Higher-order assembly of gastropodan hemocyanins as revealed by Scanning Transmission Electron Microscopy (STEM)

1989 ◽  
Vol 47 ◽  
pp. 248-249
Author(s):  
M.G. Hamilton ◽  
R.R. Rodriguez ◽  
T.T. Herskovits ◽  
J.S. Wall
Keyword(s):  
Analytical Ultracentrifugation ◽  
Large Angle ◽  
Gel Filtration ◽  
Scattered Electron ◽  
Molecular Weights ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
The Masses ◽  
Individual Particles

The hemocyanins of gastropods consist of aggregates of a cylindrical decameric subparticle that assembles into di-, tri-, tetra-, penta-, and larger multi-decameric particles with sedimentation coefficients of ca. 105 S, 130 S, 150 S, 170 S, and higher values. We are using STEM to measure the masses of individual particles and analytical ultracentrifugation to determine the distribution of sedimenting components.Hemocyanins were isolated from freshly collected hemolymph by gel filtration on BioGel A-5m columns. Samples were analyzed with schlieren optics in a Beckman Madel E ultracentrifuge. Specimens were diluted into 0.1 M HEPES, pH 8.0, 0.01 M MgC12 to a final concentration of 100 ug/mL and freeze-dried for STEM analysis. The STEM instrument was operated at 40 kV using a -140 °C cold stage. The elastically scattered electron signal from the STEM large angle annular detector was used to form the images. The specimens were imaged with 10 A pixels at a dose of 6-10 e/A2. Molecular weights of individual particles were measured as previously described.

Analysis of Molluscan Hemocyanins by Scanning Transmission Electron Microscopy and Light Scattering

1988 ◽  
Vol 46 ◽  
pp. 162-163
Author(s):  
P. S. Furcinitti ◽  
J. S. Wall ◽  
M. G. Hamilton ◽  
T. T. Herskovits
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Light Scattering ◽  
Scanning Transmission Electron Microscopy ◽  
Large Angle ◽  
Scattered Electron ◽  
Scanning Transmission Electron ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission

Hemocyanins are copper containing, oxygen binding proteins found in many invertebrate species of the phyla Arthropoda and Mollusca. Molluscan hemocyanins are cylindrical macromolecular assemblies of a basic decameric unit. The hemocyanins of two chitons, Stenonlax conspicua and Mopalia muscosa. are decamers, while those of two gastropods, Fasciolaria tulipa and Pleuroplora gieantea. are di-decamers. The hemocyanins of a third gastropod species, Busvcon contrarium can exist in a spectrum of multi-decameric forms. Molecular weights of the various types of molluscan hemocyanins were measured by absolute light scattering and Scanning Transmission Electron Microscopy (STEM) as a first step in understanding multi-decamer formation in some types of hemocyanins.Hemocyanins were prepared as previously described, dialyzed into 1M HEPES buffer, pH 7.4, containing 0.05M MgCl and freeze-dried or negatively stained for STEM analysis. Specimens were examined at the Brookhaven STEM Biotechnology Resource, which was operated at 40 kV using a -140°C cold stage. The elastically scattered electron signal from the STEM large angle annular detector was used to form the images.

scholarly journals Ionic Nanocomplexes of Hyaluronic Acid and Polyarginine to Form Solid Materials: A Green Methodology to Obtain Sponges with Biomedical Potential

Nanomaterials ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 944
Author(s):  
María Gabriela Villamizar-Sarmiento ◽  
Ignacio Moreno-Villoslada ◽  
Samuel Martínez ◽  
Annesi Giacaman ◽  
Victor Miranda ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
High Capacity ◽  
Solid Material ◽  
Fibroblast Cell ◽  
High Porosity ◽  
Design Development ◽  
Solid Materials ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission

We report on the design, development, characterization, and a preliminary cellular evaluation of a novel solid material. This material is composed of low-molecular-weight hyaluronic acid (LMWHA) and polyarginine (PArg), which generate aqueous ionic nanocomplexes (INC) that are then freeze-dried to create the final product. Different ratios of LMWHA/PArg were selected to elaborate INC, the size and zeta potential of which ranged from 100 to 200 nm and +25 to −43 mV, respectively. Turbidimetry and nanoparticle concentration analyses demonstrated the high capacity of the INC to interact with increasing concentrations of LMWHA, improving the yield of production of the nanostructures. Interestingly, once the selected formulations of INC were freeze-dried, only those comprising a larger excess of LMWHA could form reproducible sponge formulations, as seen with the naked eye. This optical behavior was consistent with the scanning transmission electron microscopy (STEM) images, which showed a tendency of the particles to agglomerate when an excess of LMWHA was present. Mechanical characterization evidenced low stiffness in the materials, attributed to the low density and high porosity. A preliminary cellular evaluation in a fibroblast cell line (RMF-EG) evidenced the concentration range where swollen formulations did not affect cell proliferation (93–464 µM) at 24, 48, or 72 h. Considering that the reproducible sponge formulations were elaborated following inexpensive and non-contaminant methods and comprised bioactive components, we postulate them with potential for biomedical purposes. Additionally, this systematic study provides important information to design reproducible porous solid materials using ionic nanocomplexes.

The BNL STEM Facility

1997 ◽  
Vol 3 (S2) ◽  
pp. 277-278
Author(s):  
Joseph S. Wall ◽  
Martha N. Simon ◽  
James F. Hainfeld
Keyword(s):  
National Laboratory ◽  
Computer Control ◽  
Large Angle ◽  
Brookhaven National Laboratory ◽  
Dark Field ◽  
Vacuum System ◽  
Vacuum Chambers ◽  
System A ◽  
Freeze Dried ◽  
Individual Particles

The STEM facility at Brookhaven National Laboratory has been in operation since Oct. ‘77, using a custom-built instrument (STEM1) with cold field emission source, 2.5Å probe, -150°C cold stage, efficient dark field detectors and computer control & data acquisition system. A specimen changing air lock and several portable vacuum chambers permit vacuum transfer of specimens from a separate vacuum system where they were freeze dried overnight.The large angle dark-field signal produced by the STEM is directly proportional to the total mass within the probed area. STEM mass mapping is based on this linear relationship and the fact that only specimen-specific atoms remain on the substrate after washing with volatile buffer and freeze drying. All images are digital and available via Internet. PC software can be provided for analysis.STEM mass accuracy ranges from a fraction of a percent on well-defined individual particles such as viruses in the 50 MDa to 10 GDa range, to ∼1% around 1 MDa and ∼10% in the 50 kDa range.

Structural analysis of biological macromolecular assemblies by scanning transmission electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 262-263
Author(s):  
M. Boublik ◽  
S. J. Tumminia ◽  
W. Hellmann ◽  
Q. Zhang ◽  
J.F. Hainfeld ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Macromolecular Complex ◽  
Functional Sites ◽  
Macromolecular Assemblies ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Annular Detector ◽  
Topographical Mapping ◽  
Particle Elimination

Separation of the resolution and contrast affecting components and the optimized placement of detectors for the collection of elastic (contrast-forming) electrons on Brookhaven dedicated STEM make it possible to quantitatively detect greater than 90% of the available elastically scattered electrons. The high contrast and superior signal-to-noise ratio associated with the STEM annular detector allow for the imaging of unstained freeze-dried biological macromolecular complexes (chromatin, viruses, nucleic acids) at radiation doses as low as 1 e/Å. Specimens prepared in this way are free of the main resolution-limiting conditions of conventional TEM i.e. staining , air drying and radiation damage. The image intensity of unstained specimens can be related to their local projected mass and used for calculation of the total mass and mass distribution within any selected particle. Elimination of staining makes it possible to use heavy metals as high-resolution markers for topographical mapping of components and/or functional sites on a particular macromolecular complex.

Radial Mass Density Analysis of Unstained Stem Images

1985 ◽  
Vol 43 ◽  
pp. 318-319
Author(s):  
P.S. Furcinitti ◽  
J.F. Hainfeld ◽  
J.J. Lipka ◽  
J.S. Wall
Keyword(s):  
Signal To Noise Ratio ◽  
Mass Density ◽  
Unit Length ◽  
Large Angle ◽  
Scanning Transmission Electron Microscope ◽  
Outer Diameter ◽  
Biological Macromolecules ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Spot 5

The high contrast and signal-to-noise ratio inherent in the Scanning Transmission Electron Microscope (STEM) makes it possible to examine unstained, freeze-dried biological macromolecules. Since the large-angle, elastically scattered STEM signal is directly proportional to the specimen mass, molecular weight or mass per unit length determinations are possible for individual macromolecules. For objects which have cylindrical or spherical symmetry the resolution lost by sparse sampling (2 Å spot, 5 or 10 Å between pixels) may be regained by employing the “Vernier Sampling” method developed by Steven et al. to rebin the data on a finer grid. A projected mass distribution is then obtained for the average values of the mass per unit area on an axis perpendicular to the symmetry axis. Assuming the particle to consist of a set of concentric cylinders of varying density, a set of simultaneous equations can be solved for the mass density at each annular ring. Thus the outer diameter and the internal radial structure of complex macromolecules Can be determined.

scholarly journals Solution and surface effects on plasma fibronectin structure.

1983 ◽  
Vol 97 (6) ◽  
pp. 1686-1692 ◽  
Author(s):  
N M Tooney ◽  
M W Mosesson ◽  
D L Amrani ◽  
J F Hainfeld ◽  
J S Wall
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ionic Strength ◽  
Scanning Transmission Electron Microscopy ◽  
Plasma Fibronectin ◽  
Scanning Transmission Electron ◽  
Neutral Ph ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission

As assessed by electron microscopy, the reported shape of the plasma fibronectin molecule ranges from that of a compact particle to an elongated, rod-like structure. In this study, we evaluated the effects of solution and surface conditions on fibronectin shape. Freeze-dried, unstained human plasma fibronectin molecules deposited at pH 7.0-7.4 onto carbon films and examined by scanning transmission electron microscopy appeared relatively compact and pleiomorphic, with approximate average dimensions of 24 nm X 16 nm. Negatively stained molecules also had a similar shape but revealed greater detail in that we observed irregular, yarn-like structures. Glutaraldehyde-induced intramolecular cross-linking did not alter the appearance of plasma fibronectin. Molecules deposited at pH 2.8, pH 9.3, or after succinylation were less compact than those deposited at neutral pH. In contrast, fibronectin molecules sprayed onto mica surfaces at pH 7, rotary shadowed, and examined by transmission electron microscopy were elongated and nodular with a contour length of 120-130 nm. Sedimentation velocity experiments and electron microscopic observations indicate that fibronectin unfolds when it is succinylated, when the ionic strength is raised at pH 7, or when the pH is adjusted to 9.3 or 2.8. Greater unfolding is observed at pH 2.8 at low ionic strength (less than 0.01) compared with material at that pH in 0.15 M NaCl solution. We conclude that (a) the shape assumed by the fibronectin molecule can be strongly affected by solution conditions and by deposition onto certain surfaces; and that (b) the images of fibronectin seen by scanning transmission electron microscopy at neutral pH on carbon film are representative of molecules in physiologic solution.

scholarly journals X-ray ptychographic mode of self-assembled CdSe/CdS octapod-shaped nanocrystals in thick polymers

2020 ◽  
Vol 53 (3) ◽  
pp. 741-747
Author(s):  
Liberato De Caro ◽  
Francesco Scattarella ◽  
Davide Altamura ◽  
Milena P. Arciniegas ◽  
Dritan Siliqi ◽  
...  
Keyword(s):  
Direct Method ◽  
Imaging Method ◽  
Free Standing ◽  
X Ray ◽  
Molecular Weights ◽  
Density Maps ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Processing Techniques ◽  
Pod Length

This work describes the application of X-ray ptychography for the inspection of complex assemblies of highly anisotropic nanocrystals embedded in a thick polymer matrix. More specifically, this case deals with CdSe/CdS octapods, with pod length L = 39 ± 2 nm and pod diameter D = 12 ± 2 nm, dispersed in free-standing thick films (24 ± 4 µm) of polymethyl methacrylate and polystyrene, with different molecular weights. Ptychography is the only imaging method available to date that can be used to study architectures made by these types of nanocrystals in thick polymeric films, as any other alternative direct method, such as scanning/transmission electron microscopy, can be definitively ruled out as a result of the large thickness of the free-standing films. The electron density maps of the investigated samples are reconstructed by combining iterative difference map algorithms and a maximum likelihood optimization algorithm. In addition, post image processing techniques are applied to both reduce noise and provide a better visualization of the material morphological details. Through this process, at a final resolution of 27 nm, the reconstructed maps allow us to visualize the intricate network of octapods inside the polymeric matrices.

X-ray microanalysis of mineralized deposits in a variety of pathological conditions in the brain

1995 ◽  
Vol 53 ◽  
pp. 866-867
Author(s):  
C. A. Ackerley ◽  
L. E. Becker
Keyword(s):  
Electron Microscope ◽  
Scanning Transmission Electron Microscope ◽  
Small Degree ◽  
X Ray ◽  
Transmission Electron ◽  
Formalin Fixed Paraffin ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Formalin Fixed Paraffin Embedded ◽  
The Brain

Although a small degree of mineralization can be a common occurrence without associated pathological symptoms, certain diseases of the brain do however exhibit distinct increases in mineralization with characteristic distributions l>2. In this study, tissues from a number of these disorders were prepared for x-ray microanalysis in several ways. Where possible, material was slam frozen on a liquid nitrogen cooled polished copper block, cryosections prepared and freeze dried in the scanning transmission electron microscope (STEM) using a cold stage prior to analysis by energy dispersive x-ray spectrometry (EDS). In addition, samples were freeze substituted for several days, embedded in LR white and cut on dry knives before analysis. Where only formalin fixed paraffin embedded materials were available, .5μ.m sections were cut and mounted on carbon planchets. The specimens were then deparaffinized with xylene and viewed with the backscatter electron detector (BEI) in the scanning electron microscope (SEM) and analyzed by EDS.

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