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

PLoS ONE ◽  
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.

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

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.

A super-resolution platform for correlative live single-molecule imaging and STED microscopy

2019 ◽  
Vol 16 (12) ◽  
pp. 1263-1268 ◽  
Author(s):  
V. V. G. Krishna Inavalli ◽  
Martin O. Lenz ◽  
Corey Butler ◽  
Julie Angibaud ◽  
Benjamin Compans ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Single Molecule Imaging ◽  
Sted Microscopy

Single-molecule spectroscopy

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.

scholarly journals Imaging Single Molecules vs. Single-Molecule Imaging: Counting Single Molecules using Dextran-Streptavidin-Antibody Clusters (DSA)

2017 ◽  
Vol 112 (3) ◽  
pp. 295a
Author(s):  
Qiaoqiao Ruan ◽  
Richard A. Haack ◽  
Zhen Lin ◽  
Patrick J. Macdonald ◽  
Kerry M. Swift ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecules ◽  
Single Molecule Imaging

scholarly journals Introduction: Super-Resolution and Single-Molecule Imaging

2017 ◽  
Vol 117 (11) ◽  
pp. 7241-7243 ◽  
Author(s):  
Julie Biteen ◽  
Katherine A. Willets
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Single Molecule Imaging

scholarly journals Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single Molecule Localization Microscopy

2021 ◽  
Author(s):  
Siddharth Matikonda ◽  
Dominic Helmerich ◽  
Mara Meub ◽  
Gerti Beliu ◽  
Philip Kollmannsberger ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Single Molecule ◽  
Computational Analysis ◽  
Super Resolution ◽  
Underlying Mechanism ◽  
New Approach ◽  
Localization Microscopy ◽  
Order Of Magnitude ◽  
Resolution Single ◽  
Single Molecule Localization Microscopy

<p>The light-promoted conversion of extensively used cyanine dyes to blue-shifted emissive products has been observed in various contexts. However, both the underlying mechanism and the species involved in this photoconversion reaction have remained elusive. Here we report that irradiation of heptamethine cyanines provides pentamethine cyanines, which, in turn, are photoconverted to trimethine cyanines. We detail an examination of the mechanism and substrate scope of this remarkable two-carbon phototruncation reaction. Supported by computational analysis, we propose that this reaction involves a singlet oxygen-initiated multi-step sequence involving a key hydroperoxycyclobutanol intermediate. Building on this mechanistic framework, we identify conditions to improve the yield of photoconversion by over an order of magnitude. We then demonstrate that cyanine phototruncation can be applied to super-resolution single-molecule localization microscopy, leading to improved spatial resolution with shorter imaging times. We anticipate these insights will help transform a common, but previously mechanistically ill-defined, chemical transformation into a valuable optical tool.</p>

scholarly journals RhoBAST - a rhodamine-binding aptamer for super-resolution RNA imaging

2020 ◽  
Author(s):  
Murat Sunbul ◽  
Jens Lackner ◽  
Annabell Martin ◽  
Daniel Englert ◽  
Benjamin Hacene ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Emission ◽  
Super Resolution ◽  
Image Resolution ◽  
Rna Aptamer ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Fluorescence Light ◽  
Rhodamine Dye ◽  
Single Molecule Localization Microscopy

AbstractRhoBAST is a novel fluorescence light-up RNA aptamer (FLAP) that transiently binds a fluorogenic rhodamine dye. Fast dye association and dissociation result in intermittent fluorescence emission, facilitating single-molecule localization microscopy (SMLM) with an image resolution not limited by photobleaching. We demonstrate RhoBAST's excellent properties as a RNA marker by resolving subcellular and subnuclear structures of RNA in live and fixed cells by SMLM and structured illumination microscopy (SIM).

scholarly journals Minimizing Structural Bias in Single-Molecule Super-Resolution Microscopy

2018 ◽  
Author(s):  
Hesam Mazidi ◽  
Jin Lu ◽  
Arye Nehorai ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Estimation Algorithm ◽  
Image Resolution ◽  
Spread Function ◽  
Localization Errors ◽  
Imaging Artifacts ◽  
Detection And Localization ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

ABSTRACTSingle-molecule localization microscopy (SMLM) depends on sequential detection and localization of individual molecular blinking events. Due to the stochasticity of single-molecule blinking and the desire to improve SMLM’s temporal resolution, algorithms capable of analyzing frames with a high density (HD) of active molecules, or molecules whose images overlap, are a prerequisite for accurate location measurements. Thus far, HD algorithms are evaluated using scalar metrics, such as root-mean-square error, that fail to quantify the structure of errors caused by the structure of the sample. Here, we show that the spatial distribution of localization errors within super-resolved images of biological structures are vectorial in nature, leading to systematic structural biases that severely degrade image resolution. We further demonstrate that the shape of the microscope’s point-spread function (PSF) fundamentally affects the characteristics of imaging artifacts. We built a Robust Statistical Estimation algorithm (RoSE) to minimize these biases for arbitrary structures and PSFs. RoSE accomplishes this minimization by estimating the likelihood of blinking events to localize molecules more accurately and eliminate false localizations. Using RoSE, we measure the distance between crossing microtubules, quantify the morphology of and separation between vesicles, and obtain robust recovery using diverse 3D PSFs with unmatched accuracy compared to state-of-the-art algorithms.

scholarly journals Developing super-resolution fluorescent microscopy for virology research

2021 ◽  
Vol 63 (11) ◽  
pp. 6-12
Author(s):  
Trong Nghia Nguyen ◽  
◽  
Thi Bich Ngoc Nguyen ◽  
Hong Nhung Tran ◽  
Duc Toan Nguyen ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Dengue Virus ◽  
Single Molecule ◽  
Fluorescent Microscopy ◽  
Super Resolution ◽  
Hemorrhagic Fever ◽  
Reconstruction Process ◽  
Virus Particles ◽  
Microscopy Technique ◽  
Localisation Microscopy

Taking advantage of the use of photoswitchable probes and high precision localisation of single molecules to surpass the diffraction limit, super-resolution fluorescence microscopy allows observing non-invasive live-cell at sub-diffraction size (<200 nm). Given the advantage of super-resolution fluorescence microscopy, our group has reconstructed the super-resolution fluorescence microscopybased on the single-molecule localisation microscopy technique with a resolution of 20 nm. In this research, the authors present the reconstruction process of the microscopy system and its application in observing hemorrhagic fever Dengue virus. Dengue virus was cultured in baby hamster kidney (BHK-21) cells and was then negative stained for transmission electron microscope (TEM) or immunofluorescent labeled for stochastic optical reconstruction microscopy (STORM). The diameter of the Dengue virus particles is 45-60 nm measured using TEM and is 84±12 nm measured using STORM. After subtraction of the length of the antibody attached to the virus particles, the diameter of Dengue virus particles measured using STORM are close to which measured using TEM. In conclusion, the authors highlight the findings of super-resolution fluorescence microscopy-based Dengue virus studies and their contributions to the understanding of Dengue virus particles. The current advances in super-resolution microscopy may open new avenues for future virology teaching and research.

scholarly journals Light Sheet Illumination for 3D Single-Molecule Super-Resolution Imaging of Neuronal Synapses

2021 ◽  
Vol 13 ◽  
Author(s):  
Gabriella Gagliano ◽  
Tyler Nelson ◽  
Nahima Saliba ◽  
Sofía Vargas-Hernández ◽  
Anna-Karin Gustavsson
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Super Resolution ◽  
Light Sheet ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Single Molecule Tracking ◽  
Resolution Imaging

The function of the neuronal synapse depends on the dynamics and interactions of individual molecules at the nanoscale. With the development of single-molecule super-resolution microscopy over the last decades, researchers now have a powerful and versatile imaging tool for mapping the molecular mechanisms behind the biological function. However, imaging of thicker samples, such as mammalian cells and tissue, in all three dimensions is still challenging due to increased fluorescence background and imaging volumes. The combination of single-molecule imaging with light sheet illumination is an emerging approach that allows for imaging of biological samples with reduced fluorescence background, photobleaching, and photodamage. In this review, we first present a brief overview of light sheet illumination and previous super-resolution techniques used for imaging of neurons and synapses. We then provide an in-depth technical review of the fundamental concepts and the current state of the art in the fields of three-dimensional single-molecule tracking and super-resolution imaging with light sheet illumination. We review how light sheet illumination can improve single-molecule tracking and super-resolution imaging in individual neurons and synapses, and we discuss emerging perspectives and new innovations that have the potential to enable and improve single-molecule imaging in brain tissue.

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