scholarly journals Blinking Statistics and Molecular Counting in direct Stochastic Reconstruction Microscopy (dSTORM)

2019 ◽  
Author(s):  
Lekha Patel ◽  
Dylan M. Owen ◽  
Edward A.K. Cohen
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Diffraction Limit ◽  
Training Data ◽  
Switching Behavior ◽  
Cellular Structures ◽  
Exact Probability ◽  
Localization Microscopy ◽  
Exact Probability Distribution ◽  
Single Molecule Localization Microscopy

AbstractMany recent advancements in single molecule localization microscopy exploit the stochastic photo-switching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging. Modeling the photo-switching behavior of a fluorophore as a continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photo-switching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a dSTORM experiment involving an arbitrary number of molecules. We demonstrate that when training data is available to estimate photo-switching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations.

scholarly journals Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM)

2021 ◽  
Author(s):  
Lekha Patel ◽  
David Williamson ◽  
Dylan M Owen ◽  
Edward A K Cohen
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Immunological Synapse ◽  
Training Data ◽  
Supplementary Information ◽  
Cellular Structures ◽  
Exact Probability ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Exact Probability Distribution ◽  
Optical Reconstruction

Abstract Motivation Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Results Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Availability and implementation Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Efficient Cross-Correlation Filtering of One- and Two-Color Single Molecule Localization Microscopy Data

2021 ◽  
Vol 1 ◽  
Author(s):  
Angel Mancebo ◽  
Dushyant Mehra ◽  
Chiranjib Banerjee ◽  
Do-Hyung Kim ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Cross Correlation ◽  
Pair Correlation ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Data Sets ◽  
Oligomeric State ◽  
Localization Microscopy ◽  
Cross Correlation Analysis ◽  
Single Molecule Localization Microscopy

Single molecule localization microscopy has become a prominent technique to quantitatively study biological processes below the optical diffraction limit. By fitting the intensity profile of single sparsely activated fluorophores, which are often attached to a specific biomolecule within a cell, the locations of all imaged fluorophores are obtained with ∼20 nm resolution in the form of a coordinate table. While rendered super-resolution images reveal structural features of intracellular structures below the optical diffraction limit, the ability to further analyze the molecular coordinates presents opportunities to gain additional quantitative insights into the spatial distribution of a biomolecule of interest. For instance, pair-correlation or radial distribution functions are employed as a measure of clustering, and cross-correlation analysis reveals the colocalization of two biomolecules in two-color SMLM data. Here, we present an efficient filtering method for SMLM data sets based on pair- or cross-correlation to isolate localizations that are clustered or appear in proximity to a second set of localizations in two-color SMLM data. In this way, clustered or colocalized localizations can be separately rendered and analyzed to compare other molecular properties to the remaining localizations, such as their oligomeric state or mobility in live cell experiments. Current matrix-based cross-correlation analyses of large data sets quickly reach the limitations of computer memory due to the space complexity of constructing the distance matrices. Our approach leverages k-dimensional trees to efficiently perform range searches, which dramatically reduces memory needs and the time for the analysis. We demonstrate the versatile applications of this method with simulated data sets as well as examples of two-color SMLM data. The provided MATLAB code and its description can be integrated into existing localization analysis packages and provides a useful resource to analyze SMLM data with new detail.

scholarly journals Direct supercritical angle localization microscopy for nanometer 3D superresolution

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anindita Dasgupta ◽  
Joran Deschamps ◽  
Ulf Matti ◽  
Uwe Hübner ◽  
Jan Becker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
State Of The Art ◽  
Dna Origami ◽  
Cellular Structures ◽  
Full Potential ◽  
Localization Microscopy ◽  
Structural Cell ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

Abstract3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. In supercritical angle localization microscopy (SALM), z-positions of single fluorophores are extracted from the intensity of supercritical angle fluorescence, which strongly depends on their distance to the coverslip. Here, we realize the full potential of SALM and improve its z-resolution by more than four-fold compared to the state-of-the-art by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying fitting routines to extract precise intensities of single emitters. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

scholarly journals Improved localization precision via restricting confined biomolecule stochastic motion in single-molecule localization microscopy

Nanophotonics ◽  
2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jielei Ni ◽  
Bo Cao ◽  
Gang Niu ◽  
Danni Chen ◽  
Guotao Liang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Direct Evidence ◽  
Diffraction Limit ◽  
Drift Correction ◽  
Stochastic Motion ◽  
Localization Microscopy ◽  
Alexa Fluor ◽  
Biological Studies ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

Abstract Single-molecule localization microscopy (SMLM) plays an irreplaceable role in biological studies, in which nanometer-sized biomolecules are hardly to be resolved due to diffraction limit unless being stochastically activated and accurately located by SMLM. For biological samples preimmobilized for SMLM, most biomolecules are cross-linked and constrained at their immobilizing sites but still expected to undergo confined stochastic motion in regard to their nanometer sizes. However, few lines of direct evidence have been reported about the detectability and influence of confined biomolecule stochastic motion on localization precision in SMLM. Here, we access the potential stochastic motion for each immobilized single biomolecule by calculating the displacements between any two of its localizations at different frames during sequential imaging of Alexa Fluor-647-conjugated oligonucleotides. For most molecules, localization displacements are remarkably larger at random frame intervals than at shortest intervals even after sample drift correction, increase with interval times and then saturate, showing that biomolecule stochastic motion is detected and confined around the immobilizing sizes in SMLM. Moreover, localization precision is inversely proportional to confined biomolecule stochastic motion, whereas it can be deteriorated or improved by enlarging the biomolecules or adding a post-crosslinking step, respectively. Consistently, post-crosslinking of cell samples sparsely stained for tubulin proteins results in a better localization precision. Overall, this study reveals that confined stochastic motion of immobilized biomolecules worsens localization precision in SMLM, and improved localization precision can be achieved via restricting such a motion.

scholarly journals Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

2020 ◽  
Author(s):  
Anindita Dasgupta ◽  
Joran Deschamps ◽  
Ulf Matti ◽  
Uwe Hübner ◽  
Jan Becker ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
State Of The Art ◽  
Dna Origami ◽  
Cellular Structures ◽  
Full Potential ◽  
Localization Microscopy ◽  
Structural Cell ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

Abstract3D single molecule localization microscopy (SMLM) is an emerging superresolution method for structural cell biology, as it allows probing precise positions of proteins in cellular structures. Supercritical angle fluorescence strongly depends on the z-position of the fluorophore and can be used for z localization in a method called supercritical angle localization microscopy (SALM). Here, we realize the full potential of SALM by directly splitting supercritical and undercritical emission, using an ultra-high NA objective, and applying new fitting routines to extract precise intensities of single emitters, resulting in a four-fold improved z-resolution compared to the state of the art. We demonstrate nanometer isotropic localization precision on DNA origami structures, and on clathrin coated vesicles and microtubules in cells, illustrating the potential of SALM for cell biology.

scholarly journals Resolving Biology Beyond the Diffraction Limit with Single-Molecule Localization Microscopy

2016 ◽  
Author(s):  
Nafiseh Rafiei ◽  
Daniel Nino ◽  
Joshua N. Milstein
Keyword(s):  
Optical Imaging ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Diffraction Limit ◽  
Review Article ◽  
Localization Microscopy ◽  
Short Overview ◽  
The Future ◽  
Microscopic World ◽  
Single Molecule Localization Microscopy

Optical imaging provides a window into the microscopic world, but the level of observable detail is ultimately limited by the wavelength of light being employed. By harnessing the physics of photoswitchable dyes and fluorescent proteins, single-molecule localization microscopy (SMLM) provides a window into the nano-world of biology. This mini-review article provides a short overview of SMLM and discusses some of its prospects for the future.

scholarly journals Verifying molecular clusters by 2-color localization microscopy and significance testing

2019 ◽  
Author(s):  
Andreas M. Arnold ◽  
Magdalena C. Schneider ◽  
Christoph Hüsson ◽  
Robert Sablatnig ◽  
Mario Brameshuber ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Sensitivity ◽  
Significance Testing ◽  
Cellular Structures ◽  
Testing Methods ◽  
P Values ◽  
New Approach ◽  
Localization Microscopy ◽  
Statistical Framework ◽  
Single Molecule Localization Microscopy

AbstractWhile single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential could not be fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the analysis of SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach it is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

scholarly journals Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy

2021 ◽  
Vol 8 ◽  
Author(s):  
Sangyoon Ko ◽  
Jiwoong Kwon ◽  
Sang-Hee Shim
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Green Emission ◽  
Switching Behavior ◽  
Light Illumination ◽  
High Concentration ◽  
Localization Microscopy ◽  
Single Molecule Localization Microscopy

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

scholarly journals Nanoscopic Stoichiometry and Single-Molecule Counting

2019 ◽  
Author(s):  
Daniel Nino ◽  
Daniel Djayakarsana ◽  
Joshua N. Milstein
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Organic Dyes ◽  
Spatial Scales ◽  
Diffraction Limit ◽  
Dna Origami ◽  
Localization Microscopy ◽  
Practical Guide ◽  
Genomic Analyses ◽  
Single Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) has the potential to revolutionize proteomic and genomic analyses by providing information on the number and stoichiometry of proteins or nucleic acids aggregating at spatial scales below the diffraction limit of light. Here we present a method for molecular counting with SMLM built upon the exponentially distributed blinking statistics of photoswitchable fluorophores, with a focus on organic dyes. We provide a practical guide to molecular counting, highlighting many of the challenges and pitfalls, by benchmarking the method on fluorescently labeled, surface mounted DNA origami grids. The accuracy of the results illustrates SMLM’s utility for optical ‘-omics’ analysis.

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