneous measurement of position and color of single fluorescent emitters using diffractive optics

2014

Self Cite

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We propose a method for simultaneously measuring the position and emission color of single fluorescent emitters based on the use of a large pitch diffraction grating in the emission light path. The grating produces satellite spots adjacent to the main spot; the relative distance between the spots is a measure for the emission wavelength. We present proof-of-principle experiments on beads and mixtures of quantum dots using a spatial light modulator for making a programmable diffraction grating. A wavelength precision of around 10 nm can be achieved for 1000 signal photons and practical background levels, while maintaining a localization precision of around 10 nm. Super-resolution microscopy based on the localization of stochastically activated single fluorescent molecules is a powerful technique for studying biological structure on the nanoscale [1][2][3]. The study of biological function on this scale requires imaging two or more interacting molecular species. These species are usually differentiated by labeling with fluorescent molecules that have different emission colors. The different species can be imaged subsequently by changing illumination and filter sets [4] or simultaneously by using multibandpass filters and a dichroic beamsplitter in the emission path for imaging two color channels simultaneously [5,6]. The latter approach allows for multiplexing three and, in some cases, four different fluorescent species. An alternative is the use of activator-reporter labeling techniques in which different activator molecules can couple to the same reporter molecule, thereby allowing for subsequent imaging of the different channels by switching the illumination wavelength only [7]. Multicolor localization approaches are also applied in single particle tracking, broadening the scope to emitters such as quantum dots (QDs) [8].A different approach to color measurement is enabled by placing a blazed diffraction grating with a fine pitch in the emission path [9,10]. In this way two subimages, corresponding to the zeroth and first diffraction orders, are imaged side by side on two camera halves. The 0th order image gives the positions of the emitters; the 1st order image is smeared out in proportion to the emission spectral width over many pixels, thus enabling spectroscopic measurements. The resulting low signal-to-background ratio (SBR), however, compromises spectral and localization precision. Moreover, differentiation between species does not necessarily require measuring the full emission spectrum. Here, we propose a new method for measuring both the position and the color of single emitters based on the use of a grating with a relatively large pitch. The grating produces a set of satellite diffraction orders directly adjacent to the 0th order, the main spot. Now the satellite order(s) are projected just a handful of pixels away instead of to the other half of the camera. A consequence is that spot broadening due to the emission spectral width is at the subpixel level. The distance between the diffraction spots Δx ...

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The Lateral and Axial Localization Uncertainty in Super‐Resolution Light Microscopy

Cited by 124 publications

References 39 publications

Quantitative evaluation of software packages for single-molecule localization microscopy

Quantitative evaluation of software packages for single-molecule localization microscopy

Localization Precision in Stepwise Photobleaching Experiments

Simultaneous measurement of position and color of single fluorescent emitters using diffractive optics

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