High-Resolution Analysis of Rapidly Frozen Biological Specimens: Capabilities and Limitations

1999 ◽  
Vol 5 (S2) ◽  
pp. 412-413
Author(s):  
S.B. Andrews ◽  
N.B. Pivovarova ◽  
J. Hongpaisan ◽  
R.D. Leapman
Keyword(s):  
Low Temperature ◽  
Low Dose ◽  
Biological Tissues ◽  
Transmission Microscopy ◽  
Energy Filtering ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Analytical Microscopy ◽  
Electron Microscopes ◽  
Mapping Techniques

The past decade has seen major advances in the analytical capability and utility of both fixed beam and scanning beam electron microscopes. In particular, scanning transmission electron microscopy (STEM) and energy-filtering transmission microscopy (EFTEM) have benefited from the development of devices and techniques—including improved electron optics, sensitive solid-state detectors and new software for imaging and electron energy loss spectroscopy (EELS)—that optimize detection of weak spectroscopic signals arising from biological specimens while minimizing specimen damage. Here we discuss and illustrate some of these advances, especially in the context of structural imaging, detection limits and mapping techniques for the biologically important elements phosphorus and calcium. Analytical microscopy of biological tissues is absolutely dependent on cryotechniques. It is generally agreed that rapid freezing and subsequent low-temperature processing, e.g., cryosectioning or direct cryotransfer of frozen-hydrated specimens, is the most reliable way to preserve the native distribution and organization of biological structures. Equally important, however, as an adjunct to spectroscopic analysis is the use of established low-temperature, low-dose techniques for recording optimized images. By limiting beam exposure, low-dose methods greatly improve the quality of images from fragile, freeze-dried preparations. In this case, the quality and information content of, e.g., cryosections are virtually as good as conventional preparations (Fig 1).

Transmission Scanning Electron Microscopy and Energy Analysis with the Siemens ELMISKOP 101

1972 ◽  
Vol 30 ◽  
pp. 450-451
Author(s):  
H. M. Thieringer
Keyword(s):  
Objective Lens ◽  
Transmission Microscopy ◽  
Image Recording ◽  
Mode Of Operation ◽  
Scanning Transmission ◽  
Electron Microscopes ◽  
And Performance ◽  
Convergent Beam ◽  
Ray Path ◽  
Beam Diffraction

It has repeatedly been show that with conventional electron microscopes very fine electron probes can be produced, therefore allowing various micro-techniques such as micro recording, X-ray microanalysis and convergent beam diffraction. In this paper the function and performance of an SIEMENS ELMISKOP 101 used as a scanning transmission microscope (STEM) is described. This mode of operation has some advantages over the conventional transmission microscopy (CTEM) especially for the observation of thick specimen, in spite of somewhat longer image recording times.Fig.1 shows schematically the ray path and the additional electronics of an ELMISKOP 101 working as a STEM. With a point-cathode, and using condensor I and the objective lens as a demagnifying system, an electron probe with a half-width ob about 25 Å and a typical current of 5.10-11 amp at 100 kV can be obtained in the back focal plane of the objective lens.

scholarly journals Confocal soft X-ray scanning transmission microscopy: setup, alignment procedure and limitations

2015 ◽  
Vol 22 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Andreas Späth ◽  
Jörg Raabe ◽  
Rainer H. Fink
Keyword(s):  
Magnetic Thin Films ◽  
Biological Tissues ◽  
Polymer Materials ◽  
Zone Plate ◽  
Contrast Imaging ◽  
Transmission Microscopy ◽  
X Ray ◽  
Alignment Procedure ◽  
High Contrast Imaging ◽  
Scanning Transmission

Zone-plate-based scanning transmission soft X-ray microspectroscopy (STXM) is a well established technique for high-contrast imaging of sufficiently transparent specimens (e.g.ultrathin biological tissues, polymer materials, archaeometric specimens or magnetic thin films) with spatial resolutions in the regime of 20 nm and high spectroscopic or chemical sensitivity. However, due to the relatively large depth of focus of zone plates, the resolution of STXM along the optical axis so far stays unambiguously behind for thicker X-ray transparent specimens. This challenge can be addressed by the implementation of a second zone plate in the detection pathway of the beam, resulting in a confocal arrangement. Within this paper a first proof-of-principle study for a confocal STXM (cSTXM) and an elaborate alignment procedure in transmission and fluorescence geometry are presented. Based on first confocal soft X-ray micrographs of well known specimens, the advantage and limitation of cSTXM as well as further development potentials for future applications are discussed.

3D Reconstruction from STEM Images of Xanthine Dehydrogenase and Insulin Receptor: Refinements and Molecular Modelling.

2000 ◽  
Vol 6 (S2) ◽  
pp. 276-277
Author(s):  
D.R. Beniac ◽  
R.Z.T. Luo ◽  
A.B. Fernandes ◽  
T. Iwasaki ◽  
C.C. Yip ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Scanning Transmission Electron Microscopy ◽  
Low Dose ◽  
Quaternary Structure ◽  
Three Dimensional ◽  
Xanthine Dehydrogenase ◽  
Dark Field ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Transmission

We have reconstructed the three-dimensional quaternary structure of the complete 480 kDa insulin receptor (IR), complexed with Nanogold - labeled insulin, and the 290 kDa Xanthine Dehydrogenase (XDH). Both molecules were imaged by low-dose, low-temperature dark field scanning transmission electron microscopy (STEM). XDH and IR were both freeze-plunged in liquid ethane, and transferred to the STEM (VG HB 601) where they were freeze dried at -130°C. Reconstruction was carried out using the method of quaternion-assisted angular reconstruction (IQAD) as described previously. XDH was further refined by an iterative process in which the IQAD produced volume was used as a reference for further refinements. Separate reconstructions of two sets of half the images indicated an inter-reconstruction resolution of 20 Å and 9 Å by phase residual criteria for the IR and XDH reconstructions, respectively.

Scanning and Transmission Microscopy of Nematocyst Batteries in Epitheliomuscular Cells of Hydra

1971 ◽  
Vol 29 ◽  
pp. 410-411 ◽  
Author(s):  
Jane A. Westfall ◽  
S. Yamataka ◽  
Paul D. Enos
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Osmium Tetroxide ◽  
External Surface ◽  
Three Dimensional ◽  
Surface Structures ◽  
Transmission Microscopy ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Scanning Electron

Scanning electron microscopy (SEM) provides three dimensional details of external surface structures and supplements ultrastructural information provided by transmission electron microscopy (TEM). Animals composed of watery jellylike tissues such as hydras and other coelenterates have not been considered suitable for SEM studies because of the difficulty in preserving such organisms in a normal state. This study demonstrates 1) the successful use of SEM on such tissue, and 2) the unique arrangement of batteries of nematocysts within large epitheliomuscular cells on tentacles of Hydra littoralis.Whole specimens of Hydra were prepared for SEM (Figs. 1 and 2) by the fix, freeze-dry, coat technique of Small and Màrszalek. The specimens were fixed in osmium tetroxide and mercuric chloride, freeze-dried in vacuo on a prechilled 1 Kg brass block, and coated with gold-palladium. Tissues for TEM (Figs. 3 and 4) were fixed in glutaraldehyde followed by osmium tetroxide. Scanning micrographs were taken on a Cambridge Stereoscan Mark II A microscope at 10 KV and transmission micrographs were taken on an RCA EMU 3G microscope (Fig. 3) or on a Hitachi HU 11B microscope (Fig. 4).

Characterization of microtubules by dark-field STEM and replication

1991 ◽  
Vol 49 ◽  
pp. 152-153
Author(s):  
S.B. Andrews ◽  
R.D. Leapman ◽  
P.E. Gallant ◽  
T.S. Reese
Keyword(s):  
Protein Interactions ◽  
Low Dose ◽  
Unit Length ◽  
Dark Field ◽  
Carbon Films ◽  
Microtubule Associated Proteins ◽  
Molecular Weights ◽  
Freeze Dried ◽  
Protein Motors ◽  
Associated Proteins

As part of a study on protein interactions involved in microtubule (MT)-based transport, we used the VG HB501 field-emission STEM to obtain low-dose dark-field mass maps of isolated, taxol-stabilized MTs and correlated these micrographs with detailed stereo images from replicas of the same MTs. This approach promises to be useful for determining how protein motors interact with MTs. MTs prepared from bovine and squid brain tubulin were purified and free from microtubule-associated proteins (MAPs). These MTs (0.1-1 mg/ml tubulin) were adsorbed to 3-nm evaporated carbon films supported over Formvar nets on 600-m copper grids. Following adsorption, the grids were washed twice in buffer and then in either distilled water or in isotonic or hypotonic ammonium acetate, blotted, and plunge-frozen in ethane/propane cryogen (ca. -185 C). After cryotransfer into the STEM, specimens were freeze-dried and recooled to ca.-160 C for low-dose (<3000 e/nm2) dark-field mapping. The molecular weights per unit length of MT were determined relative to tobacco mosaic virus standards from elastic scattering intensities. Parallel grids were freeze-dried and rotary shadowed with Pt/C at 14°.

Conformation of eukaryotic ribosomal RNAs

1983 ◽  
Vol 41 ◽  
pp. 592-593
Author(s):  
M. Boublik ◽  
G. Thornton ◽  
G. Oostergetel ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Molecular Weight ◽  
Mass Measurement ◽  
Carbon Film ◽  
Structural Complexity ◽  
Scanning Transmission Electron Microscope ◽  
Electron Microscopic ◽  
Pure Nitrogen ◽  
Freeze Dried ◽  
Scanning Transmission ◽  
Partially Unfolded

Understanding the structural complexity of ribosomes and their role in protein synthesis requires knowledge of the conformation of their components - rRNAs and proteins. Application of dedicated scanning transmission electron microscope (STEM), electrical discharge of the support carbon film in an atmosphere of pure nitrogen, and determination of the molecular weight of individual rRNAs enabled us to obtain high resolution electron microscopic images of unstained freeze-dried rRNA molecules from BHK cells in a form suitable for evaluation of their 3-D structure. Preliminary values for the molecular weight of 28S RNA from the large and 18S RNA from the small ribosomal subunits as obtained by mass measurement were 1.84 x 106 and 0.97 x 106, respectively. Conformation of rRNAs consists, in general, of alternating segments of intramolecular hairpin stems and single stranded loops in a proportion which depends on their ionic environment, the Mg++ concentration in particular. Molecules of 28S RNA (Fig. 1) and 18S RNA (not shown) obtained by freeze-drying from a solution of 60 mM NH+4 acetate and 2 mM Mg++ acetate, pH 7, appear as partially unfolded coils with compact cores suggesting a high degree of ordered secondary structure.

The Theoretical Contrast in Scanning and Conventional High Resolution Microscopes

1969 ◽  
Vol 27 ◽  
pp. 170-171
Author(s):  
E. Zeitler ◽  
M. G. R. Thomson
Keyword(s):  
Small Volume ◽  
Volume Element ◽  
Optical Processing ◽  
Image Construction ◽  
Object Interaction ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Optical Treatment ◽  
Electron Microscopes ◽  
Intensity Contrast

In the formation of an image each small volume element of the object is correlated to an areal element in the image. The structure or detail of the object is represented by changes in intensity from element to element, and this variation of intensity (contrast) is determined by the interaction of the electrons with the specimen, and by the optical processing of the information-carrying electrons. Both conventional and scanning transmission electron microscopes form images which may be considered in this way, but the mechanism of image construction is very different in the two cases. Although the electron-object interaction is the same, the optical treatment differs.

Scanning Transmission Microscopy Applied to Thick Specimen

1972 ◽  
Vol 30 ◽  
pp. 452-453
Author(s):  
H. Koike ◽  
T. Matsuo ◽  
K. Ueno ◽  
M. Suzuki
Keyword(s):  
Psoas Muscle ◽  
Image Intensifier ◽  
Chromatic Aberration ◽  
Image Contrast ◽  
Transmission Microscopy ◽  
Crystalline Materials ◽  
Thick Specimen ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Electron Microscope Image

Since the identification of single atoms was achieved by Crewe et al, scanning transmission microscopy has been put into pratical use. Recently they applied this method to the quantitative mass analysis of DNA.As pointed out previously the chromatic aberration which decreases the image contrast and quality, does not affect a scanning transmission image as it does a conventional transmission electron microscope image. Thus, the STEM method is advantageous for thick specimen. Further this method employs a high sensitive photomultiplier tube which also functions as an image intensifier. This detection method is effective for the observation of living specimens or easily damaged specimens. In this respect the scanning transmission microscope with high accelerating voltage is necessary.Since Uyeda's experiments of crystalline materials, many workers have been discussed how thick specimens can be observed by CTEM. With biological specimens, R. Szirmae reported on the decrease in the image contrast of rabbit psoas muscle sections at various accelerating voltages and specimen thicknesses.

Reduction of Specimen Contamination in Electron-Beam Instruments

1976 ◽  
Vol 34 ◽  
pp. 576-577
Author(s):  
G. Lehmpfuhl ◽  
P. J. Smith
Keyword(s):  
Electron Beam ◽  
Cold Trap ◽  
Beam Diameter ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Hydrocarbon Molecules ◽  
Electron Microscopes ◽  
Combination Of Methods ◽  
Convergent Beam ◽  
Very High

Specimens being observed with electron-beam instruments are subject to contamination, which is due to polymerization of hydrocarbon molecules by the beam. This effect becomes more important as the size of the beam is reduced. In convergent-beam studies with a beam diameter of 100 Å, contamination was observed to grow on samples at very high rates. Within a few seconds needles began forming under the beam on both the top and the underside of the sample, at growth rates of 400-500 Å/s, severely limiting the time available for observation. Such contamination could cause serious difficulty in examining a sample with the new scanning transmission electron microscopes, in which the beam is focused to a few angstroms.We have been able to reduce the rate of contamination buildup by a combination of methods: placing an anticontamination cold trap in the sample region, preheating the sample before observation, and irradiating the sample with a large beam before observing it with a small beam.

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

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