Nanoscopic Stoichiometry and Single-Molecule Counting

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

Frontiers in Bioinformatics ◽

10.3389/fbinf.2021.739769 ◽

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.

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Direct supercritical angle localization microscopy for nanometer 3D superresolution

Nature Communications ◽

10.1038/s41467-021-21333-x ◽

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.

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

Nanophotonics ◽

10.1515/nanoph-2021-0481 ◽

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.

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Direct Supercritical Angle Localization Microscopy for Nanometer 3D Superresolution

10.1101/2020.06.25.171058 ◽

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.

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

10.1101/834572 ◽

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.

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3D particle averaging and detection of macromolecular symmetry in localization microscopy

Nature Communications ◽

10.1038/s41467-021-22006-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hamidreza Heydarian ◽

Maarten Joosten ◽

Adrian Przybylski ◽

Florian Schueder ◽

Ralf Jungmann ◽

...

Keyword(s):

Single Molecule ◽

Signal To Noise Ratio ◽

Fluorescent Labeling ◽

Dna Origami ◽

Particle Analysis ◽

Nuclear Pore ◽

Localization Microscopy ◽

Structural Template ◽

Microscopy Techniques ◽

Single Molecule Localization Microscopy

AbstractSingle molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data.

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

10.1101/094839 ◽

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.

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Probing Biosensing Interfaces With Single Molecule Localization Microscopy (SMLM)

Frontiers in Chemistry ◽

10.3389/fchem.2021.655324 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xiaoyu Cheng ◽

Wei Yin

Keyword(s):

Single Molecule ◽

Molecular Level ◽

Diffraction Limit ◽

Far Field ◽

Biological Sensing ◽

Localization Microscopy ◽

Chemical Catalysis ◽

Nanostructured Interfaces ◽

Single Molecule Localization Microscopy ◽

Conventional Light Microscopy

Far field single molecule localization microscopy (SMLM) has been established as a powerful tool to study biological structures with resolution far below the diffraction limit of conventional light microscopy. In recent years, the applications of SMLM have reached beyond traditional cellular imaging. Nanostructured interfaces are enriched with information that determines their function, playing key roles in applications such as chemical catalysis and biological sensing. SMLM enables detailed study of interfaces at an individual molecular level, allowing measurements of reaction kinetics, and detection of rare events not accessible to ensemble measurements. This paper provides an update to the progress made to the use of SMLM in characterizing nanostructured biointerfaces, focusing on practical aspects, recent advances, and emerging opportunities from an analytical chemistry perspective.

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Improved resolution in single-molecule localization microscopy using QD-PAINT

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00572-4 ◽

2021 ◽

Vol 53 (3) ◽

pp. 384-392

Author(s):

Yeonho Chang ◽

Do-Hyeon Kim ◽

Kai Zhou ◽

Min Gyu Jeong ◽

Soyeon Park ◽

...

Keyword(s):

Single Molecule ◽

Organic Dyes ◽

Photophysical Properties ◽

Photon Number ◽

Molecular Structures ◽

Great Promise ◽

Resolution Limit ◽

Localization Microscopy ◽

Nanoscale Topography ◽

Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.

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Single-Molecule Imaging of Active Mitochondrial Nitroreductases using a Photo-Crosslinking Fluorescent Sensor

10.26434/chemrxiv.7992980.v1 ◽

2019 ◽

Author(s):

Zacharias Thiel ◽

Pablo Rivera-Fuentes

Keyword(s):

Subcellular Localization ◽

Fluorescent Probe ◽

Single Molecule ◽

Mammalian Cells ◽

Fluorescent Sensor ◽

Biological Functions ◽

Localization Microscopy ◽

Drug Activation ◽

Nitroreductase Activity ◽

Single Molecule Localization Microscopy

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines and play a role in detoxification and pro-drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super-resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo-crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two-color, threedimensional, single-molecule localization microscopy. These experiments allowed us to image the sub-mitochondrial organization of microdomains of nitroreductase activity.<br>

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