scholarly journals Parallel, linear, and subnanometric 3D tracking of microparticles with Stereo Darkfield Interferometry

2021 ◽  
Vol 7 (6) ◽  
pp. eabe3902
Author(s):  
Martin Rieu ◽  
Thibault Vieille ◽  
Gaël Radou ◽  
Raphaël Jeanneret ◽  
Nadia Ruiz-Gutierrez ◽  
...  
Keyword(s):  
High Precision ◽  
High Throughput ◽  
Single Molecule ◽  
Particle Tracking ◽  
Focal Plane ◽  
Tracking System ◽  
Three Dimensional ◽  
3D Tracking ◽  
Single Molecule Force Spectroscopy ◽  
A New Technique

While crucial for force spectroscopists and microbiologists, three-dimensional (3D) particle tracking suffers from either poor precision, complex calibration, or the need of expensive hardware, preventing its massive adoption. We introduce a new technique, based on a simple piece of cardboard inserted in the objective focal plane, that enables simple 3D tracking of dilute microparticles while offering subnanometer frame-to-frame precision in all directions. Its linearity alleviates calibration procedures, while the interferometric pattern enhances precision. We illustrate its utility in single-molecule force spectroscopy and single-algae motility analysis. As with any technique based on back focal plane engineering, it may be directly embedded in a commercial objective, providing a means to convert any preexisting optical setup in a 3D tracking system. Thanks to its precision, its simplicity, and its versatility, we envision that the technique has the potential to enhance the spreading of high-precision and high-throughput 3D tracking.

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 Sample preparation method to improve the efficiency of high-throughput single-molecule force spectroscopy

2019 ◽  
Vol 5 (4) ◽  
pp. 176-183 ◽  
Author(s):  
Lei Jin ◽  
Li Kou ◽  
Yanan Zeng ◽  
Chunguang Hu ◽  
Xiaodong Hu
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Single Molecule ◽  
Preparation Method ◽  
Force Spectroscopy ◽  
Experimental Result ◽  
Sample Preparation Method ◽  
Dna Stretching ◽  
Single Molecule Force Spectroscopy ◽  
Preparation Methods

Abstract Inefficient sample preparation methods hinder the performance of high-throughput single-molecule force spectroscopy (H-SMFS) for viscous damping among reactants and unstable linkage. Here, we demonstrated a sample preparation method for H-SMFS systems to achieve a higher ratio of effective target molecules per sample cell by gas-phase silanization and reactant hydrophobization. Digital holographic centrifugal force microscopy (DH-CFM) was used to verify its performance. The experimental result indicated that the DNA stretching success ratio was improved from 0.89% to 13.5%. This enhanced efficiency preparation method has potential application for force-based DNA stretching experiments and other modifying procedures.

scholarly journals High-throughput in-focus differential interference contrast imaging of three-dimensional orientations of single gold nanorods coated with a mesoporous silica shell

RSC Advances ◽  
2020 ◽  
Vol 10 (50) ◽  
pp. 29868-29872
Author(s):  
Geun Wan Kim ◽  
Seokyoung Yoon ◽  
Jung Heon Lee ◽  
Ji Won Ha
Keyword(s):  
Mesoporous Silica ◽  
High Throughput ◽  
Gold Nanorods ◽  
Focal Plane ◽  
Three Dimensional ◽  
Silica Shell ◽  
Differential Interference Contrast ◽  
Contrast Imaging ◽  
3D Space ◽  
Dic Microscopy

Spherical AuNRs@mSiO2 have randomly oriented AuNR cores in 3D space, which could be resolved on the same focal plane by interference-based DIC microscopy.

Single-Molecule Force Spectroscopy of β-Peptides That Display Well-Defined Three-Dimensional Chemical Patterns

2011 ◽  
Vol 133 (11) ◽  
pp. 3981-3988 ◽  
Author(s):  
Claribel Acevedo-Vélez ◽  
Guillaume Andre ◽  
Yves F. Dufrêne ◽  
Samuel H. Gellman ◽  
Nicholas L. Abbott
Keyword(s):  
Single Molecule ◽  
Three Dimensional ◽  
Force Spectroscopy ◽  
Single Molecule Force Spectroscopy

scholarly journals Symmetrically-dispersed spectroscopic single-molecule localization microscopy

2019 ◽  
Author(s):  
K. Song ◽  
Y. Zhang ◽  
B. Brenner ◽  
C. Sun ◽  
H. F. Zhang
Keyword(s):  
Spectral Analysis ◽  
Single Molecule ◽  
Particle Tracking ◽  
Three Dimensional ◽  
Spatial Localization ◽  
Single Particle Tracking ◽  
Localization Microscopy ◽  
First Time ◽  
Spectral Channels ◽  
Single Molecule Localization Microscopy

AbstractSpectroscopic single-molecule localization microscopy (sSMLM) achieved simultaneously imaging and spectral analysis of single molecules for the first time. Current sSMLM fundamentally suffers from reduced photon budget because of dividing photons from individual stochastic emission into spatial and spectral channels. Therefore, both spatial localization and spectral analysis only use a portion of the total photons, leading to reduced precisions in both channels. To improve the spatial and spectral precisions, we present symmetrically-dispersed sSMLM or SDsSMLM to fully utilize all photons from individual stochastic emissions in both spatial and spectral channels. SDsSMLM achieved 10-nm spatial and 0.8-nm spectral precisions at a total photon budget of 1000. Comparing with existing sSMLM using a 1:3 splitting ratio between spatial and spectral channels, SDsSMLM improved the spatial and spectral precisions by 42% and 10%, respectively, under the same photon budget. We also demonstrated multi-color imaging in fixed cells and three-dimensional single-particle tracking using SDsSMLM.

scholarly journals Three-dimensional tracking of tethered particles for probing nanometer-scale single-molecule dynamics using plasmonic microscope

2021 ◽  
Author(s):  
Guangzhong Ma ◽  
Zijian Wan ◽  
Yunze Yang ◽  
Wenwen Jing ◽  
Shaopeng Wang
Keyword(s):  
Single Molecule ◽  
High Spatial Resolution ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Axial Direction ◽  
Nanometer Scale ◽  
3D Tracking ◽  
Single Molecule Level ◽  
Plasmonic Imaging ◽  
Molecule Dynamics

Three-dimensional (3D) tracking of surface-tethered single-particle reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in axial direction, for measuring the dynamics of biomolecules with spatial scale of several nanometers. Here we present a plasmonic imaging technique that can track the motion of ~100 tethered particles in 3D simultaneously with sub-nanometer axial precision at millisecond time resolution. By tracking the 3D coordinates of tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzyme. We further show that the particle motion pattern can be used to identify specific and non-specific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.

scholarly journals A New System for Three-Dimensional Tracking of Motile Microorganisms

2000 ◽  
Vol 66 (5) ◽  
pp. 2238-2242 ◽  
Author(s):  
Roland Thar ◽  
Nicholas Blackburn ◽  
Michael Kühl
Keyword(s):  
Tracking System ◽  
Three Dimensional ◽  
Dark Field ◽  
3D Tracking ◽  
Free Swimming ◽  
Size Classes ◽  
Resolution Limits ◽  
Dark Field Illumination ◽  
Swimming Microorganisms ◽  
New System

ABSTRACT A new three-dimensional (3D)-tracking system with optimized dark-field illumination is presented. It allows simultaneous 3D tracking of several free-swimming microorganisms with diameters of >10 μm. Resolution limits and illumination efficiencies for different size classes of microorganisms are treated analytically. First applications for 3D tracking of protists are demonstrated.

scholarly journals A Three-Dimensional Tracking Method with the Self-Calibration Functions of Coaxiality and Magnification for Single Fluorescent Nanoparticles

2019 ◽  
Vol 10 (1) ◽  
pp. 131
Author(s):  
Shuai Mao ◽  
Jin Shen ◽  
Yajing Wang ◽  
Wei Liu ◽  
Jinfeng Pan
Keyword(s):  
Tracking System ◽  
Three Dimensional ◽  
The Self ◽  
Fluorescent Nanoparticles ◽  
3D Tracking ◽  
Tracking Method ◽  
Self Calibration ◽  
Position Detection ◽  
Vector Measurement ◽  
Detection Unit

A self-calibrating variable magnification three-dimensional (3D) tracking system for single fluorescent nanoparticles is proposed. The system was based on astigmatic micro-imaging and has a simple configuration incorporating a dual-spot position detection unit ray transfer matrix. By analyzing this matrix and utilizing the beam vector measurement of a dual-spot position detection unit, it was demonstrated that the proposed tracking system had the self-calibration functions of coaxiality and magnification; thus, it could accurately track the adopted fluorescent nanoparticles through zoom microscopical measurements in 3D. The available measurements of the proposed system and accuracy were experimentally validated.

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