scholarly journals Subunit structure of junctional feet in triads of skeletal muscle: a freeze-drying, rotary-shadowing study.

1984 ◽  
Vol 99 (5) ◽  
pp. 1735-1742 ◽  
Author(s):  
D G Ferguson ◽  
H W Schwartz ◽  
C Franzini-Armstrong
Keyword(s):  
Three Dimensional ◽  
Freeze Fracture ◽  
Subunit Structure ◽  
Thin Sections ◽  
Large Structures ◽  
Foot Structure ◽  
Freeze Dried ◽  
Thin Sectioning ◽  
En Face ◽  
A Subunit

Isolated heavy sarcoplasmic reticulum vesicles retain junctional specializations (feet) on their outer surface. We have obtained en face three-dimensional views of the feet by shadowing and replicating the surfaces of freeze-dried isolated vesicles. Feet are clearly visible as large structures located on raised platforms. New details of foot structure include a four subunit structure and the fact that adjacent feet do not abut directly corner to corner but are offset by half a subunit. Feet aligned within rows were observed to be rotated at a slight angle off the long axis of the row creating a center-to-center spacing (32.5 nm) slightly less than the average diagonal of the feet (35.3 nm). Comparison with previous information from thin sections and freeze-fracture showed that this approach to the study of membranes faithfully preserves structure and allows better visualization of surface details than either thin-sectioning or negative-staining.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

An inexpensive water volume control device for ultramicrotomy

1990 ◽  
Vol 48 (3) ◽  
pp. 732-733
Author(s):  
E.G. Kokko ◽  
B. Gowen ◽  
C.R. Jahnke
Keyword(s):  
Three Dimensional ◽  
Control Device ◽  
Volume Control ◽  
Water Volume ◽  
Thin Sections ◽  
Precise Control ◽  
Transmission Electron ◽  
Thin Sectioning ◽  
Main Disadvantage ◽  
Diamond Knife

Numerous techniques and related devices have been reported for preparing serial thin-sections with an ultramicrotome for transmission electron microscopy. The section pickup method, a continuous serial thin-sectioning technique developed by Farenbach (1984), is helpful for: (a) three-dimensional EM reconstruction (Todd et.al. 1989) and (Farenbach 1984), (b)section thickness determinations of thick (LM) or thin (EM) sections using the “section piling - resectioning” technique of Sakai (1980), (c) insuring that important or rare EM sections (such as from a reembedded 0.5 urn LM section) are picked up, or (d) for routine section pickup on formvar-coated slot grids. Farenbach’s technique makes use of a pedestal, located within a diamond knife trough, to hold the slot grids. It calls for a peristaltic pump to maintain precise control of the water level within the trough. The main disadvantage of his technique is the relatively high cost (approx. $ 500 US) for a pump capable of these accurate and two-way flow adjustments of small volumes of liquid.

The interpretation of granitic textures from serial thin sectioning, image analysis and three-dimensional reconstruction

1995 ◽  
Vol 59 (395) ◽  
pp. 203-211 ◽  
Author(s):  
D. N. Bryon ◽  
M. P. Atherton ◽  
R. H. Hunter
Keyword(s):  
Image Analysis ◽  
Three Dimensional ◽  
Three Dimensional Reconstruction ◽  
Mineral Phase ◽  
Thin Sections ◽  
Thin Sectioning ◽  
Potential Problems ◽  
Dimensional Reconstruction ◽  
Textural Development

AbstractTextural development of the felsic phases in two granodioritic rocks from the zoned Linga superunit of the Peruvian Coastal Batholith has been characterized using serial thin sectioning, image analysis and three-dimensional reconstruction. The study has shown how each mineral phase contributed to the texture during the formation and development of a contiguous crystal framework and during subsequent restriction, isolation and occlusion of the melt-filled porosity. The work highlights the important factors and potential problems in the use of serial thin sections and imaging in the analysis of complex polyphase rock textures.

scholarly journals Analysis of the structure of intramembrane particles of the mammalian urinary bladder.

1980 ◽  
Vol 86 (2) ◽  
pp. 514-528 ◽  
Author(s):  
J D Robertson ◽  
J Vergara
Keyword(s):  
Urinary Bladder ◽  
Glass Surface ◽  
Hexagonal Lattice ◽  
Freeze Fracture ◽  
Transitional Epithelium ◽  
Thin Sections ◽  
Intramembrane Particles ◽  
Thin Sectioning ◽  
Space Constant ◽  
Dominant Structure

The luminal and discoid vacuole membranes of the superficial cell layer of the transitional epithelium of the mammalian urinary bladder have been studied by thin-sectioning and freeze-fracture-etch (FFE) electron microscope methods. For the FFE studies membranes were deposited on a cationized glass surface, covered by a thin copper disc, and fractured under liquid N2. Specimens were etched at -100 degrees C and replicated at -190 degrees C. A model of the lattice membrane derived from thin sections was used to predict the heights of the fracture faces above the glass surface. A hexagonal pattern of globular intramembrane particles spaced 160 A apart was seen in the external fracture (EF) face plaques as previously described and regarded as the dominant structure. However, very extensive areas of another pattern, seen before in only limited areas, have beeen found in the EF faces. The pattern consists of a smooth hexagonal lattice with the same space constant as the globular one but a different structure. By image analysis it consists of overlapping domains bordered by shared but incomplete metal rims. Each domain has a central spot of metal encircled by a shadow. The surface of the smooth lattice is partly complementary to the corresponding protoplasmic fracture (PF) face which shows a similar hexagonal lattice with the same space constant. The height of the smooth EF lattice above the glass substrate is the same as the plane of the center of the lipid bilayer predicted by the model. The mean heights of the particles of the globular EF lattice are greater than the total thickness of the membrane as predicted by the model and confirmed by measurements. The globular EF lattice is not complementary and it is concluded that the globular particles do not exist in the native membrane but arise artifactually during the preparatory procedures.

Thin sectioning, freeze fracturing, energy dispersive x-ray analysis, and chemical analysis in the study of inclusions in seed protein bodies: almond, Brazil nut, and quandong

1978 ◽  
Vol 56 (17) ◽  
pp. 2050-2061 ◽  
Author(s):  
John N. A. Lott ◽  
Mark S. Buttrose
Keyword(s):  
Chemical Analysis ◽  
Freeze Fracture ◽  
Prunus Dulcis ◽  
Energy Dispersive ◽  
Protein Bodies ◽  
Brazil Nut ◽  
Fixed Tissue ◽  
Thin Sections ◽  
X Ray ◽  
Freeze Dried

Protein bodies from almond (Prunus dulcis), Brazil nut (Bertholletia excelsa), and quandong (Santalum acuminatum) have been studied in thin sections of fixed and embedded tissue, in freeze-fracture replicas of unfixed tissue, by chemical analysis of tissue for P, K, Mg, and Ca, and by energy dispersive x-ray (EDX) analysis of both sections of glutaraldehyde-fixed tissue and freeze-dried tissue powders. The protein bodies in all three species contained globoid crystals, protein crystalloids, and proteinaceous matrix regions. Results of EDX analyses were consistent with globoid crystals being rich in phytin. Variation in both the structure and the elemental composition of globoids was common. In almond some globoids were lobed rather than spherical, and large globoid crystals often contained considerable calcium whereas small globoid crystals contained little if any calcium. The globoid crystals of Brazil nut often contained barium in addition to P, K, Ca, and Mg. Protein crystalloids of Brazil nut were compound crystals. Protein bodies of quandong seed, which is largely endosperm rather than embryo, were unexceptional.

scholarly journals Definitive Evidence for the existence of tight junctions in invertebrates

1980 ◽  
Vol 86 (3) ◽  
pp. 765-774 ◽  
Author(s):  
NJ Lane ◽  
HJ Chandler
Keyword(s):  
Tight Junctions ◽  
Freeze Fracture ◽  
Permeability Barrier ◽  
Thin Sections ◽  
Definitive Evidence ◽  
Common Precursor ◽  
En Face ◽  
Spider Central Nervous System ◽  
Exogenous Compounds ◽  
Colloidal Lanthanum

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.

The extrarhabdomeral cytoskeleton in photoreceptors of Diptera. II. Plasmalemmal undercoats

1984 ◽  
Vol 220 (1220) ◽  
pp. 353-359 ◽  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Genetic Analysis ◽  
Freeze Fracture ◽  
Integral Membrane Proteins ◽  
Basal Region ◽  
Thin Sections ◽  
En Face ◽  
Thiol Proteases ◽  
Planar Membranes

The plasmalemmal undercoats of those regions of the photoreceptors of the blowfly Lucilia that flank the central extracellular space of each ommatidium are described from en face and transverse thin sections. Labile structures were stabilized before fixation for electron microscopy by using an inhibitor of thiol proteases, Ep-475, as described in the previous paper (Blest et al., Proc. R. Soc. Lond . B 220, 339-352, 1984). Membranes of R 1-6 are underlain by a closely associated, randomly organized filamentous meshwork. That of the basal region of R 7 is highly organized, and consists of very long, about 8 nm filaments running parallel to each other and to the longitudinal ommatidial axis; these ‘backbone’ filaments are tightly adherent to the plasma membrane, and are spaced some 190-200 nm apart. They are linked by abundant transverse filaments that form a reticulum between them. The degree of ordering of the reticulum in life is not clear, but some well-preserved profiles suggest that it may be high. Replicas obtained by the freeze-fracture technique show that extrarhabdomeral membranes have dense populations of intramembrane particles, just as they do in Drosophila where a genetic analysis has shown them to consist largely of rhodopsin. It is proposed as a working hypothesis that these planar membranes can be regarded as flat equivalents of the microvillar membranes, that some fraction of the integral membrane proteins may be immobilized by bonding to the plasmalemmal undercoat, and that the latter may help to constrain both the translational and rotational movements of rhodopsin molecules.

scholarly journals A freeze-fracture and lanthanum tracer study of the complex junction between Sertoli cells of the canine testis.

1978 ◽  
Vol 76 (1) ◽  
pp. 57-75 ◽  
Author(s):  
C J Connell
Keyword(s):  
Tight Junctions ◽  
Sertoli Cells ◽  
Freeze Fracture ◽  
Intercellular Junctions ◽  
Septate Junction ◽  
Septate Junctions ◽  
Thin Sections ◽  
En Face ◽  
Lanthanum Tracer ◽  
Complex Junction

What appear to be true septate junctions by all techniques currently available for the cytological identification of intercellular junctions are part of a complex junction that interconnects the Sertoli cells of the canine testis. In the seminiferous epithelium, septate junctions are located basal to belts of tight junctions. In thin sections, septate junctions appear as double, parallel, transverse connections or septa spanning an approximately 90-A intercellular space between adjacent Sertoli cells. In en face sections of lanthanum-aldehyde-perfused specimens, the septa themselves exclude lanthanum and appear as electron-lucent lines arranged in a series of double, parallel rows on a background of electron-dense lanthanum. In freeze-fracture replicas this vertebrate septate junction appears as double, parallel rows of individual or fused particles which conform to the distribution of the intercellular septa. Septate junctions can be clearly distinguished from tight junctions as tight junctions prevent the movement of lanthanum tracer toward the lumen, appear as single rows of individual or fused particles in interlacing patterns within freeze-fracture replicas, and are seen as areas of close membrane apposition in thin sections. Both the septate junction and the tight junction are associated with specializations of the Sertoli cell cytoplasm. This is the first demonstration in a vertebrate tissue of a true septate junction.

Ultrastructural Investigations of the Contractile Filaments of Amoebae

1974 ◽  
Vol 32 ◽  
pp. 188-189
Author(s):  
P.L. Moore
Keyword(s):  
Cytoplasmic Streaming ◽  
Three Dimensional ◽  
Freeze Fracture ◽  
Amoeboid Movement ◽  
Different Types ◽  
Chemical Conditions ◽  
Complete Interpretation

Previous freeze fracture results on the intact giant, amoeba Chaos carolinensis indicated the presence of a fibrillar arrangement of filaments within the cytoplasm. A complete interpretation of the three dimensional ultrastructure of these structures, and their possible role in amoeboid movement was not possible, since comparable results could not be obtained with conventional fixation of intact amoebae. Progress in interpreting the freeze fracture images of amoebae required a more thorough understanding of the different types of filaments present in amoebae, and of the ways in which they could be organized while remaining functional.The recent development of a calcium sensitive, demembranated, amoeboid model of Chaos carolinensis has made it possible to achieve a better understanding of such functional arrangements of amoeboid filaments. In these models the motility of demembranated cytoplasm can be controlled in vitro, and the chemical conditions necessary for contractility, and cytoplasmic streaming can be investigated. It is clear from these studies that “fibrils” exist in amoeboid models, and that they are capable of contracting along their length under conditions similar to those which cause contraction in vertebrate muscles.

Lead aspartate: a new en bloc stain for electron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 34-35
Author(s):  
J.R. Walton
Keyword(s):  
Electron Microscopy ◽  
Calcium Ion ◽  
Enzyme Reaction ◽  
Lead Citrate ◽  
Reaction Products ◽  
En Bloc ◽  
Thin Sections ◽  
Lead Salts ◽  
Thin Sectioning ◽  
High Alkalinity

In electron microscopy, lead is the metal most widely used for enhancing specimen contrast. Lead citrate requires a pH of 12 to stain thin sections of epoxy-embedded material rapidly and intensively. However, this high alkalinity tends to leach out enzyme reaction products, making lead citrate unsuitable for many cytochemical studies. Substitution of the chelator aspartate for citrate allows staining to be carried out at pH 6 or 7 without apparent effect on cytochemical products. Moreover, due to the low, controlled level of free lead ions, contamination-free staining can be carried out en bloc, prior to dehydration and embedding. En bloc use of lead aspartate permits the grid-staining step to be bypassed, allowing samples to be examined immediately after thin-sectioning.Procedures. To prevent precipitation of lead salts, double- or glass-distilled H20 used in the stain and rinses should be boiled to drive off carbon dioxide and glassware should be carefully rinsed to remove any persisting traces of calcium ion.

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