Negative staining studied by TEM and STEM mass measurements: Preliminary observations on collagen sheets negatively stained with uranyl acetate

Quantitative mass measurements by dark-field scanning transmission EM and conventional bright-field transmission EM have been used to determine the increase in mass brought about by negative staining. pN-collagen (which forms sheets of uniform thickness and known mass per unit area) was used as a test specimen; the negative stain was uranyl acetate (1%, pH 4.4). The mass increase corresponded to the addition of roughly 8 uranyl acetate molecules per nm² for lightly negatively stained specimens; for heavily stained specimens, it was 30 molecules or more. The appearance of the image was related to the mass increase. This preliminary study shows that mass measurements can provide a basis for the quantitative interpretation of images from negatively stained specimens.     

Electron microscopic studies of lipopolysaccharides from phase I and phase II Coxiella burnetii

Lipopolysaccharides from phase I (LPSI) Coxiella burnetii Ohio and Nine Mile strains and from phase II (LPSII) Nine Mile stain were negatively and positively and examined with the electron microscope. The ultrastructure of LPSI and LPSII positively stained with uranyl formate or uranyl acetate was ribbon-like. When negatively stained with uranyl acetate, LPSI was ribbon-like but LPSII exhibited hexagonal lattice structures. However, LPSII stained negatively with sodium phosphotungstate and ammonium molybdate exhibited hexagonal lattice ultrastructures which were not identical to those observed when negatively stained with uranyl acetate. The hexagonal lattice structures formed in vitro were due to the interactions of LPSII and the staining reagents rather than to protein-LPS interactions. The differences in the ultrastructures of LPSI and LPSII are undoubtedly based on variations in their chemical composition.     

A study of staining for electron microscopy using collagen as a model system—VI. Uranyl nitrate negative staining patterns and their correlation with sequence data

Collagen is used as a model system to study the mechanism of negative staining. Negative staining patterns from reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) were compared with chemical data by a computer-aided correlation procedure. The stain used was uranyl nitrate, pH 3.2 and 4.9. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant stain-excluding factor determining the small-scale distribution of stain along the collagen fibril. Some contribution of positive staining can also be demonstrated by the analysis described here.     

A study of staining for electron microscopy using collagen as a model system—V. The effect of suberimidate fixation on negative staining patterns

The effects of the fixative dimethylsuberimidate (DMS) on negative staining patterns were studied using reconstituted fibrils of type I calf skin collagen (of known amino acid sequence) as a model system and comparing electron-optical data and chemical data by a computer-aided correlation procedure. The results show that the ‘bulkiness’ (average cross-sectional area or ‘plumpness’) of amino acid side chains is the dominant factor in determining the stain-excluding property of a DMS-fixed negatively stained collagen fibril as it is in unfixed collagen. Some contribution of positive staining can also be demonstrated after DMS-fixation by partial correlation analysis. Other evidence suggests that (unlike glutaraldehyde) DMS does not produce any morphological alterations to the negative staining pattern.     

A study of staining for electron microscopy using collagen as a model system—VIII. Simulation of the negative staining pattern

Simulated negative staining patterns of collagen fibrils were prepared for visual display by a graphical procedure in which amino acid side-chains along the staggered molecules were weighted according to their stain-excluding capacity. The simulated patterns were then compared directly with electron-optical images of collagen fibrils negatively stained with sodium phosphotungstate or lithium tungstate. These visual comparisons confirm previous observations that satisfactory matching occurs when side-chains are weighted according to their ‘bulkiness’ (average cross-sectional area or ‘plumpness’). Optimal matching at the edges of the overlap zones occurred when a hairpin-like conformation was assumed for the N-terminal telopeptides and a condensed conformation for the hydrophobic part of the C-terminal telopeptides. The negative staining pattern is known to include some element of positive staining; visual matching suggests that this additional uptake of positive staining ions occurs predominantly in the more accessible gap zone in a fibril D-period. A slight mismatching between observed and simulated patterns can be understood if the gap zone suffers greater axial shrinkage than the overlap zone when specimens are prepared for electron microscopy.     

Biochemical and ultrastructural characterization of a high molecular weight soluble Mg2+ -ATPase from human erythrocytes

The isolation and purification of a 600,000 Mr cytosolic Mg2+ -ATPase from human erythrocytes is described. The electrophoretic properties of the native and sodium dodecyl sulphate-dissociated protein are presented and compared with those of the erythrocyte protein cylindrin . The Mg2+-ATPase has a single subunit of Mr 100,000 and it has an isoelectric point of 4.9. From transmission electron microscopy of negatively stained specimens, it is proposed that the Mg2+-ATPase is hexameric, containing two superimposed trimers of the 100,000 Mr subunit, which gives rise to a 13 nm pseudohexagonal particle with a central 3 nm cavity. Varying the orientation of the protein in the negative stain also produces images that are not hexagonal. When orientated on-edge, the protein produces a double-disc image, which is most clearly defined under acidic negative staining conditions with uranyl acetate, when some aggregation of the protein is produced. The ultrastructure of the Mg2+-ATPase is shown to be distinctly different from that of cylindrin . A comparative discussion of the negatively stained transmission electron microscopical images of the Mg2+-ATPase, mitochondrial F1-ATPase and several other oligomeric proteins and enzymes is presented.     

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.     

A comparative negative staining study of aqueous suspensions of sphingomyelin

Bovine brain sphingomyelin liposomes have been studied by TEM when negatively stained at 4, 22 and 60δC with uranyl acetate,sodium phosphotungstate and amonium molybdate. The liposomes images vary slightly in the different negative stains, yet overall agreement exists once the different permeability and ionic propreties of the stains and the orietation of the liposomes are taken into account. Sodium phosphotungstate possesses an undesirable aggregative action on sphingomylin liposomes, but no aggregation has been encountered with the other stains. The liposomes images at 4 and 22δC are very similar, since both temperatures are beneath the crystalline-liqiud crystalline phase transition temperature (Tc). At 60δC, wich is above the Tc for sphingomyelin, the liposomal particles appear to be much more flexible and accordingly present a more varied shape than at the lower temperatured. The overall conformation of the sphingomyelin liposome is thought to be a ca 50 nm flattened single bilayer vesicle. Nevertheless, data are presented which suggest that some single bilayer disc-like micelles of sphingomyelin are also present.The larger multi-lamellar particular structures or myelion bodies, which are present when solid sphingomyelin is simply disperesed in water, have been studied by negative staining at 22 and 60δC. At 22δC, sphingomylin myelin bodies contain bilayers which exhibit the 26.5 nm undulatory P⨿β′pre-transition phase. The periodic feature is revealed particularly clearly by uranyl acetate. Considerable image complexity is usually present, because of overlapping information from more than one bilayer. Myelin bodies are occasionally split open during the negative staining and the single bilayer regions then reveal the image periodicity with superior clarity. Ammonium molybdate reveals the Pβ′ pre-transition phase undulations rather faintly owing to the permeation of this across the phospholipid bilayers. Sodium phosphotungstate has not been found to reveal this structural feature of the phospholipid bilayer. Ar 22δC some liposomes from spontaneously from the larger lipid bodies, but at 60δC their vesicularization occurs very much more rapidly.     

The negative staining pattern of collagen fibrils as studied by electron microscopy. The effect of fixation

The collagen negative staining pattern was studied by correlating electron-optical data with chemical data. Stains used were: phosphotungstic acid, pH 3.2 and 7.0, lithium tungstate, pH 7.2, methylamine tungstate, pH 6.6, and uranyl nitrate, pH 3.2 and 4.9. The principal factor determining the small-scale distribution of stain in such patterns is local exclusion by ‘bulky’ amino acid side-chains. A small superimposed positive staining contribution can also be detected. The effects of diimidoesters and tannic acid on negative staining patterns were also investigated.     

Preservation of alveolar type II pneumocyte lamellar bodies for electron microscopic studies.

Simultaneous fixation with glutaraldehyde and osmium tetroxide, followed by an uranyl acetate (UA) treatment before dehydration and embedding (Hirsch and Fedorko 1968) ensures a very good preservation of lamellar bodies (LB's) as well as of the cellular membranes in type II pneumocyte. The uranyl acetate treatment appeared to be the most efficient step of the procedure. The morphological aspect of lamellar bodies after such a preparation was similar to that observed after freeze-etching of lipid retaining methods. Moreover, the Hirsch-Fedorko procedure is very simple and can easily be used for routine ultrastructural and radioautographic studies. On the other hand, it appeared that the uranyl acetate phospholipid "complex" is very sensitive to the pH of chemical solutions used after sectioning. The "complex" is variously dissolved by alkaline solutions, photographic developers or stains. The best preservation of ultrastructure was obtained with neutral or acidic developers and acidic stains.     

Structure of α-tropomyosin magnesium paracrystals: II. Simulation of staining patterns from the sequence and some observations on the mechanism of positive staining

Electron micrographs of magnesium paracrystals of α-tropomyosin stained with uranyl acetate show a repeating pattern of 14 dark bands. Previous studies (Caspar et al., 1969; Ohtsuki, 1974) have shown that the molecules in the paracrystal lie antiparallel with their ends near two prominent white bands. These white lines divide the pattern into two zones containing nine and five dark bands, respectively, with the longer zone corresponding to the overlap between C termini. The present study shows that the intensity of the prominent white lines is reduced after digesting tropomyosin with carboxypeptidase A. This implies that, even in supposedly positively stained material, the white lines result from the exclusion of residual negative stain by the local thickening associated with the overlap of the ends of consecutive parallel molecules (NC overlap). Computer image processing and least-squares analysis have been employed to relate the positively stained patterns observed in both digested and undigested material to molecular positions and the amino acid sequence. Over a range of different staining criteria, it is shown that the pattern is fitted best when the C termini overlap by 176 ± 5 residues and the ends of consecutive parallel molecules overlap by 11 ± 5 residues. Uranyl ions appear to bind to carboxyl groups in the structure unless they form salt bridges with basic residues or they lie in the innermost positions of the tropomyosin coiled coil. Systematic differences between predicted and observed patterns near the molecular ends suggest that the conformation of the NC overlap may not be completely α-helical. A model with a globular N terminus and an extended C terminus is more consistent with the observed staining patterns and also offers an explanation for some other observations.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Electron microscopy of G-banded human mitotic chromosomes

Trypsin-treated human metaphase chromosomes stained with Giemsa and uranyl acetate showed clear, reproducible band structures under the transmission electron microscope (TEM). The banding pattern observed with TEM corresponded very closely to the G-band pattern visualized by light microscopy. The TEM images were used for karyotype analyses. Trypsin-treated chromosomes stained with uranyl acetate alone also showed clear G-bands under TEM. Shadow casting in addition to uranyl acetate staining revealed more structural detail of the chromosomes. Chromosome fibers, 200 Å–300 Å in diameter, were observed in the interband regions. Most chromosomes showed the major G-bands under the higher TEM magnification wit0out any trypsin treatment.     

PREFERENTIAL STAINING OF NUCLEIC ACID-CONTAINING STRUCTURES FOR ELECTRON MICROSCOPY

Oriented fibres of extracted nucleohistone were employed as test material in a study of satisfactory fixation, embedding, and staining methods for structures containing a high proportion of nucleic acid. Fixation in buffered osmium tetroxide solution at pH 6, containing 10^-2 M Ca⁺⁺, and embedding in Araldite enabled sections of the fibres to be cut in which the orientation was well preserved. These could be strongly stained in 2 per cent aqueous uranyl acetate, and showed considerable fine structure. Certain regions in the nuclei of whole thymus tissue could also be strongly stained by the same procedure, and were identical with the regions stained by the Feulgen procedure in adjacent sections. Moreover, purified DNA was found to take up almost its own dry weight of uranyl acetate from 2 per cent aqueous solution. Strongest staining of whole tissue was obtained with very short fixation times-5 minutes or so at 0°C. Particularly intense staining was obtained when such tissue stained in uranyl acetate was further stained with lead hydroxide. Although the patterns of staining by lead hydroxide alone and by uranyl acetate were similar in tissues fixed for longer times (½ hour to 2 hours, at 0°C or 20°C), in briefly fixed material the DNA-containing regions appeared relatively unstained by lead hydroxide alone, whilst often there was appreciable staining of RNA-containing structures. Observations on the staining of some viruses by similar techniques are also described.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.     

Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal.

Mycoplasma pneumoniae was grown on Formvar- and carbon-coated electron microscope grids and treated with the nonionic detergent Triton X-100 to gently remove the membrane and cytoplasm. The detergent mixture was composed of 0.5% Triton X-100 in SSR-2 broth base. After this treatment, the grids were rinsed in a mixture of 0.1 M KCl, 5 mM MgCl2, and 6 mM potassium phosphate buffer (pH 7.05) and negatively stained with uranyl acetate. The Triton X-100-resistant remains of M. pneumoniae after gentle removal of the membrane and cytoplasm consisted of fibrous structures oriented similarly to the undisrupted cells. The thin fibers displayed a negative staining quality and diameter analogous to that of rabbit muscle F-actin. The fibrous moieties ended in rodlike condensations which appeared striated in negatively stained and shadowed preparations. These striations were regular, and the majority of rod structures had lengths of 220 to 300 nm and widths of 50 to 80 nm. Specific antibody to rabbit muscle actin, produced in guinea pigs, was used in indirect immunofluorescence of the M. pneumoniae colonies. Fluorescence was detected, with concentrations at the colony center and at the tips of filamentous cells.     

Quaternary structure of the isolated restriction endonuclease EndoR.Bgl I from Bacillus globigii as revealed by electron microscopy.

The restriction endonuclease EndoR · BglI was purified nearly to homogeneity. BglI samples, when negatively stained with 4% uranyl acetate, show two different particle projections in the electron microscope. Projection A has an outer diameter of 22.5 ± 0.8 nm and is composed of six intensity maxima arranged in a ring; the centre of the ring exhibits slightly visible additional substructures. Projection B is also a ring; its outer diameter is 23.8 ± 0.7 nm; it does not show detailed fine structure, aside from a probable 10-fold rotational symmetry. Variations of the negative staining technique (single carbon layer, 2% uranyl acetate; ‘sandwich’ preparation with 4% uranyl acetate) revealed additional fine structural details for both projections. From the electron microscopic observations, a model of the enzyme particle was developed containing 20 identical, biologically active monomers of molecular weight around 61,000, arranged as a pentagonal dodecahedron. Tilting experiments established this structure decisively by interconversion of the different appearances of given particles in the expected way. By sodium dodecyl sulphate/polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis in a continuous molecular sieve gradient and evaluation of negatively stained enzyme particles, a molecular weight of the monomer of 61,000 was estimated, resulting in a total enzyme particle molecular weight of 1.2 × 10⁶ also determined by linear sucrose density-gradient centrifugation.     

Isolation and characterization of keratin-like proteins from cultured cells with fibroblastic morphology

Intermediate filaments (IF) isolated from a variety of cultured cells, conventionally described as fibroblasts, are composed predominantely of proteins of molecular weights of 54,000 and/or 55,000. Less than 15% of the protein found in native IF preparations from these cells is composed of three to four polypeptides of molecular weights 60,000- 70,000. We have investigated some biochemical and immunological properties of these proteins isolated from BHK-21 and mouse 3T3 cells. They are capable of forming paracrystals that exhibit a light/dark banding pattern when negatively stained with uranyl acetate. The dark bands are composed of longitudinally aligned approximately 2-nm-diam filaments. The center-to-center spacing between either dark or light bands is 37-40 nm. These dimensions are consistent with the secondary structure of IF polypeptides and suggest that the dark bands represent lateral alignment of alpha-helical coiled-coil domains. Immunoblotting, secondary structure, as well as amino acid composition data indicate that the 60,000-70,000-mol-wt paracrystal polypeptides are similar to keratin. Thus, polypeptides with biochemical and immunological properties of epidermal keratin are present in cells normally considered to be fibroblasts.     

Histochemical characterization of the lead intranuclear inclusion bodies

A battery of histological and histochemical techniques was applied on the lead intranuclear bodies that have resuted in the kidneys of adult Wistar male rats receiving lead acetate in their diet to determine their nature. The intranuclear inclusion bodies have stained strongly with xanthene, anthraquinone, and trisulfonated basophilic dyes and weakly with dyes containing both positive and negative radicals, and they have responded negatively to acidophilic cationic dyes. They have also reacted positively to proteins and lead stains, but weakly to lipid stains, and negatively to Feulgen and methyl green pyronin techniques. The intranuclear bodies proved to be lead lipoprotein complexes containing sulfyhydryl groups and are basic in nature with orthochromatic, eosinophilic, argyrophilic, osmophilic, fuchsinophilic, and sudanophilic characteristics.     

Contrasting of Lowicryl K4M thin sections

Summary A method is presented for increasing the contrast of cellular structures on ultrathin sections from tissues embedded in Lowicryl K4M. The method, designated UA/MC adsorption staining, is based on the uranyl acetate/methyl cellulose staining of thawed cryosections. Ultrathin Lowicryl K4M sections were exposed to a uranyl acetate/methyl cellulose solution and the excess solution was removed with filter paper, leaving the remainder to air dry on the section. Sections on the grids were then directly observed in the electron microscope. Parameters such as methyl cellulose and uranyl acetate concentrations, duration of staining, temperature and pH were all assessed for their effect on subsequent contrast formation. Conditions were achieved which yielded intense contrast of cellular membranes, basement membranes and extracellular matrix components usually not apparent in Lowicryl K4M thin sections routinely counter-stained with uranyl acetate and lead acetate. The enhancement of the contrast of these structures does not obscure colloidal gold particles used for immunocytochemistry or lectin labeling, thus making the UA/MC adsorption staining method useful for increasing membrane contrast in routine post-embedding immuno- and lectin cytochemistry on Lowicryl K4M thin sections.