scholarly journals Obtaining 3D Super-resolution Information from 2D Super-resolution Images through a 2D-to-3D Transformation Algorithm

2017 ◽  
Author(s):  
Andrew Ruba ◽  
Wangxi Luo ◽  
Joseph Kelich ◽  
Weidong Yang
Keyword(s):  
Single Molecule ◽  
Rotational Symmetry ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cells ◽  
Limited Information ◽  
Spatiotemporal Resolution ◽  
Superresolution Microscopy ◽  
Localization Analysis ◽  
Probability Information

AbstractCurrently, it is highly desirable but still challenging to obtain three-dimensional (3D) superresolution information of structures in fixed specimens as well as dynamic processes in live cells with a high spatiotemporal resolution. Here we introduce an approach, without using 3D superresolution microscopy or real-time 3D particle tracking, to achieve 3D sub-diffraction-limited information with a spatial resolution of ≤ 1 nm. This is a post-localization analysis that transforms 2D super-resolution images or 2D single-molecule localization distributions into their corresponding 3D spatial probability information. The method has been successfully applied to obtain structural and functional information for 25-300 nm sub-cellular organelles that have rotational symmetry. In this article, we will provide a comprehensive analysis of this method by using experimental data and computational simulations.

scholarly journals 3D tracking of extracellular vesicles by holographic fluorescence imaging

2020 ◽  
Vol 6 (45) ◽  
pp. eabc2508
Author(s):  
Matz Liebel ◽  
Jaime Ortega Arroyo ◽  
Vanesa Sanz Beltrán ◽  
Johann Osmond ◽  
Ala Jo ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Fluorescent Detection ◽  
Live Cells ◽  
Fluorescent Light ◽  
3D Tracking ◽  
Anisotropic Motion ◽  
Lag Times

Fluorescence microscopy is the method of choice in biology for its molecular specificity and super-resolution capabilities. However, it is limited to a narrow z range around one observation plane. Here, we report an imaging approach that recovers the full electric field of fluorescent light with single-molecule sensitivity. We expand the principle of digital holography to fast fluorescent detection by eliminating the need for phase cycling and enable three-dimensional (3D) tracking of individual nanoparticles with an in-plane resolution of 15 nm and a z-range of 8 mm. As a proof-of-concept biological application, we image the 3D motion of extracellular vesicles (EVs) inside live cells. At short time scales (<4 s), we resolve near-isotropic 3D diffusion and directional transport. For longer lag times, we observe a transition toward anisotropic motion with the EVs being transported over long distances in the axial plane while being confined in the horizontal dimension.

scholarly journals Extending resolution of structured illumination microscopy with sparse deconvolution

2021 ◽  
Author(s):  
Weisong Zhao ◽  
Shiqun Zhao ◽  
Liuju Li ◽  
Xiaoshuai Huang ◽  
Shijia Xing ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Cell ◽  
Frame Rate ◽  
Live Cells ◽  
Photon Flux ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution

Abstract The spatial resolutions of live-cell super-resolution microscopes are limited by the maximum collected photon flux. Taking advantage of a priori knowledge of the sparsity and continuity of biological structures, we develop a deconvolution algorithm that further extends the resolution of super-resolution microscopes under the same photon budgets by nearly twofold. As a result, sparse structured illumination microscopy (Sparse-SIM) achieves ~60 nm resolution at a 564 Hz frame rate, allowing it to resolve intricate structural intermediates, including small vesicular fusion pores, ring-shaped nuclear pores formed by different nucleoporins, and relative movements between the inner and outer membranes of mitochondria in live cells. Likewise, sparse deconvolution can be used to increase the three-dimensional resolution and contrast of spinning-disc confocal-based SIM (SD-SIM), and operates under conditions with the insufficient signal-to-noise-ratio, all of which allows routine four-color, three-dimensional, ~90 nm resolution live-cell super-resolution imaging. Overall, sparse deconvolution may be a general tool to push the spatiotemporal resolution limits of live-cell fluorescence microscopy.

scholarly journals Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

2019 ◽  
Author(s):  
Elias A. Halabi ◽  
Dorothea Pinotsi ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Time Lapse ◽  
Switching Behavior ◽  
Spatiotemporal Resolution ◽  
New Information ◽  
Human Neurons ◽  
Long Time ◽  
Intracellular Organelles

Photoswitchable molecules have found multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allowed us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

scholarly journals Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

2018 ◽  
Author(s):  
Elias A. Halabi ◽  
Dorothea Pinotsi ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Time Lapse ◽  
Switching Behavior ◽  
Spatiotemporal Resolution ◽  
New Information ◽  
Human Neurons ◽  
Long Time ◽  
Intracellular Organelles

Photoswitchable molecules have found multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allowed us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

scholarly journals Photoregulated Fluxional Fluorophores for Live-Cell Super-Resolution Microscopy with No Apparent Photobleaching

2019 ◽  
Author(s):  
Elias A. Halabi ◽  
Dorothea Pinotsi ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Time Lapse ◽  
Switching Behavior ◽  
Spatiotemporal Resolution ◽  
New Information ◽  
Human Neurons ◽  
Long Time ◽  
Intracellular Organelles

Photoswitchable molecules have found multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allowed us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

scholarly journals PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking

2012 ◽  
Vol 20 (5) ◽  
pp. 4957 ◽  
Author(s):  
Ignacio Izeddin ◽  
Mohamed El Beheiry ◽  
Jordi Andilla ◽  
Daniel Ciepielewski ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Single Molecule ◽  
Three Dimensional ◽  
Super Resolution ◽  
Resolution Imaging

scholarly journals Particle localization using local gradients and its application to nanometer stabilization of a microscope

2021 ◽  
Author(s):  
Anatolii V. Kashchuk ◽  
Oleksandr Perederiy ◽  
Chiara Caldini ◽  
Lucia Gardini ◽  
Francesco Saverio Pavone ◽  
...  
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Universal Method ◽  
Superresolution Microscopy ◽  
Accurate Localization ◽  
Micro Particles ◽  
Localization Algorithms ◽  
Gradient Calculation ◽  
Particle Localization ◽  
Local Gradient

Accurate localization of single particles plays an increasingly important role in a range of biological techniques, including single molecule tracking and localization-based superresolution microscopy. Such techniques require fast and accurate particle localization algorithms as well as nanometer-scale stability of the microscope. Here, we present a universal method for three-dimensional localization of single labeled and unlabeled particles based on local gradient calculation of microscopy images. The method outperforms current techniques in high noise conditions, and it is capable of nanometer accuracy localization of nano- and micro-particles with sub-ms calculation time. By localizing a fixed particle as fiducial mark and running a feedback loop, we demonstrate its applicability for active drift correction in sensitive nanomechanical measurements such as optical trapping and superresolution imaging. A multiplatform open software package comprising a set of tools for local gradient calculation in brightfield and fluorescence microscopy is shared to the scientific community.

scholarly journals 3D Interferometric Lattice Light-Sheet Imaging

2020 ◽  
Author(s):  
Bin Cao ◽  
Guanshi Wang ◽  
Jieru Li ◽  
Alexandros Pertsinidis
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Detection Sensitivity ◽  
Live Cells ◽  
Background Suppression ◽  
Axial Resolution ◽  
Volumetric Imaging ◽  
Spatio Temporal ◽  
And Function

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1σ) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach

2018 ◽  
Vol 20 (12) ◽  
pp. 8088-8098 ◽  
Author(s):  
Rajeev Yadav ◽  
H. Peter Lu
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Super Resolution ◽  
Live Cells ◽  
Imaging Approach ◽  
Resolution Imaging ◽  
Function Relationship ◽  
Bleaching Step ◽  
Relationship Of ◽  
Resolution Single

Correlating single-molecule fluorescence photo-bleaching step analysis and single-molecule super-resolution imaging, our findings for the clustering effect of the NMDA receptor ion channel on the live cell membranes provide a new and significant understanding of the structure–function relationship of NMDA receptors.

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