Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy

The domainal substructure and molecular conformation of human fibrinogen have been investigated by evaluating scanning transmission electron microscopic images of freeze-dried or negatively contrasted native fibrinogen (fractions I-4 and I-9), glutaraldehyde-treated fibrinogen, or plasmic core fragments D₁ and E₂. Although some unstained freeze-dried native or glutaraldehyde-treated fibrinogen molecules were relatively compact and even occasionally spheroidal, typical images were elongated symmetrical tridomainal structures 460 Å ± 20 Å in length; frequently they were bent into a variety of elongated though non-linear arrangements. Their identification as monomolecular forms of fibrinogen by scanning transmission electron microscopic mass measurements resolved uncertainties relating to the identity of such objects as single molecules. The central domains of fraction I-4 molecules had a greater mass than those of fraction I-9 (1.01 × 10⁵M_rversus 7.5 × 10 M_r, respectively). This difference accounted for the observed mass difference between fraction I-4 and fraction I-9 molecules (i.e. 3.27 × 10⁵M_rversus 2.97 × 10⁵M_r, respectively) and suggested that the COOH-terminal region of the Aα chain (major portions of which are always absent from fraction I-9 molecules) is situated within the mass integration radius for the central domain. When the COOH-terminal region of the Aα chain was present it appeared in negative stain as a thread-like structure originating between the middle and outer domains and extending toward the central domain, sometimes appearing to wind around the long axis.The outer domains of negatively stained molecules resembled negatively stained images of fragment D₁ and could frequently be resolved into at least two discrete subdomains, forming an oblong structure usually canted at an angle of ～120 ° to 150 ° relative to the long axis. Our findings are consistent with prevailing tridomainal structural models of fibrinogen and suggest that these molecules are flexible and may exist in unfolded configurations, or as relatively compact, partially or completely folded forms.     

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.     

Morphometric analysis of the surface cells of rabbit corneal epithelium by scanning electron microscopy

The corneal surface of female New Zealand white rabbits (1.9-2.6 kg) was examined at x500 magnification by scanning electron microscopy. A total of 112 micrographs, taken as sequential sets from the center to the edge of the corneal surface from 8 different animals, was analyzed using a digitizer pad. Each cell was identified by the number of immediately bordering cells and by the nature of its electron reflex (light, medium, dark). Analysis of areas of the cells by number of bordering cells (number of cell sides) reveals a wide range of areas and skewed distributions especially when the number of sides is 5 or less. Overall, the cell-surface area increases as the number of cell sides increases. However, analyses of the mean surface areas for cells with different numbers of sides and additionally grouped by electron reflex suggests the existence of three separate populations of cells at the corneal surface. The possible etiology and dynamics of this complex cell mosaic are discussed in relation to circadian rhythms and to resurfacing of the cornea following mechanical trauma, ultraviolet radiation, and toxic chemical exposure.     

Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy

The immunogold method is widely used to localize, identify, and distinguish cellular antigens. There are, however, some pitfalls that can lead to nonspecific binding, particularly in cytoskeletal studies with gold probes prepared from small gold particles. We present a list of suggestions for minimizing nonspecific binding, with particular attention to two problems identified in this study. First, we find that the method used to prepare the colloidal gold particles affects the degree of nonspecific binding. Second, the standard BSA-stabilized small gold probes evidently possess exposed regions that bind to the proteins of cytoskeletal preparations. This was investigated in whole-mount cytoskeletal preparations of cultured cells by use of light microscopy, transmission electron microscopy, and photoelectron microscopy of silver-enhanced specimens. Gold probes were made from approximately 5-nm particles generated by reduction of HAuCl4 with three different reducing agents: white phosphorus, sodium borohydride, and citrate-tannic acid. All three preparations stabilized in the conventional way showed significant levels of nonspecific binding, which was highest with citrate-tannic acid. This problem was largely solved with all three types of probes by including fish gelatin in the probe buffer, by substituting fish gelatin for the BSA stabilizer used to prepare the probes, or by pre-adsorption methods. Application of these techniques resulted in clear immunogold labeling patterns with minimal nonspecific background.     

High-voltage electron microscopy of human diploid fibroblasts during ageing in vitro. Morphometric analysis of mitochondria

Since recent studies have suggested a diminished mitochondrial functional capacity in late-passage ('old') compared to early-passage ('young') normal fibroblasts and fibroblasts from the Hutchinson-Gilford (progeria) syndrome of premature ageing, we analysed whole-cell preparations on the high voltage electron microscope to look for mitochondrial and related defects. All strains examined showed considerable heterogeneity in cell size and intracellular morphology. Mitochondria were readily seen in all cells, predominantly as long slender rods with frequent branching, but occasional circular and saccular forms were also evident. Various parameters of mitochondrial mass including mean number, weight, and total length of mitochondria per cell weight tended to increase in old and progeria cells, but only the former attained statistical significance due to the heterogeneity and consequent variance. A significant finding was the decreased width of mitochondria in old and progeria cells. Cystic blebs were evident in mitochondria of some cells with an apparent increase in old and progeria fibroblasts. These blebs appeared to be due to weakening of the inner membrane, allowing dilatation of the outer membrane which otherwise appeared intact. The number of osmiophilic inclusions per cell weight, particularly lipofuscin granules and autophagic vacuoles, was significantly increased in old and progeria cells. In conclusion, despite some morphological changes, mitochondria of old and progeria cells maintain a structurally and bioenergetically adequate mass compatible with continued cellular viability.     

High-resolution transmission electron microscopy of adult human peritubular dentine

Summary Single crystals from adult human peritubular dentine were studied by high-resolution transmission electron microscopy. Periodic fringe patterns were obtained from which the exact shape of the inorganic crystals were deduced. The crystals were found to have a mean length of 36.00±1.87 nm, a mean width of 25.57±1.37 nm, and a mean thickness of 9.76±0.69 nm. They consisted of platelets with a mean width-to-thickness ratio of 2.61, each being a flattened hexagonal prism of hydroxyapatite. Such conclusions are based upon a) the electron diffraction patterns that we obtained, and b) our comparison of the values of the periodic, equidistant fringes seen along different planes of sectioning with the corresponding theoretical values for hydroxyapatite.     

Analyses of phosphorylase kinase by transmission and scanning transmission electron microscopy

Under conventional electron microscopy negatively stained phosphorylase kinase exhibits a bilobal structure resembling two bridged opposing parentheses. In this predominant particle orientation, usually only one bridge is observed; however, in many particles two bridges can be seen. Scanning transmission electron microscopy of unstained phosphorylase kinase shows very similar structures, with a particle mass equivalent to that of the hexadecameric holoenzyme. Partial digestion of the enzyme with chymotrypsin, which preferentially hydrolyzes the alpha-subunits, causes no significant changes in the structure; however, when both the alpha and beta subunits are degraded by trypsin, single lobed particles appear, i.e. the connecting bridges are missing. Mass analysis of scanning transmission electron microscopy images of trypsinized enzyme indicates that the protease does, in fact, split the particle into halves. Transmission electron microscopy of an alpha gamma delta complex isolated after incubation of the holoenzyme with LiBr shows only small particles approximately one-fourth the size of the holoenzyme. Thus, integrity of the beta subunit may be necessary in order for the two lobes of phosphorylase kinase to be bridged. These data also indicate that the subunits are arranged as a bridged dimer of octamers 2 (alpha 2 beta 2 gamma 2 delta 2).     

Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage

Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.     

Conventional transmission electron microscopy

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.Transmission electron microscopy (TEM) has been an important technology in cell biology ever since it was first used in the early 1940s. The most frequently used TEM application in cell biology entails imaging stained thin sections of plastic-embedded cells by passage of an electron beam through the sample such that the beam will be absorbed and scattered, producing contrast and an image (see

Odontoblast processes in human dentin revealed by fluorescence labeling and transmission electron microscopy

In the present undertaking, the distribution of odontoblast processes in human dentin was determined through the DiI carbocyanine dye fluorescent staining of the cell membrane, while F-actin was identified by rhodamine-phalloidin. Confocal laser scanning microscopy revealed intense labeling for both agents in inner dentin, while transmission electron microscopy (TEM) identified dentinal tubules including odontoblast processes in this area, each process being surrounded by a cell membrane and containing an abundance of filamentous structures. Electron-dense "lamina limitans" lined the dentinal tubules. Individual cell processes became narrower toward the middle area, and their overall numbers decreased as well under TEM. Labeling for F-actin was absent in both middle and outer dentin, while faint labeling for DiI was visible along the dentinal tubules as far as the dentino-enamel junction (DEJ), where it was also recognized within the tubules themselves. Under TEM, the dentinal tubules lined with electron-dense structures were, in fact, empty in the middle and outer dentin. Immediately below the DEJ, however, the tubules manifested dense concentrations of fine granular material. Our study, therefore, appears to suggest that odontoblast processes do not extend beyond the inner dentin of fully erupted human premolars.     

Image analysis of Artemia salina ribosomes by scanning transmission electron microscopy.

A dedicated scanning transmission electron microscope (STEM) at Brookhaven National Laboratory was used to visualize unstained freeze-dried ribosomal particles under conditions which considerably reduce the specimen distortion inherent in the heavy metal staining and air-drying preparative steps used in routine transmission electron microscopy (TEM). From high-resolution STEM images it is possible to determine molecular mass and the mass distribution within individual ribosomal particles and perform statistical evaluation of the data. Analysis of digitized STEM images of Artemia salina ribosomes provided evidence that a standard preparation of these eukaryotic ribosomes consists of a population of heterogenous particles. Because of the integrity of rRNAs established by agarose gel electrophoresis, variations in the composition and structure of the 80S monosomes and the large (60S) and small (40S) ribosomal subunits, as monitored by their mass, were attributed to the loss of ribosomal proteins, from the large subunits in particular. These results are relevant not only to the degree of ribosomal biological activity, but should also be taken into consideration for particle selection in the reconstruction of the "native" eukaryotic ribosome 3-D model.     

Visualization of ceramide channels by transmission electron microscopy

Functional studies have shown that the sphingolipid ceramide, self-assembles in phospholipid membranes to form large channels capable of allowing proteins to cross the membrane. Here these channels are visualized by negative stain transmission electron microscopy. The images contain features consistent with stain-filled pores having a roughly circular profile. There is no indication of tilt, and the results are consistent with the formation of right cylinders. The sizes of the pores range from 5 to 40nm in diameter with an asymmetric distribution indicating no apparent upper size limit. The size distribution matches well with the distribution of sizes calculated from electrophysiological measurements.     

Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy

An atypical peg-like terminal constriction (“peg”) on metaphase chromosomes of the plant genus Oziroë could be identified as a nucleolus organizing region (NOR) by detecting 45S rDNA with correlative light microscopy (LM) and scanning electron microscopy (SEM) in situ hybridization (ISH). Using high-resolution 3D analytical SEM, the architecture and DNA distribution of the peg-like NOR were characterized as typical for chromosomes, albeit with significantly smaller chromomeres. ISH procedure was improved for SEM concerning signal localization, labeling efficiency, and structural preservation, allowing 3D SEM analysis of the peg-like NOR structure and rDNA distribution for the first time. It could be shown that implementation of FluoroNanogold markers is an attractive tool that allows efficient immunodection in both LM and SEM. A model is proposed for the peg structure and its mode of condensation.     

Quantitative analysis of knock-on and thermal damage of biological specimens by electron irradiation in transmission electron microscopy

High-energy electrons are able to transfer momentum to nuclei, which results in displacement on to the interstitial lattice sites with a maximum transferred energy of 4 · 10⁴ eV for carbon at 100 keV. Moreover, most of the energy dissipated in energy losses is converted into heat, which results in melting and evaporation.The specimen temperature rise was calculated by the heat conduction theory and confirmed by the specimen drift due to the thermal damage. The damage can be reduced by a small area of illumination, the use of a metal-coated microgrid and small area scanning.A further displacement due to the knock-on collision and the resulting etching rate of biological specimens was measured. The damage is proportional to the current density in c cm^-2 at the specimen. The allowable maximum dose was obtained from the measurement of an etching rate with the weight loss and dry density of the specimens.It was found that the images affected by the electron irradiation, in which -H, C-H, C-N bonds break molecules in proteinaceous biological specimens are removed, and the remaining molecules are changed to stable carbon-rich molecules by deposition, polymerization and contamination. In addition, defect images were observed in high contrast, when compared with unaffected images taken with a small area scanning method.     

Visualizing aquatic bacteria by light and transmission electron microscopy

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.     

Integrated fluorescence and transmission electron microscopy

Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.     

Sutural mineralization of rat calvaria characterized by atomic-force microscopy and transmission electron microscopy

The application of transmission electron microscopy (TEM) and atomic-force microscopy (AFM) aid the acquisition of detailed structural information on the process of hard tissue formation. The sutural mineralization of rat calvaria is taken as a model for a collagen-related mineralization system. After cryofixation or chemical fixation an anhydrous tissue preparation technique with no staining procedures is used. The atomic-force microscope and the transmission electron microscope are used for structural analysis of the mineralizing region of the sutural tissue. With the application of AFM the collagen macroperiod is shown to be well represented in the unmineralized sutural tissue. At the mineralization front the collagen fibrils are found to be thickened and to change to a characteristic stacked platelet structure. Using TEM the macroperiod is faintly visible before mineral crystallites have formed and is more prominent after the apatite crystallization has started in the fibrils. In this step a needle-like structure of the newly formed apatitic crystals is visible.