Structural analysis of biological macromolecular assemblies by scanning transmission electron microscopy

Author(s):  
M. Boublik ◽  
S. J. Tumminia ◽  
W. Hellmann ◽  
Q. Zhang ◽  
J.F. Hainfeld ◽  
...  

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.

Author(s):  
M. G. R. Thomson

The variation of contrast and signal to noise ratio with change in detector solid angle in the high resolution scanning transmission electron microscope was discussed in an earlier paper. In that paper the conclusions were that the most favourable conditions for the imaging of isolated single heavy atoms were, using the notation in figure 1, either bright field phase contrast with β0⋍0.5 α0, or dark field with an annular detector subtending an angle between ao and effectively π/2.The microscope is represented simply by the model illustrated in figure 1, and the objective lens is characterised by its coefficient of spherical aberration Cs. All the results for the Scanning Transmission Electron Microscope (STEM) may with care be applied to the Conventional Electron Microscope (CEM). The object atom is represented as detailed in reference 2, except that ϕ(θ) is taken to be the constant ϕ(0) to simplify the integration. This is reasonable for θ ≤ 0.1 θ0, where 60 is the screening angle.


Author(s):  
R.D. Leapman ◽  
K.E. Gorlen ◽  
C.R. Swyt

The determination of elemental distributions by electron energy loss spectroscopy necessitates removal of the non-characteristic spectral background from a core-edge at each point in the image. In the scanning transmission electron microscope this is made possible by computer controlled data acquisition. Data may be processed by fitting the pre-edge counts, at two or more channels, to an inverse power law, AE-r, where A and r are parameters and E is energy loss. Processing may be performed in real-time so a single number is saved at each pixel. Detailed analysis, shows that the largest contribution to noise comes from statistical error in the least squares fit to the background. If the background shape remains constant over the entire image, the signal-to-noise ratio can be improved by fitting only one parameter. Such an assumption is generally implicit in subtraction of the “reference image” in energy selected micrographs recorded in the CTEM with a Castaing-Henry spectrometer.


Author(s):  
G. Botton ◽  
G. L’Espérance ◽  
M.D. Ball ◽  
C.E. Gallerneault

The recently developed parallel electron energy loss spectrometers (PEELS) have led to a significant reduction in spectrum acquisition time making EELS more useful in many applications in material science. Dwell times as short as 50 msec per spectrum with a PEELS coupled to a scanning transmission electron microscope (STEM), can make quantitative EEL images accessible. These images would present distribution of elements with the high spatial resolution inherent to EELS. The aim of this paper is to briefly investigate the effect of acquisition time per pixel on the signal to noise ratio (SNR), the effect of thickness variation and crystallography and finally the energy stability of spectra when acquired in the scanning mode during long periods of time.The configuration of the imaging system is the following: a Gatan PEELS is coupled to a CM30 (TEM/STEM) electron microscope, the control of the spectrometer and microscope is performed through a LINK AN10-85S MCA which is interfaced to a IBM RT 125 (running under AIX) via a DR11W line.


Nanomaterials ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 944
Author(s):  
María Gabriela Villamizar-Sarmiento ◽  
Ignacio Moreno-Villoslada ◽  
Samuel Martínez ◽  
Annesi Giacaman ◽  
Victor Miranda ◽  
...  

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.


2013 ◽  
Vol 19 (4) ◽  
pp. 1050-1060 ◽  
Author(s):  
Lewys Jones ◽  
Peter D. Nellist

AbstractThe aberration-corrected scanning transmission electron microscope has great sensitivity to environmental or instrumental disturbances such as acoustic, mechanical, or electromagnetic interference. This interference can introduce distortions to the images recorded and degrade both signal noise and resolution performance. In addition, sample or stage drift can cause the images to appear warped and leads to unreliable lattice parameters being exhibited. Here a detailed study of the sources, natures, and effects of imaging distortions is presented, and from this analysis a piece of image reconstruction code has been developed that can restore the majority of the effects of these detrimental image distortions for atomic-resolution data. Example data are presented, and the performance of the restored images is compared quantitatively against the as-recorded data. An improvement in apparent resolution of 16% and an improvement in signal-to-noise ratio of 30% were achieved, as well as correction of the drift up to the precision to which it can be measured.


Author(s):  
A. Engel ◽  
J. W. Wiggins ◽  
David Woodruff

Six modes of transmission electron microscopy are compared by a numerical simulation of the image formation assuming perfectly coherent illumination and ignoring the influence of radiation damage and noise. The comparison includes five modes of conventional electron microscopy (CEM): axial bright field, Unwin's phase plate, central stop dark field, tilted-beam dark field and conical illumination dark field, and the annular detector mode of the scanning transmission electron microscope (STEM).


Author(s):  
P.S. Furcinitti ◽  
J.F. Hainfeld ◽  
J.J. Lipka ◽  
J.S. Wall

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.


Author(s):  
E. J. Kirkland ◽  
R. F. Loane ◽  
J. Silcox

The multislice method (e.g. Goodman and Moodie) of simulating bright field conventional transmission electron microscope (BF-CTEM) images of crystalline specimens can be extended to simulation of scanning transmission electron microscope (STEM) images of similar specimens in the annular dark field (ADF) mode. According to the reciprocity theorem (Pogany and Turner and Cowley) BF-CTEM would be equivalent to BF STEM with a point detector. Such a detector (STEM) however would yield an exceedingly small signal to noise ratio. Thus, STEM has found more use in the ADF mode (e.g. Crewe et al.) exploiting the large contrast arising from heavy atoms. In BF imaging (CTEM and STEM) the constrast is roughly proportional to the scattering amplitude f α Z3/4 whereas in DF (CTEM and STEM) imaging it is roughly proportional to the scattering cross σ α Z3/2 where Z is atomic number, a form that is advantageous foatom discrimination.


Author(s):  
M.G. Hamilton ◽  
R.R. Rodriguez ◽  
T.T. Herskovits ◽  
J.S. Wall

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.


2014 ◽  
Vol 20 (4) ◽  
pp. 1246-1253 ◽  
Author(s):  
Debora Keller ◽  
Stephan Buecheler ◽  
Patrick Reinhard ◽  
Fabian Pianezzi ◽  
Darius Pohl ◽  
...  

AbstractThis work presents a systematic study that evaluates the feasibility and reliability of local band gap measurements of Cu(In,Ga)Se2 thin films by valence electron energy-loss spectroscopy (VEELS). The compositional gradients across the Cu(In,Ga)Se2 layer cause variations in the band gap energy, which are experimentally determined using a monochromated scanning transmission electron microscope (STEM). The results reveal the expected band gap variation across the Cu(In,Ga)Se2 layer and therefore confirm the feasibility of local band gap measurements of Cu(In,Ga)Se2 by VEELS. The precision and accuracy of the results are discussed based on the analysis of individual error sources, which leads to the conclusion that the precision of our measurements is most limited by the acquisition reproducibility, if the signal-to-noise ratio of the spectrum is high enough. Furthermore, we simulate the impact of radiation losses on the measured band gap value and propose a thickness-dependent correction. In future work, localized band gap variations will be measured on a more localized length scale to investigate, e.g., the influence of chemical inhomogeneities and dopant accumulations at grain boundaries.


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