Super-resolution displacement mapping of unbound single molecules reveals nanoscale heterogeneities in intracellular diffusivity

AbstractIntracellular diffusion underlies vital processes of the cell. However, it remains difficult to elucidate how an average-sized protein diffuses in the cell with good spatial resolution and sensitivity. Here we report single-molecule displacement/diffusivity mapping (SMdM), a super-resolution strategy that enables the nanoscale mapping of intracellular diffusivity through the local statistics of instantaneous displacements of freely diffusing single molecules. We thus show that diffusion in the cytoplasm and in the nucleus to both be spatially heterogeneous at the nanoscale, and such variations in local diffusivity correlate strongly with the ultrastructure of the actin cytoskeleton and the chromosome, respectively. Moreover, we identify the net charge of the diffuser as a key determinant of diffusion rate: intriguingly, the possession of positive, but not negative, net charges significantly impedes diffusion, and the exact degree of slowdown is determined by the specific subcellular environments.

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Single-molecule spectroscopy

10.1093/oso/9780198768609.003.0006 ◽

2017 ◽

Author(s):

Michel Orrit

Keyword(s):

Nonlinear Optics ◽

Single Molecule ◽

Single Molecules ◽

Super Resolution ◽

Optical Methods ◽

Single Molecule Spectroscopy ◽

New Techniques ◽

Single Nanoparticle ◽

Cell Biologist ◽

Sensitivity And Selectivity

This chapter gives an overview of the main optical methods used to detect and study single molecules and other small objects (nano-objects). Much of the work so far has exploited the excellent sensitivity and selectivity of fluorescence, but several new techniques, mostly based on nonlinear optics, have recently reached the single-molecule or single-nanoparticle regime. The chapter briefly discusses some results with reference to published reviews. Single-molecule techniques have now been incorporated into the arsenal of the physico-chemist and the cell biologist. However, the recent development of super-resolution techniques and of new labels suggests that further progress can be expected from measurements on single nano-objects in the next few years.

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Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) Microscopy for Ultra-superresolution Imaging of Dendra2-HA transfected NIH3T3 cells

10.1101/2020.05.16.099077 ◽

2020 ◽

Author(s):

Partha Pratim Mondal

Keyword(s):

Distribution Function ◽

Single Molecule ◽

Gaussian Function ◽

Single Molecules ◽

Super Resolution ◽

Underlying Mechanism ◽

Image Resolution ◽

Size Spectrum ◽

Nih3t3 Cells ◽

Microscopy Technique

To be able to resolve molecular-clusters it is crucial to access vital informations (such as, molecule density and cluster-size) that are key to understand disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precisions (LPs)) to reconstruct super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single molecules) that degrade image resolution. Ideally it should be possible to identify the brightest molecules (also termed as, the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. POSSIBLE microscopy explores this possibility by introducing narrow probability size-distribution of single molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single molecule filtering is carried out by the Gaussian distribution function to filter out unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single molecule images with an average PSF sizes of σ ± Δσ = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques.

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Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy for ultra-superresolution imaging

PLoS ONE ◽

10.1371/journal.pone.0242452 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0242452

Author(s):

Partha Pratim Mondal

Keyword(s):

Single Molecule ◽

Gaussian Function ◽

Single Molecules ◽

Super Resolution ◽

Underlying Mechanism ◽

Image Resolution ◽

Size Spectrum ◽

Single Molecule Imaging ◽

Microscopy Technique ◽

Disease Biology

To be able to resolve molecular-clusters it is crucial to access vital information (such as, molecule density, cluster-size, and others) that are key in understanding disease progression and the underlying mechanism. Traditional single-molecule localization microscopy (SMLM) techniques use molecules of variable sizes (as determined by its localization precision (LP)) to reconstruct a super-resolution map. This results in an image with overlapping and superimposing PSFs (due to a wide size-spectrum of single-molecules) that undermine image resolution. Ideally, it should be possible to identify the brightest molecules (also termed as the fortunate molecules) to reconstruct ultra-superresolution map, provided sufficient statistics is available from the recorded data. Probabilistic Optically-Selective Single-molecule Imaging Based Localization Encoded (POSSIBLE) microscopy explores this possibility by introducing a narrow probability size-distribution of single-molecules (narrow size-spectrum about a predefined mean-size). The reconstruction begins by presetting the mean and variance of the narrow distribution function (Gaussian function). Subsequently, the dataset is processed and single-molecules are filtered by the Gaussian function to remove unfortunate molecules. The fortunate molecules thus retained are then mapped to reconstruct an ultra-superresolution map. In-principle, the POSSIBLE microscopy technique is capable of infinite resolution (resolution of the order of actual single-molecule size) provided enough fortunate molecules are experimentally detected. In short, bright molecules (with large emissivity) holds the key. Here, we demonstrate the POSSIBLE microscopy technique and reconstruct single-molecule images with an average PSF sizes of σ ± Δσ = 15 ± 10 nm, 30 ± 2 nm & 50 ± 2 nm. Results show better-resolved Dendra2-HA clusters with large cluster-density in transfected NIH3T3 fibroblast cells as compared to the traditional SMLM techniques. Cluster analysis indicates densely-packed HA molecules, HA-HA interaction, and a surge in the number of HA molecules per cluster post 24 Hrs of transfection. The study using POSSIBLE microscopy introduces new insights in influenza biology. We anticipate exciting applications in the multidisciplinary field of disease biology, oncology, and biomedical imaging.

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Single-molecule spectroscopy and imaging over the decades

Faraday Discussions ◽

10.1039/c5fd00149h ◽

2015 ◽

Vol 184 ◽

pp. 9-36 ◽

Cited By ~ 40

Author(s):

W. E. Moerner ◽

Yoav Shechtman ◽

Quan Wang

Keyword(s):

Single Molecule ◽

Optical Switching ◽

Fluorescent Protein ◽

Single Molecules ◽

Three Dimensional ◽

Super Resolution ◽

Laser Frequency ◽

High Resolution Spectroscopy ◽

Focal Volume ◽

Ensemble Averaging

As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent 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 1990s, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from 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. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies 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. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many talented scientists all over the world have extended our knowledge of the nanoscale and many microscopic mechanisms previously hidden by ensemble averaging.

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Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

Nature Communications ◽

10.1038/s41467-021-22002-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jagadish Sankaran ◽

Harikrushnan Balasubramanian ◽

Wai Hoh Tang ◽

Xue Wen Ng ◽

Adrian Röllin ◽

...

Keyword(s):

Single Molecule ◽

Temporal Dynamics ◽

Growth Factor Receptor ◽

Super Resolution ◽

Actin Binding ◽

Biological Knowledge ◽

Data Set ◽

Epidermal Growth ◽

Molecular Brightness ◽

Super Resolution Microscopy

AbstractSuper-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

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Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

International Journal of Molecular Sciences ◽

10.3390/ijms22041903 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1903

Author(s):

Ivona Kubalová ◽

Alžběta Němečková ◽

Klaus Weisshart ◽

Eva Hřibová ◽

Veit Schubert

Keyword(s):

Single Molecule ◽

Imaging Techniques ◽

Chromatin Condensation ◽

Super Resolution ◽

Stimulated Emission ◽

Wide Field ◽

Structured Illumination Microscopy ◽

Metaphase Chromosomes ◽

Localization Microscopy ◽

Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

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Calibration-Free Single-Molecule Absolute Quantification Using Super-resolution Microscopy

Analytical Chemistry ◽

10.1021/acs.analchem.1c00390 ◽

2021 ◽

Vol 93 (15) ◽

pp. 6195-6204

Author(s):

Guang Li ◽

Qiqing Zhang

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Absolute Quantification ◽

Calibration Free ◽

Super Resolution Microscopy

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Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

Nature Communications ◽

10.1038/s41467-020-20863-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Alan M. Szalai ◽

Bruno Siarry ◽

Jerónimo Lukin ◽

David J. Williamson ◽

Nicolás Unsain ◽

...

Keyword(s):

Single Molecule ◽

Total Internal Reflection ◽

Single Molecules ◽

Fluorescence Microscope ◽

Internal Reflection ◽

Total Internal Reflection Fluorescence ◽

Axial Position ◽

Localization Microscopy ◽

Localization Precision ◽

Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

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