DNA Origami Calibrators for Counting Fluorophores on Single Particles by Flow Cytometry

Small Methods ◽  
2022 ◽  
pp. 2101364
Author(s):  
Denis Selnihhin ◽  
Kim I. Mortensen ◽  
Jannik B. Larsen ◽  
Jens B. Simonsen ◽  
Finn Skou Pedersen
Keyword(s):  
Flow Cytometry ◽  
Dna Origami ◽  
Single Particles

scholarly journals Conventional, High-Resolution and Imaging Flow Cytometry: Benchmarking Performance in Characterisation of Extracellular Vesicles

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 124
Author(s):  
Jaco Botha ◽  
Haley R. Pugsley ◽  
Aase Handberg
Keyword(s):  
Flow Cytometry ◽  
High Resolution ◽  
Extracellular Vesicles ◽  
Single Particles ◽  
Multiple Parameters ◽  
Imaging Flow Cytometry ◽  
Highly Sensitive ◽  
Individual Project ◽  
Benchmarking Performance ◽  
High Throughput Manner

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.

scholarly journals Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Florian Stehr ◽  
Johannes Stein ◽  
Julian Bauer ◽  
Christian Niederauer ◽  
Ralf Jungmann ◽  
...  
Keyword(s):  
Particle Tracking ◽  
Fusion Proteins ◽  
Organic Dyes ◽  
Single Particle Tracking ◽  
Dna Origami ◽  
Single Particles ◽  
Inherent Limitation ◽  
Cellular Processes ◽  
Fluorescently Labeled Oligonucleotides ◽  
Observation Times

AbstractMonitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales.

Determination of Dynamical Characteristics of Thrombocytes Through Initial Stage of Their Aggregation

2009 ◽  
Vol 4 (2) ◽  
pp. 69-77
Author(s):  
Irina Kolesnikova ◽  
Vyacheslav Nekrasov ◽  
Tatyana Sherstova ◽  
Galina Tsvetovskaya ◽  
Elena Chikova ◽  
...  
Keyword(s):  
Experimental Data ◽  
Flow Cytometry ◽  
Steric Factor ◽  
Aggregation Kinetics ◽  
Theoretical Treatment ◽  
Single Particles ◽  
Dynamical Characteristics ◽  
Diagnostic Potential ◽  
Initial Stage

In the work, a method of application of scanning flow cytometry for investigation of the aggregation dynamics of invitro activated thrombocytes is presented. As a result, the rate constant of creation of initial aggregates, – dimers, – from single thrombocytes, and the effective steric factor of aggregation, which characterized the thrombocytes activation degree, are determined. The study reveals diagnostic potential of the scanning flow cytometer in determination of relative concentrations of aggregates and non-aggregated thrombocytes in a sample by measuring light-scattering signals from single particles and evolution of the distribution function of the particles on the signals. Theoretical treatment of the experimental data is based on modeling the particles aggregation kinetics with the use of Smoluchovski equation.

3-D reconstruction of molecular shape from microcrystal thin sections

1983 ◽  
Vol 41 ◽  
pp. 748-749
Author(s):  
S. Cusack ◽  
J.-C. Jésior
Keyword(s):  
Three Dimensional ◽  
Molecular Shape ◽  
Biological Molecules ◽  
Fourier Space ◽  
Single Particles ◽  
Thin Sections ◽  
Standard Methods ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dimensional Reconstruction

Three-dimensional reconstruction techniques using electron microscopy have been principally developed for application to 2-D arrays (i.e. monolayers) of biological molecules and symmetrical single particles (e.g. helical viruses). However many biological molecules that crystallise form multilayered microcrystals which are unsuitable for study by either the standard methods of 3-D reconstruction or, because of their size, by X-ray crystallography. The grid sectioning technique enables a number of different projections of such microcrystals to be obtained in well defined directions (e.g. parallel to crystal axes) and poses the problem of how best these projections can be used to reconstruct the packing and shape of the molecules forming the microcrystal.Given sufficient projections there may be enough information to do a crystallographic reconstruction in Fourier space. We however have considered the situation where only a limited number of projections are available, as for example in the case of catalase platelets where three orthogonal and two diagonal projections have been obtained (Fig. 1).

Computer Assisted Image Reconstruction of Oligomer Structure

1976 ◽  
Vol 34 ◽  
pp. 472-473
Author(s):  
A.M. Jones ◽  
A. Max Fiskin
Keyword(s):  
Three Dimensional ◽  
Depth Of Field ◽  
Computer Assisted ◽  
Projection Data ◽  
Two Dimensional ◽  
Single Particles ◽  
Dimensional Reconstruction ◽  
Computer Assisted Image ◽  
The Fourier Transform ◽  
Space Approach

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.

New challenges to image processing posed by cryo-Electron microscopy of single particles

1991 ◽  
Vol 49 ◽  
pp. 422-423
Author(s):  
Joachim Frank
Keyword(s):  
Electron Microscopy ◽  
Phase Contrast ◽  
Particle Orientation ◽  
Single Particles ◽  
Contrast Transfer Function ◽  
Virtual Absence ◽  
Cryo Electron Microscopy ◽  
Spatial Frequencies ◽  
New Challenges ◽  
Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

Microstructure and reactivity of small metal particles: The role of Ce additives

1992 ◽  
Vol 50 (1) ◽  
pp. 318-319
Author(s):  
L.D. Schmidt ◽  
K. R. Krause ◽  
J. M. Schwartz ◽  
X. Chu
Keyword(s):  
Atmospheric Pressure ◽  
Noble Metals ◽  
Mixed Oxides ◽  
Catalytic Properties ◽  
Chemical Shifts ◽  
Catalytic Converter ◽  
Structural Evolution ◽  
Single Particles ◽  
Small Metal Particles

The evolution of microstructures of 10- to 100-Å diameter particles of Rh and Pt on SiO2 and Al2O3 following treatment in reducing, oxidizing, and reacting conditions have been characterized by TEM. We are able to transfer particles repeatedly between microscope and a reactor furnace so that the structural evolution of single particles can be examined following treatments in gases at atmospheric pressure. We are especially interested in the role of Ce additives on noble metals such as Pt and Rh. These systems are crucial in the automotive catalytic converter, and rare earths can significantly modify catalytic properties in many reactions. In particular, we are concerned with the oxidation state of Ce and its role in formation of mixed oxides with metals or with the support. For this we employ EELS in TEM, a technique uniquely suited to detect chemical shifts with ∼30Å resolution.

Structure of rattlesnake venom lectin determined by single particle high-resolution electron microscopy

1996 ◽  
Vol 54 ◽  
pp. 62-63
Author(s):  
Peter D. Moisiuk ◽  
Daniel R. Beniac ◽  
Ross A. Ridsdale ◽  
Martin Young ◽  
Bhushan Nagar ◽  
...  
Keyword(s):  
High Resolution Electron Microscopy ◽  
Lipid Monolayer ◽  
Outer Diameter ◽  
X Ray Diffraction ◽  
Single Particles ◽  
Crotalus Atrox ◽  
3D Reconstructions ◽  
X Ray ◽  
Rattlesnake Venom ◽  
Covalently Linked

Venom from the rattlesnake Crotalus atrox contains a mixture of enzymes that induce a localized effect leading to hemorrhaging, necrosis and edema. As a member of the crotalid family of snake venoms, Crotalus atrox venom contains a C-type lectin that will agglutinate blood cells in a Ca2+-dependent fashion. The lectin is a hydrophilic protein, consisting of two covalently linked, 135 amino acid residues, identical subunits that are rich in aspartic acid, glutamic acid and lysine. Sequence homology with known carbohydrate recognition domains (CRDs) indicates that rattlesnake venom lectin (RSLV) contains a CRD motif that is not linked to accessory domains. Preliminary X-ray diffraction and sedimentation analysis has indicated that lectin from Crotalus atrox forms decamers composed of two five-fold symmetric pentamers. Single particles of RSVL imaged at – 171°C displayed two distinct orientations on the specimen support (Figure a) following incubation in a crystallization Teflon well, coated with a lipid monolayer consisting of phosphatidylcholine and monosialoganglioside. When lying in an end-on orientation, the lectin exhibited a “pentagonal ring” with an outer diameter of 6.7 nm and an inner hollow core of 1.7 nm. A side orientation was also seen, whereby a thickness of 5.8 nm was measured for the lectin. Image processing of 2280 single particles placed in 100 classes (Figure b) led to 3D reconstructions of RSVL (Figure c). Density limited 3D reconstructions showed the lectin to be made of two five-fold symmetrical rings covalently linked between the five subunits that constitute each ring of this homodimer. These results are consistent with sedimentation and preliminary X-ray diffraction analysis on the shape of RSVL and provide the framework for structural verification by 2D electron crystallography.

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