Thin Sections : I. A Study of Section Thickness and Physical Distortion Produced during Microtomy

Knowledge of the thickness of sections is important for proper interpretation of electron micrographs. Therefore, the thicknesses of sections of n-butyl methacrylate polymer were determined by ellipsometry, and correlated with the color shown in reflected light. The results are: gray, thinner than 60 mµ; silver, 60 to 90 mµ; gold, 90 to 150 mµ; purple, 150 to 190 mµ; blue, 190 to 240 mµ; green, 240 to 280 mµ; and yellow, 280 to 320 mµ. These results agree well with optical theory and with previous published data for thin films. Sections, after cutting, are 30 to 40 per cent shorter than the face of the block from which they were cut. Only a small improvement results from allowing the sections to remain in the collecting trough at room temperature. Heating above room temperature, however, reduces this shortening, with a corresponding improvement in dimensions and spatial relationships in the sections. When the thickness of the section is considered in interpreting electron micrographs instead of considering the section to be two-dimensional, a more accurate interpretation is possible. The consideration of electron micrographs as arising from projections of many profiles from throughout the whole thickness of the section explains the apparent lack of continuity often observed in serial sections. It is believed that serial sections are actually continuous, but that the change in size of structure through the thickness of one section and the consideration of only the largest profile shown in the micrograph can account for the lack of continuity previously observed.     

INTERPRETATIONS OF ELECTRON MICROGRAPHS OF SINGLE AND SERIAL SECTIONS

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

Interpretation of electron micrographs of single and serial sections

A method of securing serial sections for electron microscopy is described. Serial sections present certain anomalies of interpretation of a nature such that a complete and detailed three-dimensional reconstruction of the sectioned tissue cannot be made. These anomalies are discussed, as well as those which have been encountered in the interpretation of single sections. Observations of the following kinds have been made in an attempt to elucidate the interpretation of single and serial sections: differing methods of mounting adjacent sections, observation of the same section by high-angle stereoscopy, and examination of sections which have been shadowed prior to and subsequent to electron microscopy. It is found that the appearance of sections is independent of the choice of side to be placed against the formvar films. Stereoscopy shows that the appearance of fine structures is strongly dependent upon the direction of the penetrating electron beam with respect to the plane of the structures. Stereoscopy, combined with shadowing, shows quantitatively that extensive sublimation of polymer occurs upon normal exposure in the electron microscope. Observation of sections shadowed prior to electron microscopy indicates that varying amounts of material are removed between sections by the action of microtomy; i.e., it is probable that the sum of the thicknesses of several serial sections is considerably less than the total thickness of material removed from the block. It is believed that this effect, combined with the effect of sublimation, aids in explaining the failure of adjacent sections to exhibit continuity in their detailed structures.     

ULTRASTRUCTURAL INTERRELATIONSHIPS OF NERVE PROCESSES AND SMOOTH MUSCLE CELLS IN THREE DIMENSIONS

The muscularis externa of the intestinal wall of frogs was fixed in osmium tetroxide, embedded in Vestopal-W, serially sectioned for electron microscopy, and stained with uranyl acetate. A method to obtain individually mounted and properly positioned serial sections is described. The three-dimensional techniques used during the course of this investigation demonstrate that it is possible to examine carefully relatively large areas of tissue on individual serial sections with the electron microscope and subsequently to construct montages of electron micrographs of pertinent areas from each section. Several carefully rendered interrelationships of nerve processes and smooth muscle cells in three dimensions are exhibited and described. Recent studies of other neuro-effector relationships are discussed in relation to the present status of the nature and organization of the autonomic nervous system in visceral organs.     

FINE STRUCTURE STUDIES OF PHLEUM ROOT MERISTEM CELLS. I. MITOCHONDRIA

Avers , Charlotte J. (Douglass Coll., Rutgers—The State U., New Brunswick, N.J.) Fine structure studies of Phleum root meristem cells. I. Mitochondria. Amer. Jour. Bot. 49(9): 996–1003. Illus. 1962.—Mitochondrial numbers were computed from thick sections embedded in paraffin and stained with iron hematoxylin, from methacrylate embedded, 0.5 μ-thick sections stained with osmium, and from electron micrographs of ultrathin sections fixed in KMnO₄. The mean mitochondrial counts per average cell (20 × 20 × 10μ) were: 121 ± 4 in paraffin sections, 600 ± 40 in methacrylate sections, and 991 ± 96 from electron micrographs. These numbers were higher than the Janus green B count of 91 ± 2 per cell found during an earlier study. In each case, arithmetical conversion factors were used to calculate total numbers of mitochondria per cell since section thicknesses were less than that of whole cell length or depth. Determinations of numbers of mitochondria probably varied depending on section thickness and specific staining procedures used, but the higher count from electron micrographs was assumed to be reasonable on a volumetric basis. The cytoplasmic volume of the average cell is about 2500 μ³ and the volume of a single average mitochondrion is about 0.1μ^3. On this basis, the variety of structures and their relatively sparse distribution seems possible despite the apparently high numbers found. In addition to mitochondria, the numbers of various cellular components per unit cytoplasmic area were determined. These data showed that mitochondria were about 6 times more numerous than proplastids, about 4.5 times more numerous than Golgi bodies, and even more frequent when compared with vesicles, dense bodies, or microbodies. No correlation was found between cytoplasmic area and numbers of organelles per unit area. Photographic evidence was presented for the occurrence in plant cells of the hepatic cell, polymorphic “lysosome” described by Ashford and Porter. The controversial nature of the lysosome is discussed.     

Electron microscope observations on intracellular virus-like particles associated with the cells of the Lucké renal adenocarcinoma

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mmicro) having a thick capsule and a dense inner body (35 to 40 mmicro) that is eccentrically placed within the central cavity (70 to 80 mmicro). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.     

Ultrastructural localization of neurophysin-like immunoreactivity in the rat posterior pituitary. An alternative method using frozen-dried tissue fixed with OsO4 vapor.

Electron microscopic identification of elements containing neurophysin-like immunoreactivity can be accomplished in rat posterior pituitary that has been frozen-dried and fixed with OsO4 vapor. Alternating serial ultrathin sections are placed on grids and glass slides. The sections on the slides are stained for neurophysin using immunofluorescence histochemistry, and the resultant images are superimposed on electron micrographs from the adjacent sections. The method provides several advantages for the localization of neuropeptide immunoreactivity in nervous tissue.     

ULTRASTRUCTURE OF THE PYRENOID OF TREBOUXIA IN RAMALINA MENZIESII TUCK.1,2

Trebouxia sp., the phycobiont of the lichen Ramalina menziesii Tuck., was examined with the electron microscope. Its pyrenoid is characterized structurally as being permeated by interconnected vesiculate membrane systems associated with osmiophilic globules termed pyrenoglobuli. The variety of membrane structure associated with the pyrenoid has been documented with electron micrographs. A model based on serial sections was constructed to show the extent of vesiculation, to demonstrate that the pyrenoglobuli within the pyrenoid matrix are invariably adjacent to the thylakoid membranes within that, matrix, and to emphasize the recurrent thylakoid membrane to pyrenoglobuli relationship. A comparison of the glutaraldehyde-osmium fixation image of the pyrenoglobuli and pyrenoid matrix with the permanganate fixation image has been included. The structure of the pyrenoid matrix in thin section was examined and appears essentially granular.     

Über bandartige Strukturen in den Chromozentren vonLupinus polyphyllus

In the nuclei ofLupinus polyphyllus strongly marked chromocentres occur. In electron micrographs of anther cell nuclei these chromocentres appear either as a homogeneous network or subdivided into two distinct regions, a network-like (NR) and a banded one (GR). In both a 100 Å fiber is the basic unit. The GR is composed of 4 to 6 parallely arranged electron dense bands (240–280 Å wide) and interbands (260–300 Å wide) of low electron density. They appear to correspond to a cylindrical structure with disc-like components and connective sections. These observations are discussed in relation to chromosome structure during interphase and mitosis.

Herrn Prof. Dr. L.Geitler zur Vollendung seines 80. Lebensjahres gewidmet.     

W10BSmL, a mutant of Euglena gracilis var. bacillaris lacking plastids

Organized proplastid structures are absent from dark-grown and light-grown cells of Euglena gracilis Klebs var. bacillaris Cori mutant W10BSmL, based on electron micrographs of serial sections of entire cells. Fluorescence due to normal plastid DNA is undetectable in these cells after treatment with the DNA fluorochrome 4'6-diamidino-2-phenylindole (DAPI). Serial sections through a newly described compartmentalized osmiophilic structure in Euglena cells are presented.     

Image reconstruction using electron micrographs of insect flight muscle. Use of thick transverse sections to supplement data from tilted thin longitudinal sections.

Three-dimensional reconstruction using electron micrographs of thin sections is a powerful technique for determining cross-bridge structure. Tilt restrictions in the electron microscope prevent data collection beyond tilt angles of 60 degrees, giving rise to a "missing cone" of transform data. We show here how much of this data can be obtained using micrographs of thick transverse sections, and the effect this data has on reconstructed images of the insect flight muscle MYAC layer. As a byproduct, the analysis showed that section thinning resulting from prolonged electron irradiation had occurred in the thin longitudinal section used for the previously published MYAC layer reconstruction (Taylor et al., 1984). Comparison of projection density maps calculated from the thin longitudinal section reconstruction and the thick section data show that the data within the missing cone that is not accessible by tilting sharpens the boundaries of the components, flattens the density profile across the thick filament, and enlarges the molecular envelope of the thin filament. We conclude that the reconstructed images of the MYAC layer provide a picture of the structural principles underlying the system but that transform data within the missing cone are necessary to describe accurately the envelopes and profiles of these structural elements.     

Gap junction structures. VIII. Membrane cross-sections.

Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.     

Steam pretreatment of lignocellulosic material for enhanced enzymatic hydrolysis

Pretreatment methods were compared with steam explosion, and differing views on the relative importance of mechanical and chemical effects were outlined. Hydrolysis was desirable; pyrolysis was undesirable. The effects of initial moisture content on steam consumption, mechanism and rate of heat transfer, pentosan solubilization, and subsequent glucose yield were summarized. The insignificant effect, after treatment at 240 degrees C, of 90% pressure bleed-down before explosion on subsequent simultaneous saccharification and fermentation (SSF) yields was described. Treatment at 190 degrees C with complete bleed-down (no explosion), when compared with that at 240 degrees C with explosion from full pressure, showed at least as good solubilizatoin of pentosan, enzymatic hydrolysis, and SSF but showed greater pentosan destruction for the same degree of pentosan removal. Water washing of unexploded steamed aspenwood chips was at least as efficient as that of similarly treated but exploded chips. Scanning electron micrographs of unexploded chips showed extensive rupturing of vessel pit membranes and other morphological features associated with steam-exploded wood. Neither the explosion nor the high temperatures (above 190 degrees C) are necessary.     

The Z lattice in canine cardiac muscle

Filtered images of mammalian cardiac Z bands were reconstructed from optical diffraction patterns from electron micrographs. Reconstructed images from longitudinal sections show connecting filaments at each 38-nm axial repeat in an array consistent with cross-sectional data. Some reconstructed images from cross sections indicate two distinctly different optical diffraction patterns, one for each of two lattice forms (basket weave and small square). Other images are more complex and exhibit composite diffraction patterns. Thus, the two lattice forms co-exist, interconvert, or represent two different aspects of the same details within the lattice. Two three-dimensional models of the Z lattice are presented. Both include the following features: a double array of axial filaments spaced at 24 nm, successive layers of tetragonally arrayed connecting filaments, projected fourfold symmetry in cross section, and layers of connecting filaments spaced at intervals of 38 nm along the myofibril axis. Projected views of the models are compared to electron micrographs and optically reconstructed images of the Z lattice in successively thicker cross sections. The entire Z band is rarely a uniform lattice regardless of plane of section or section thickness. Optical reconstructions strongly suggest two types of variation in the lattice substructure: (a) in the arrangement of connecting filaments, and (b) in the arrangement of units added side-to-side to make larger myofilament bundles and/or end-to-end to make wider Z bands. We conclude that the regular arrangement of axial and connecting filaments generates a dynamic Z lattice.     

Superficial Warming of Epoxy Blocks for Cutting of 25-150 μm Sections to be Resectioned in the 40-90 nm Range

An improved method is described in which tissue areas can be initially identified in thick sections by light microscopy and isolated for subsequent ultrathin sections and observation by electron microscopy. This is achieved by embedding in hard Epon which can be sectioned at 25-150 μm on a sliding microtome after softening the blockface by applying a 60-70 C tacking iron to its surface immediately before each section is taken. The thick sections are then mounted on plastic slides to enable light microscopic selection of areas to be observed by electron microscopy. The selected areas are remounted on faced Epon blanks and resectioned at less than 50 nm. This technique makes it possible to obtain thick sections while maintaining an Epon hard enough for good serial ultrathin sections.     

OBSERVATIONS ON THE ASSOCIATION OF HELICAL POLYRIBOSOMES AND FILAMENTS WITH VINCRISTINE-INDUCED CRYSTALS IN EARLE'S L-CELL FIBROBLASTS

Earle's L-929 fibroblasts from cultures treated with 5–10 µg/ml of vincristine sulfate have a large number of eosinophilic, proteinaceous crystals in their cytoplasm. In electron micrographs, large arrays of helical polyribosomes, stacks of Golgi lamellae, and membranes of granular endoplasmic reticulum are seen in the cytoplasm of these cells. "Stalks" of fine granular material, approximately 300 A wide, are often seen in association with the arrays of the helical polyribosomes. In many sections rows of helical polyribosomes and filaments emerging from individual polyribosomes are seen in intimate contact with the crystals. A gradual reduction in the number of crystals and crystal-bearing cells is seen in cultures removed from the drug-containing medium and reincubated in fresh medium. In electron micrographs, these reincubated cells show large aggregates of filamentous material in the cytoplasm, and in many sections filaments are seen in continuity with the crystals.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

The restoration of electron micrographs blurred by drift and rotation

We have investigated the restoration of electron micrographs exhibiting blurring due to drift and rotation. Blurring due to drift arises in micrographs taken of a specimen which is moving relative to the image plane. A related problem is that of rotational blurring which arises in micrographs of thin sections of helical particles viewed in cross section. The twist of the particle within the finite thickness of the section causes the image to appear rotationally blurred about the helical axis. Restoration algorithms were evaluated by applying them to the restoration of blurred model images degraded by additive Gaussian noise. Model images were also used to investigate how an incorrect estimate of the point spread function describing the blur would effect the restoration. Images were, if necessary, geometrically transformed to a space in which the point spread function of the blur can be considered as linear and space invariant as, under these conditions, the restoration algorithms are greatly simplified. In the case of the rotationally blurred images this procedure was accomplished by transforming the image to polar coordinates. The restoration techniques were successfully applied to blurred micrographs of bacteriophage T4 and crystals of catalase. The quality of the restoration was judged by comparisons of the restored images to undegraded images. Application to micrographs of rotationally blurred cross sections of helical macrofibers of sickle hemoglobin resulted in a reduction in the amount of rotational blurring.     

An electron microscopic study of the intestinal villus. II. The pathway of fat absorption

The intestinal pathway for absorbed fat was traced in thin sections of intestinal villi from rats fed corn oil by stomach tube after a fast of 24 to 40 hours. For electron microscopy the tissues were fixed in chilled buffered osmium tetroxide and embedded in methacrylate. For light microscopy, other specimens from the same animals were fixed in formal-calcium, mordanted in K(2)Cr(2)O(7), and embedded in gelatin. Frozen sections were stained with Sudan black B or Sudan IV. About 20 minutes after feeding, small fat droplets (65 mmicro maximal diameter) appear in the striated border between microvilli. At the same time fat particles are seen within pinocytotic vesicles in the immediately subjacent terminal web. In later specimens the fat droplets are generally larger (50 to 240 mmicro) and lie deeper in the apical cytoplasm. All intracellular fat droplets are loosely enveloped in a thin membrane, the outer surface of which is sometimes studded with the fine particulate component of the cytoplasm. This envelope, apparently derived from the cell surface by pinocytosis, has at this stage evidently become a part of the endoplasmic reticulum. Just above the nucleus numerous fat droplets lie clustered within the dilated cisternae of the Golgi complex. As absorption progresses fat droplets appear in the intercellular spaces of the epithelium, in the interstitial connective tissue spaces of the lamina propria, and in the lumen of the lacteals. All of these extracellular fat droplets are devoid of a membranous envelope. The picture of fat absorption as reconstructed from these studies involves a stream of fat droplets filtering through the striated border, entering the epithelial cell by pinocytosis at the bases of the intermicrovillous spaces, and coursing through the endoplasmic reticulum to be discharged at the sides of the epithelial cell into extracellular spaces. From the epithelial spaces, the droplets move into the lamina propria and thence into the lymph. If the lumen of the endoplasmic reticulum is considered as continuous with the extracellular phase, then the entire pathway of fat absorption may be regarded as extracellular. However, it is impossible to evaluate from the electron microscopic evidence thus far available the quantitative importance of particulate fat absorption by the mechanism described.     

LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI : I. DNA and Proteins

If thin sections of Escherichia coli, labeled uniformly with tritium, are radioautographed calculations, based on the distribution of section sizes show that the number of H³ decays per section should be very close to a Poisson distribution. We might, therefore, expect that the distribution of radioautographic grain counts among random cross-sections should follow a Poisson distribution. It can then be inferred that a deviation from a Poisson indicates a high concentration of label in a preferred region. This region can then be identified by analysis of serial section and comparison with electron micrographs. Sections of cells labeled with leucine-H3 gave a Poisson distribution of grain counts, and it was concluded that proteins were distributed fairly uniformly throughout the cell. The situation was not changed if labeled cells were placed in chloramphenicol or if very short pulses of label were used. When Escherichia coli is grown in presence of chloramphenicol a major morphological change concerns the nuclear region: it becomes more regular in outline, nearly spherical, and occupies a smaller proportion of the cell length. The previously described association between DNA labeled with thymidine-H3 and the nuclear region was confirmed by showing that the distribution of the label in the cell followed exactly the morphological changes of the nuclear region. It was also shown that the concentration of DNA in the nuclear region was at least 45 times higher than that of the cytoplasm. Several morphological features of cells grown in chloramphenicol and examined in the electron microscope are discussed.