scholarly journals Nuclear accessibility of β-actin mRNA is measured by 3D single-molecule real-time tracking

2015 ◽  
Vol 209 (4) ◽  
pp. 609-619 ◽  
Author(s):  
Carlas S. Smith ◽  
Stephan Preibisch ◽  
Aviva Joseph ◽  
Sara Abrahamsson ◽  
Bernd Rieger ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Messenger Rna ◽  
Three Dimensional ◽  
Spatial Inhomogeneity ◽  
Live Cells ◽  
Nuclear Pore ◽  
Cell Nuclei ◽  
Entire Volume ◽  
Time Required

Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization.

Distinct roles of nuclear basket proteins in directing the passage of mRNA through the nuclear pore

2021 ◽  
Vol 118 (37) ◽  
pp. e2015621118
Author(s):  
Yichen Li ◽  
Vasilisa Aksenova ◽  
Mark Tingey ◽  
Jingjie Yu ◽  
Ping Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Nuclear Export ◽  
Copy Number ◽  
Messenger Rna ◽  
High Speed ◽  
Three Dimensional ◽  
Live Cells ◽  
Nuclear Pore ◽  
Stoichiometric Ratio

The in vivo characterization of the exact copy number and the specific function of each composite protein within the nuclear pore complex (NPC) remains both desirable and challenging. Through the implementation of live-cell high-speed super-resolution single-molecule microscopy, we first quantified the native copies of nuclear basket (BSK) proteins (Nup153, Nup50, and Tpr) prior to knocking them down in a highly specific manner via an auxin-inducible degron strategy. Second, we determined the specific roles that BSK proteins play in the nuclear export kinetics of model messenger RNA (mRNA) substrates. Finally, the three-dimensional (3D) nuclear export routes of these mRNA substrates through native NPCs in the absence of specific BSK proteins were obtained and further validated via postlocalization computational simulations. We found that these BSK proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs within live cells. The absence of Tpr from the NPC predominantly reduces the probability of nuclear mRNAs entering the NPC for export. Complete depletion of Nup153 and Nup50 results in an mRNA nuclear export efficiency decrease of approximately four folds. mRNAs can gain their maximum successful export efficiency as the copy number of Nup153 increased from zero to only half the full complement natively within the NPC. Lastly, the absence of Tpr or Nup153 seems to alter the 3D export routes of mRNAs as they pass through the NPC. However, the removal of Nup50 alone has almost no impact upon mRNA export route and kinetics.

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 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 Real-time 3D reconstruction from single-photon lidar data using plug-and-play point cloud denoisers

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Julián Tachella ◽  
Yoann Altmann ◽  
Nicolas Mellado ◽  
Aongus McCarthy ◽  
Rachael Tobin ◽  
...  
Keyword(s):  
3D Reconstruction ◽  
Real Time ◽  
Single Photon ◽  
Three Dimensional ◽  
Lidar Data ◽  
Computational Framework ◽  
Processing Times ◽  
Outdoor Scenes ◽  
3D Scene ◽  
Time Required

Abstract Single-photon lidar has emerged as a prime candidate technology for depth imaging through challenging environments. Until now, a major limitation has been the significant amount of time required for the analysis of the recorded data. Here we show a new computational framework for real-time three-dimensional (3D) scene reconstruction from single-photon data. By combining statistical models with highly scalable computational tools from the computer graphics community, we demonstrate 3D reconstruction of complex outdoor scenes with processing times of the order of 20 ms, where the lidar data was acquired in broad daylight from distances up to 320 metres. The proposed method can handle an unknown number of surfaces in each pixel, allowing for target detection and imaging through cluttered scenes. This enables robust, real-time target reconstruction of complex moving scenes, paving the way for single-photon lidar at video rates for practical 3D imaging applications.

scholarly journals 3D Multicolor Nanoscopy at 10,000 Cells a Day

2019 ◽  
Author(s):  
Andrew E S Barentine ◽  
Yu Lin ◽  
Miao Liu ◽  
Phylicia Kidd ◽  
Leonhard Balduf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Mammalian Cells ◽  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
High Volume ◽  
Volume Data ◽  
Cell Nuclei ◽  
Fully Integrated ◽  
Resolution Imaging

ABSTRACTDiffraction-unlimited single-molecule switching (SMS) nanoscopy techniques like STORM /(F)PALM enable three-dimensional (3D) fluorescence imaging at 20-80 nm resolution and are invaluable to investigate sub-cellular organization. They suffer, however, from low throughput, limiting the output of a days worth of imaging to typically a few tens of mammalian cells. Here we develop an SMS imaging platform that combines high-speed 3D single-molecule data acquisition with an automated, fully integrated, high-volume data processing pipeline. We demonstrate 2-color 3D super-resolution imaging of over 10,000 mammalian cell nuclei in about 26 hours, connecting the traditionally low-throughput super-resolution community to the world of omics approaches.

scholarly journals Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 167
Author(s):  
Samuel L. Junod ◽  
Jason Saredy ◽  
Weidong Yang
Keyword(s):  
Fluorescence Microscopy ◽  
Single Molecule ◽  
Nuclear Import ◽  
High Speed ◽  
Single Point ◽  
Live Cells ◽  
Nuclear Pore ◽  
Transport Dynamics ◽  
Technical Advances ◽  
Live Cell Microscopy

Understanding the detailed nuclear import kinetics of adeno-associated virus (AAV) through the nuclear pore complex (NPC) is essential for the application of AAV capsids as a nuclear delivery instrument as well as a target for drug development. However, a comprehensive understanding of AAV transport through the sub-micrometer NPCs in live cells calls for new techniques that can conquer the limitations of conventional fluorescence microscopy and electron microscopy. With recent technical advances in single-molecule fluorescence microscopy, we are now able to image the entire nuclear import process of AAV particles and also quantify the transport dynamics of viral particles through the NPCs in live human cells. In this review, we initially evaluate the necessity of single-molecule live-cell microscopy in the study of nuclear import for AAV particles. Then, we detail the application of high-speed single-point edge-excitation sub-diffraction (SPEED) microscopy in tracking the entire process of nuclear import for AAV particles. Finally, we summarize the major findings for AAV nuclear import by using SPEED microscopy.

scholarly journals Reconstructing spatial transport distributions in the nuclear pore complex from 2D images—how reliable is it?

2017 ◽  
Author(s):  
Li-Chun Tu ◽  
Maximiliaan Huisman ◽  
Yu-Chieh Chung ◽  
Carlas Smith ◽  
David Grunwald
Keyword(s):  
Single Molecule ◽  
Nuclear Pore Complex ◽  
Dimensional Space ◽  
Critical Evaluation ◽  
Three Dimensional ◽  
Cylindrical Symmetry ◽  
Nuclear Pore ◽  
Time Resolved ◽  
Functional Relationships ◽  
Model Dependency

AbstractImaging single molecules in living cells and reconstituted cell systems has resulted in a new understanding of the dynamics of nuclear pore complex functions over the last decade. It does, however, fall short on providing insights into the functional relationships between the pore and nucleocytoplasmic cargo in three-dimensional space. This limited ability is the result of insufficient resolution of optical microscopes along the optical axis and limited fluorescent signal due to the short timescales involved in nuclear transport (fractions of a second). To bypass current technological limitations, it was suggested that highly time-resolved 2D single molecule data could be interpreted as projected cargo locations and could subsequently be transformed into a spatial cargo distribution by assuming cylindrical symmetry 1. Such cargo distributions would provide valuable insights into the NPC-mediated transport in cells. This method, termed 3D-SPEED, has attracted large interest inside and beyond the nuclear pore field, but has also been sharply critiqued for a lack of critical evaluation. Here we present such an evaluation, testing the robustness, reconstruction quality and model-dependency.

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

2019 ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cross Sections ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Three Dimensional ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Photometric Method ◽  
Nuclear Pore

Single-molecule localization microscopy (SMLM) 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 SIMPLER, a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence (TIRF) microscope. SIMPLER requires no hardware modification whatsoever to a conventional TIRF microscope, and complements any 2D SMLM method to deliver 3D images with nearly isotropic nanometric resolution. Examples of the performance of SIMPLER include the visualization of the nuclear pore complex through dSTORM with sub-20 nm resolution and of microtubule cross-sections resolved with sub-10 nm through DNA-PAINT.

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