bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyethylene glycol (PEG) embedding and subsequent de‐embedding as a method for the structural and immunocytochemical examination of biological specimens by electron microscopy
Authors: Kondo Hisatake
Published: February 2005

A simple and reliable method to make resinless sections for electron microscopy was recently developed by using polyethylene glycol (PEG) as a transient embedding media. In this paper the practical procedure of this PEG method is described in detail. Normal ultrastructure of several types of in‐situ cells in resinless sections is demonstrated. The cytoplasmic matrix of all in‐situ cells examined is revealed to consist of the microtrabecular lattice. A result from application of this technique to immuno‐electron microscopy is also illustrated. This method is shown to have potential in overcoming the problem of intracellular penetration of macromolecular antibodies. Several artifacts caused by failures in specimen preparations are displayed. The real or artifactual nature of the microtrabecula is briefly discussed.
Zone‐axis diffraction patterns by the “Tanaka” method
Authors: Eades J. A.
Published: February 2005

The “Tanaka” method is one of several techniques that make it possible to obtain zone‐axis electron diffraction patterns in a transmission electron microscope without the restriction in the field of view that limits normal convergent‐beam diffraction patterns.

The method employs a convergent‐beam of electrons focused to a probe in a plane that does not coincide with the specimen. The selected area aperture can then be used to eliminate all but one of the diffracted beams to obtain the desired pattern. Practical details of operation and values of operating parameters are discussed.

The Tanaka method is a useful addition to the techniques available to the electron microscopist, especially since no instrumental modification is required.
An iodized mounting medium for coal particles
Authors: Gomez C. O. , Strickler D. W. , Austin L. G.
Published: February 2005

A high electron‐density embedding medium was developed for SEM observation of inorganic constituents of organic or carbonaceous particles. The components used are a common epoxy resin in which iodoform is dissolved before the addition of the hardener. An iodoform content of 10% by weight proved satisfactory for obtaining excellent contrast between the matrix and embedded carbonaceous particles in the SEM. The system has been successfully applied in the preparation of polished specimens of coal particles. There is no interference between the iodine and any of the most abundant or most important coal mineral components, but it was found that the epoxy resin contained chlorine as a contaminant.
Parallax measurements on stereomicrographs of hydrated single molecules: Their accuracy and precision at high magnification
Authors: Ruben George C. , Marx Kenneth A.
Published: February 2005

Stereoimaging of hydrated single complex macromolecules requires thin freeze‐etch platinum‐carbon replicas (⩽200 Å) and that the transmission electron microscope (TEM) be equipped with a tilt‐rotation eucentric goniometer stage. The original parallax equation is an accurate approximation for high‐magnification work, micrographs (105 ×) being less than 0.3% in error. In addition, we have derived formulas for high‐magnification work to measure heights, lateral distances, and the object tilt angle for an object not lying flat on the film surface. The accuracy of the height measurements is evaluated on spermidine‐condensed DNA toruses. By using the maximum error equation derived from the original parallax equation, we discuss methods to improve the height measurement precision (95% fractile) to the 5–10 Å range.
A microcomputer program in “BASIC” for the accurate determination of the height of small particles
Authors: Lea P. J.
Published: February 2005

A formula is derived to enable the calculation of the true height of an object, such as a shadowed latex bead, from electron micrographs. Knowing only the angle of shadowing and the length of the evaporated shadow, and by substituting these values in the derived formula, a microcomputer may be programmed to carry out the necessary computations. An example of such a microcomputer program is given. The correct determination of the height of particles by electron microscopy using the shadowing technique is one of the most accurate methods available for the determination of small particle height.
A study of living plant specimens by low‐temperature scanning electron microscopy
Authors: Kaneko Yasuko , Matsushima Hisashi , Wada Masao , Yamada Mitsuhiko
Published: February 2005

Young fresh Tradescantia reflexa stamen hair cells were used to clarify the optimal conditions for direct viewing and taking photographs with a scanning electron microscope (SEM) equipped with a cryo‐system. The rate of protoplasmic streaming in the cells was measured under an optical microscope after examining and photographing them in the SEM over a period of a few minutes. Almost the same rate of streaming (5.5 μm/second, 20°C) was observed in nonirradiated control cells and irradiated cells photographed in the SEM using an accelerating voltage of 10 kV with the cryo‐stage at a temperature of – 15°C. (The specimen holder and specimen were not at this temperature, but, rather, probably somewhat higher.) Fresh plant organs, tissues, and cells were also tested under the same conditions. The fine structure was well preserved in detail. The procedures were as follows: (1) prompt attachment of fresh plant materials on an aluminum specimen holder with double‐faced adhesive Scotch tape or a small amount of plastic adhesive for woodcraft; (2) setting the holder on the cryo‐stage cooled to –15°C in advance and rapid evacuation; and (3) quick SEM examination and photography (within several minutes). The advantages of this method are summarized as follows: (1) high possibility of viewing living materials; (2) minimal artifacts: freedom from chemical fixation and additional procedures utilized in ordinary SEM specimen preparation; and (3) simplicity, speediness, and economy in preparation for viewing. Since the specimens were not likely to be frozen during quick examination and photography, this method might well be called “low‐temperature SEM” (LT‐SEM) as distinguished from “cryo‐SEM”.
Sectioning spherical aluminum oxide particles for transmission electron microscopy
Authors: Tucker Dennis S. , Jenkins Elijah J. , Hren John J.
Published: February 2005

A technique was developed to aid in the interpretation of nucleation and growth characteristics of otherwise electron‐opaque Al2O3 particles. The technique involved embedding a dense collection of particles in an Epon mixture and sectioning with a diamond knife on an ultramicrotome. Serial sections were utilized to obtain three‐dimensional information. It was found that ultramicrotomy does not introduce artifacts in ceramics which would otherwise affect measurements of the crystallite size or phase fraction, nor does it affect the crystallographic data significantly.

It was observed that α‐Al2O3 nucleated at the surface of isolated particles and in the neck region of sintered particles. Other data, obtained from diffraction and in situ hot‐stage studies, indicated that each polycrystalline α‐Al2O3 particle transformed to a nearly single crystal of γ‐Al2O3, which extended into adjacent particles under favorable circumstances.
The fatty acid monolayer technique for preparing frozen‐hydrated specimens
Authors: Chang Chung‐Fu , Ohno Tamotsu , Glaeser Robert M.
Published: February 2005

A major difficulty in preparing frozen‐hydrated specimens from solutions is to control an optimal thickness of water remaining on the EM grid just before it is immersed in liquid nitrogen. In this report, we describe a modification of the fatty acid monolayer technique for preparing frozen‐hydrated specimens. With the use of this technique, all excess solvent is removed when a behenic acid (a fatty acid) monolayer film is picked up over the surface of the electron microscope grid. Low‐dose electron diffraction patterns and optical diffraction patterns of low‐dose micrographs of bacteriorhodopsin (bR) of Halobacterium halobium thus prepared show strong reflection orders at a resolution of ∽4 Å and ∽10 Å, respectively. Because the preparation of thin, hydrated specimens by the behenic acid monolayer technique does not depend upon evaporation of water, a dramatic change of ion concentration and pH is not expected to occur. The method should be applicable to specimens which need to be kept in a specific buffer and/or at a specific ionic strength.
A technique for controlling the placement of the thin area in electrojetpolished tem foils of tungsten
Authors: Fekete J. R. , Gibala R.
Published: February 2005

Recently we have been making extensive use of TEM observations of grain boundaries in a research program concerned with the deformation behavior of tungsten bicrystals. A major problem in this research involved the preparation of thin foils which contained the grain boundary in the electron tranparent region, since standard cell type electrojetpolishing techniques do not allow adequate control of the placement of the thin area. Since the grain boundary comprises a very small percentage of a foil, good thin area placement control is critical to obtaining useful samples consistently. Masking techniques which use electrically insulating lacquers are not precise enough to guarantee the position of the thin area. However, we have found that one can use the dishing characteristic of the jetpolisher (i.e., the fact that the center of the foil thins faster than the perimeter) in conjunction with the masking technique to predict the position of the thin area within a preferentially thinned region created by the masking procedure outlined below. This is the essence of the procedure outlined below.
Protein casting: A new replication technique for observing fluid surface structures
Authors: Garber M. A. , McManus W. , Bennett B. , Hunter R. L.
Published: February 2005

Certain insoluble copolymers form characteristic liquid crystalline structures at water/copolymer interfaces. The formation of these structures, discernable with phase contrast microscopy, correlates with biological activities as immunological adjuvants. In order to study such structures at the electron microscopic level, it was necessary to develop a preparation technique which preserved the water/polymer interface. A technique was devised, here called “protein casting,” which uses highly concentrated bovine serum albumin (BSA) in water, subsequently fixed with glutaraldehyde, to form a replica of the water/polymer interface. These casts appeared highly accurate at low magnifications, and resolved to less than 1ønm. This technique seems ideal for accurate replication of surfaces formed by nonmiscible liquids and should prove helpful in studies of other materials in aqueous suspension.
An image processing method for scanning electron microscopy based on the information transmission theory
Authors: Kanaya Koichi , Oho Eisaku , Naka Michiaki , Koyanagi Takehiko , Sasaki Toshihide
Published: February 2005

Based on the information transmission theory, topographic image signals in scanning electron microscopy are used to evaluate contrast, gradient, acutance, and Laplacian operator, the total of which represent the image sharpness of an edge line.

One may consider the impulse and step functions as an input to the Gaussian system function of a low‐pass filter, the impulse and step response functions possibly representing a single spot and image contrast of an edge profile, respectively. It is shown that the response function of acutance defined as the power of the gradient normalized by density is a more realistic representation of image edge sharpness. Also, edge sharpness can be greatly enhanced by utilizing the Laplacian operator through digital image processing for a disk specimen model with a rounded edge.

Contrast increased by specimen tilt, and an edge effect due to side‐scattered electrons, as well as the signal attenuation by specimen collection, are consistently obtained as the response function in the system.

The exact measurement of spot size and edge‐to‐edge resolution, and image sharpness improvement, are derived by digital image processing.
Aldehyde fixatives: Quantification of acid‐producing reactions
Authors: Johnson Timothy J. A.
Published: February 2005

Under conditions commonly used for the preservation of tissue for electron microscopy, substantial amounts of hydrogen ions were produced when either formaldehyde or glutaraldehyde was added to solutions of amines or proteins, or to tissue homogenates. In glycine‐aldehyde reactions, hydrogen ions were generated in linear proportion to the glycine concentration over the range of 0.001 M to 0.100 M glycine. In the presence of 1% formaldehyde or 1% glutaraldehyde, 0.3–0.9 equivalents of acid were produced per mole of primary amine. This variation is a function of the specific primary amine used. Acid production increased with increasing formaldehyde concentration but did not increase appreciably with increasing glutaraldehyde concentration. The concentration of amines in tissue is high. These amines are probably responsible for much of the acid generated when aldehydes react with tissue homogenates. In amine‐aldehyde reactions, the positive charge originally associated with the parent amine is simultaneously neutralized with the formation of hydrogen ions. Thus, in tissues exposed to aldehydes, there is a decrease in the overall positive charge which is related to the amount of acid generated. The inherent buffering capacity of liver and muscle was not sufficient to prevent large pH decreases when aldehydes were added to homogenates of these tissues. These data suggest that significant but transient pH decreases may occur within cells exposed to aldehydes. To minimize the aldehyde‐induced pH decreases, a buffer should be chosen so that its pKa is 0.2–0.3 pH units less than the pH desired for a particular experiment. In addition, buffer concentrations of at least 0.10 M are suggested for fixatives in order to provide adequate buffering capacity during tissue preservation.
Motorization of the dupont‐sorvall mt‐1 ultramicrotome
Authors: Caceci Thomas
Published: February 2005

A Dupont‐Sorvall MT‐1 ultramicrotome, normally a manually operated instrument, was equipped with a small motor. Ultrathin sections were successfully cut at rates from one to five sections per minute. This lowcost conversion to automatic operation offers an alternative to the purchase of a new ultramicrotome, as well as making a classic instrument more flexible.
Direct determination of ultrathin section thickness using latex particles
Authors: Jésior Jean‐Claude
Published: February 2005

A method is proposed to determine ultrathin section thickness by means of noncentral sections obtained from latex spheres. The resulting spherical segments provide the parameters necessary to calculate very simply the section thickness without further treatment. The method is illustrated by an example using the grid‐sectioning technique and could be generalized to classical microtomy.
Tracing axons in the human brain: A method utilizing light and TEM techniques
Authors: Sadun Alfredo A. , Schaechter Judith D.
Published: February 2005

The neuroanatomy of experimental animals has been investigated through a variety of histological and physiological techniques. Recently, more sophisticated methods have permitted a better understanding of the complex connections of animal brains. In contrast, the methods available for the direct examination of the human brain have remained relatively primitive.

We have developed a staining method (paraphenylene‐diamine method: PPD) which bridges the techniques of light and electron microscopy. The method permits the tracing of degenerated fibers in the human brain even after very long survival periods. With this method we have documented several visual pathways not previously described in man. We have also demonstrated, in several species, that products of axonal degeneration remain far longer than previously supposed. The PPD technique is relatively simple, reliable, provides high resolution, works in human brains, and can be used in conjunction with TEM.
Complementary freeze fracture replication: An example of its use in the study of horizontal cell gap junctions of the carp retina
Authors: Tonosaki Akira , Washioka Hiroshi , Nakamura Hidenori , Negishi Koroku
Published: February 2005

Dark‐adapted carp retinas were fixed with glutaraldehyde, frozen in liquid nitrogen slush, and their complementary replicas were harvested using a bipolar‐vacuum evaporater. On montage pictures, plasmic (PFF) and ectoplasmic fracture faces (EFF) of the external horizontal cells (EHC) were marked, and PFF of gap junction (GJ) areas were scrutinized by virtue of particulate aggregation visible at relatively low magnification. Summation of digitized values obtained from a complementary pair of replicas was taken to represent all existent GJ areas in an examined EHC surface. A similar method was applied to large horizontal processes (LHP) that are located in the inner‐most part of the internal nuclear layer and currently accepted to be extensions of EHC. The occurrence of fractured faces of plasma membrane was smaller in EHC than LHP. The percentage of GJ areas was about ten times larger in EHC than LHP. Examining complementary replicas at relatively low magnification appears to be useful for facilitating quantitative analysis of intercellular coupling by GJ structures.
A method of direct particle deposition on a carbon planchet to study mineral dust in human lung by scanning electron microscopy
Authors: Kimizuka Goro , Wang Nai‐San
Published: February 2005

Following Na‐hypochlorite digestion of lung tissue, mineral particles extracted in the chloroform layer were deposited directly on a pre‐smoothed carbon planchet for combined scanning electron microscopy and X‐ray energy dispersive spectrometry (SEM and XEDS). Total mineral particle counts were obtained, and detailed physical characteristics of the fibrous particles were documented at 600, 1,500, 4,500 and 9,000 x in three lungs without, and one lung with, histories of occupational exposure. This preparation method was simple, collected more than 99% of identifiable mineral particles in the chloroform layer, gave excellent object to background contrast without heavy metal coatings, and was suitable for XEDS. Comparable fibrous particles from the chloroform layer could also be studied by selected‐area electron diffraction to complement the results of XEDS. By this method, we found particles or fibers larger than 0.1 μm were readily counted and measured at 4,500 x. At 600 x, ferruginous bodies were found to be more than twice in number than when sought for by light microscopy. It was determined that 4,500 x is the most efficient magnification to examine and diagnose this type of specimen. The present study illustrates the importance of determining the most efficient magnification to be utilized in particle counts.
Preparation of fish tissues for electron microscopy
Authors: Olson Kenneth R.
Published: February 2005

Many of the techniques applied to study the morphology of lower vertebrates were originally developed for analysis of mammalian tissues. However, not all of these methods, especially those involving tissue perfusion, can be directly applied to piscine tissues because of the considerable physiological differences between fish and mammals. The present paper examines these physiological variables in fish and describes how they might affect morphological studies that employ tissue perfusion. A method for perfusion of fish tissues is described in detail. This procedure can be used for administration of fixatives, chemical markers, and vascular replicating resins.
A deposition technique for the imaging and analysis of protein interactions with metal and semiconductor surfaces
Authors: Panitz J. A. , Andrews C. L. , Bear D. G.
Published: February 2005

A new technique for placing biological molecules on metal, insulator, and semiconductor surfaces is described. The procedure requires only 10 μl of solution containing molecules at a concentration of 0.1—10 μg/ml. The use of a buffer that does not affect metal substrates, the possibility of fixing the molecules in solution prior to deposition, and the ability to minimize surface tension forces during air drying are other features of the new protocol. Simultaneous deposition on TEM grids and highly curved substrates permits biomolecular adsorption on technologically interesting materials to be visualized in the transmission electron microscope.
A method for abolishing the nonspecific binding artifacts produced by the peroxidase‐antiperoxidase complex (PAP) in immunostained mucous epithelia
Authors: Sowinski Janice M. , Mackay Robert L. , Nicosia Santo V.
Published: February 2005

The presence of nonspecific staining artifacts is a potential problem in ultrastructural immunocytochemistry. In the course of staining for lysozyme in the human and rabbit endocervix, the peroxidase‐antiperoxidase complex (PAP) was found to bind to mucous granules in a selective and nonspecific manner. Sections etched in 10% aqueous H2O2, incubated for 5 minutes in 1:50 PAP in Tris‐buffered saline (TBS) and subsequently treated with 3, 3′‐diaminobenzidine‐H2O2, revealed a staining precipitate within the matrix of mucous granules. The same selective and nonspecific staining could also be visualized when horseradish peroxidase (1 mg/ml) was substituted for PAP in the immunocytochemical sequence. Thus, this staining method made the reliable demonstration of the desired antigen difficult. The affinity of PAP for mucous granules can be completely eliminated by subjecting sections, previously etched in H2O2 to expose the antigenic sites, to a 3‐minute incubation in immunoglobulin (1:10 human IgA, 1:5 human IgG, or 1:5 antihuman IgG) immediately prior to using PAP in the standard immunocytochemical staining sequence. The use of this modification to Sternberger's unlabeled antibody‐enzyme method is recommended because it allows for the elimination of all nonspecific staining artifacts without interfering with specific localization by the primary antibody. The mechanism that causes nonspecific binding of peroxidase to mucous granule constituents is unclear, although carbohydrate binding may play a role in the interaction between mucous granules and peroxidase.
The use of histograms for transmission electron microscopy: Defocus and astigmatism correction at high resolution
Authors: Krakow William
Published: February 2005

A real‐time method for optimizing the defocus of a conventional transmission electron microscope in the phase contrast imaging mode has been investigated using image histogram data. This method can also be used to minimize the objective lens astigmatism. It will be shown both theoretically and empirically, using a digital television frame store, that a histogram will give the largest peak when an image has a broad and flat contrast transfer function. This method has distinct advantages of speed and minimal computational requirements over obtaining the power spectrum of an image.
Real time uses of color for electron microscopy via a television‐rate digital frame store
Authors: Krakow William
Published: February 2005

An on‐line television‐rate digital frame store device is utilized to provide color representations of a wide range of electron microscope images and image data. Various types of hardware devices in the frame store coupled with software manipulations via the host computer make rapid image acquisition, modification, measurement, and full‐color display possible in real time either from micrographs or directly from an electron microscope. Lookup tables used in conjunction with grey‐level image memories can be controlled from a menu display to provide a wide range of color‐coding schemes and sequencing. It is also possible to use color graphics overlays and alpha numeric displays along with full‐color image displays. This paper will describe many of the recent applications of color developed for electron microscopy studies of materials.
Layered crystal thin films
Authors: Hines R. L. , Patterson C. E.
Published: February 2005

A rigid peripheral clamp with an annular heater is used to bond layered crystals to grids with a thermoplastic adhesive. The layered crystals are subsequently cleaved with adhesive tape while bonded to the grid. The final layered crystal thin film covering a grid opening is exposed only to room air and is free from adhesive or fluid contamination. The results of preparation of several layered crystals, including muscovite mica, molybdenite, and talc, are described.
A method for preparing powdered specimens for transmission electron microscopy
Authors: Carr Martin J.
Published: February 2005

A new method of preparing TEM samples from powders is described. The method consists of compacting epoxy‐powder composite mixtures in special molds by centrifuging prior to polymerization. The compact is then ion milled by standard methods. Samples prepared in this way are currently being used to study dislocation structures developed during processing of ceramic powders.
Silver impregnation applied to striated muscle: The sarcoplasmic reticulum
Authors: Scales Donald J. , Yasumura Thomas
Published: February 2005

The classical black reaction developed by Camillo Golgi is shown to impregnate the tubules and fenestrations of the sarcoplasmic reticulum (SR) in striated muscle. This is a double impregnation of chromate and silver, which usually fills extracellular spaces. The method is difficult insofar as long incubation times are required, and location of the successfully “stained” SR in plastic‐embedded tissue blocks is unpredictable. The light microscope is absolutely necessary to find the good regions which can then be cut from the blocks in 1‐μm‐thick sections and examined in the electron microscope. Stereo pairs give the best results since these resolve overlap problems common to thick sections. A variety of artifacts are illustrated which can help avoid erroneous interpretations. The Golgi‐“stained” SR shows this elusive network with unsurpassed contrast and should benefit the morphological studies of muscle‐membrane enthusiasts.
Scanning electron microscopic observations on deembedded biological tissue sections: Comparison of different fixatives and embedding materials
Authors: Russell Scott D. , Daghlian Charles P.
Published: February 2005

Conventional plant histological specimens fixed in formalin‐acetic acid‐alcohol, chromic acid‐acetic acid‐formaldehyde, or glutaraldehyde‐osmium and embedded in either paraffin or plastic are examined as possible rapid methods for providing an alternative image of cellular structure by using scanning electron microscopy. Using the mitotic figures of actively growing onion root tips as a study specimen, the organization of the nucleus and spindle apparatus is reasonably well preserved as compared with isolated mitotic spindles and studies of mitosis in endosperm tissue. Relief of internal structure in this technique is obtained through the coagulant nature of the fixative. Used judiciously, this technique can reveal aspects of the three‐dimensional nature of internal tissue structure that may otherwise be difficult to discern.
The preparation of ultrathin frozen sections for immunocytochemistry at the electron microscope level
Authors: Christensen A. Kent , Komorowski Thomas E.
Published: February 2005

A description of ultrathin frozen sectioning is presented. Small pieces of lightly‐fixed but unembedded tissue are cryoprotected in 80% sucrose, mounted on chucks and frozen in liquid nitrogen. Frozen sections approximately 100 nm thick for electron microscopy are cut at about −90°C, using a cryosectioning unit mounted on a conventional ultramicrotome. Sections are picked up from the surface of the knife with a droplet of 80% sucrose, and are applied to membrane‐coated EM grids at room temperature. The mounted sections are then used for EM immunocytochemistry. Essentially the same sectioning procedure can provide frozen sections 1 μm thick for light microscope immunocytochemistry.
Some characteristics of epoxy embedding resins and how they affect contrast, cell organelle size, and block shrinkage
Authors: Mollenhauer Hilton H. , Droleskey Robert E.
Published: February 2005

The effects of epoxy resin formulation on tissue contrast and organelle size were evaluated by examining thin sections of rat liver in the electron microscope and by doing a simple morphometric analysis of peroxisome and nuclear size. Contrast was greatest for tissues embedded in the Quetol formulation of Kushida and least for tissues embedded in the Ladd formulation. Epon 812, Epon‐Araldite, and Spurr formulations gave results intermediate to those of the Quetol and Ladd formulations. Organelle size was greatest for tissues embedded in the Ladd formulation and smallest for tissues embedded in the Spurr formulation. The maximum size differences were approximately 15%. Epon 812, Epon‐Araldite, and Quetol formulations gave results (for organelle size) intermediate to those of the Ladd and Spurr formulations. The gross shrinkage properties of the epoxy resin blocks were all the same and, therefore, could not account for the differences in organelle size.
A procedure to prepare cross‐sectional samples for TEM
Authors: Rivaud Lydia
Published: February 2005

Cross‐sectional transmission electron microscopy (XTEM) is an imaging technique particularly suited to the study of layered structures. For integrated electronic devices it has become a common practice to use XTEM to assess the shape and crystallinity of component layers as well as defect structures introduced by processing. A procedure for preparing samples to be viewed by XTEM is described.
Identification of bacteria by studying one section under light microscopy, scanning, and transmission electron microscopy
Authors: Saglie F. R. , Sa Ferreira J. C. , Smith C. T. , Valentin P. L. , Carranza F. A. , Newman M. G.
Published: February 2005

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.
Electron beam radiation damage to organic inclusions in ice as an analytical tool for polymer science
Authors: Talmon Yeshayahu , Narkis Moshe , Silverstein Michael
Published: February 2005

A method is proposed to enhance contrast between microdomains of two‐component polymer systems using electron beam radiation damage. The technique is based on the different damage patterns to crosslinking‐type and scission‐type polymer particles embedded in ice. Whereas the former lose little mass and cavities form in the ice around them, the latter lose much more mass, become cellular in appearance, and occasionally swell. Applications of the technique are demonstrated by revealing the microstructure of two‐stage poly(butylacrylate)/polystyrene latices, their size distributions, and the presence of single‐component particles in the two‐component system.
The process of amorphization induced by electron irradiation in alloys
Authors: Fujita Hiroshi
Published: February 2005

Amorphization induced by electron irradiation in Ni–50 at% Ti crystals has been investigated by electron microscopy. In order to clarify successive stages of amorphization, micrographs were taken in vairous regions where the total dose of electrons was continuously changed. Contrast was manipulated by changing the reflecting condition. Results obtained are summarized as follows: (a) Amorphization occurs inhomogenously; (b) there is a critical size, i.e., about 5 nmø in the crystals, for observing sharp lattice images in small blocks; (c) lattice images change to short and wavy fringe contrasts when the block size becomes smaller than the critical one, and finally they disappear; and (d) boundary contrast of the blocks rapidly decreases with decreasing size, and is hardly observed when the size becomes smaller than the critical one. Based on the results, the cause of abnormal fringe contrasts in fine blocks, the amorphization process, and atomic structures of amorphous materials are discussed.
Merits of high resolution electron microscopy in the study of alloy structures
Authors: Kuo K. H. , Ye H. Q. , Li D. X.
Published: February 2005

We found that high resolution electron microscopy has the following merits in the study of alloy structures: (1) New phases too small to be studied by x‐rays, such as σ‐related phases, may be studied; (2) local and highly disordered structures, such as domain boundaries and domains of only a few unit cells, are more easily studied; and (3) it is easier to order solute atoms, such as Ti atoms columns in Cu4Ti.
Automatic measurement of scanning beam diameter using an on‐line digital computer
Authors: Oho Eisaku , Kobayashi Masaaki , Sasaki Toshihide , Adachi Koichi , Kanaya Koichi
Published: February 2005

A method for the measurement of electron probe beam diameter by digital image processing has recently been published. The purpose of the present report is to describe the development of an automatic system for beam diameter measurement. To complete this system, a method based on a theory which combines automation and high‐resolution conditions is proposed. In practice, the beam diameter is measured from the STEM image of a crystalline hole in a gold thin film, utilizing an on‐line computer system equipped with newly developed digital processing programs linked to a SEM in the transmission mode. The functions of the programs include statistical processing, matching, noise removal, interpolation, selection, and rotation. By combining these functions, the scanning beam diameter is accurately measured, in spite of difficulties, under most electron microscope operating conditions. The user simply appoints the edge included in the STEM image.
A comparison of unidirectional and rotary metal evaporation shadowing by computer simulation
Authors: Colquhoun William , Sokol Roger
Published: February 2005

This work employs a Fortran program to simulate rotary shadowing, that is, metal evaporation deposits in which the substrate is rotating during the deposition process. The results are compared with computer simulations of unidirectional shadowing published in a previous paper. (See Colquhoun et al., 1985). The analysis shows that a substantial improvement in resolution is possible with rotary shadowing. The improved information retrieval amounts to ∼15% with a shadow angle of 33° and ∼37% with a shadow angle of 8°. Unidirectional shadowing results in a grain linearity; the computer simulations show that this undesirable effect is eliminated by the rotary process.

These advantages of rotary shadowing are discussed along with the more straight forward interpretability of the rotary shadowed images. These advantages are weighed against the higher contrast generated by the unidirectional shadowing process.
Advances in ultrarapid freezing for the preservation of cellular ultrastructure
Authors: Gilkey John C. , Staehelin L. Andrew
Published: February 2005

Most of our current knowledge of cellular ultrastructure is derived from studies of chemically fixed and chemically cryoprotected preparations. In the first part of this review, we document the many artifacts associated with chemical techniques that render them unsuitable for further refinement of our understanding of cellular ultrastructure. The best method currently available for the preservation of cellular ultrastructure is ultrarapid freezing. The second part of this review is a consideration of the physics of ice crystal formation in biological systems, which suggests that ice crystals will be present in any frozen, uncryoprotected specimen. We define an ultrarapidly frozen preparation as one in which the ice crystals are so small as to be invisible at the electron microscopic level. Improvements in the ease of application and reliability of ultrarapid freezing techniques have reached the point that these techniques can be used by anyone requiring the best achievable preservation of cellular ultrastructure. In the third part of this review, we describe and critique the five methods of ultrarapid freezing in current use.
A method for sectioning and handling frozen sections for scanning electron microscopy
Authors: Mowry Thomas M. , Whalon Mark E. , Klomparens Karen
Published: February 2005

Glutaraldehyde‐fixed insects were embedded in Tissue‐TekR and sectioned on a liquid‐nitrogen‐cooled stage. The sections were sandwiched between two layers of microscope lens paper and all postsectioning treatments were carried out on this sandwich, including dehydration and critical‐point drying. In some cases, the sections were placed on filter membranes and lyophilized. These procedures produced intact specimens which maintained internal morphology as well as inter‐ and intracellular integrity without expensive or specialized equipment.
Surfactants as resin modifiers and their effect on sectioning
Authors: Mollenhauer Hilton H.
Published: February 2005

Ease of cutting thin sections with glass knives is markedly improved if the embedding resin contains a surfactant such as lecithin. With lecithin, it is possible to cut 50–100 thin sections from the same place on the knife edge even after facing off the block with 1–2‐μm‐thick sections. Image quality is similar to that of the unmodified resin if the resin formula is optimized. If not, some chatter or a “mottled” appearance of the tissue image may be present. Lecithin does not significantly affect sectioning with a diamond knife or the appearance of the section in the microscope. The increased ease of sectioning with glass presumably will be translated to diamond knives in the form of an increased useful life of the cutting edge.
Ultra‐high voltage electron microscopy: Past, present, and future
Authors: Fujita Hiroshi
Published: February 2005

High voltage electron microscopy has shown numerous advantages for the study of natural science, including biology, but it is especially useful in materials science. The most important advantage for materials science is in‐situ experiments on detailed processes of the same phenomena that occur in bulk materials. For such in‐situ experiments, the specimens should be thicker than a few microns to observe the behavior of lattice effect. The maximum observable thickness of the specimens and other advantages markedly increase with increasing accelerating voltage, and since 1965, two 3 MV instruments have been installed. The present paper is mainly concerned with these 3 MV electron microscopes and their applications to new research fields.
Rotary shadowing with platinum‐carbon in biological electron microscopy: A review of methods and applications
Authors: Chandler Douglas E.
Published: February 2005

Rotary shadowing when combined with such specimen preparation techniques as quick freezing and deep etching, critical point drying, and glycerol spraying is a highly versatile method of visualizing cell and macromolecular ultrastructure. This review outlines the procedures commonly used to prepare specimens for rotary shadowing and evaluates the relative merits of rotary shadowing when compared to unidirectional shadowing and negative staining.
Modification of a TEM‐goniometer specimen holder to enable beam current measurements in a (S)TEM for use in quantitative X‐ray microanalysis
Authors: Stols A. L. H. , Smits H. T. J. , Stadhouders A. M.
Published: February 2005

In order to have available a specimen holder suited to measure the beam current as is often required in quantitative electron probe X‐ray microanalysis, the rod of a low background beryllium specimen holder of a transmission electron microscope was modified. The tip was electrically insulated from the mass of the microscope and connected electrically to the central contact of a BNC connector mounted on the specimen holder handle. With this modified specimen holder the current absorbed by the specimen and/or the specimen holder could be measured easily and accurately. The modified specimen holder has been used to measure the beam current stability of an analytical electron microscope under various conditions. Data were obtained for tungsten as well as lanthanum hexaboride cathodes. Small changes to other types of specimen tips made it possible to exchange these for the low background tip.
Three‐dimensional reconstruction from serial section images by computer graphics
Authors: Baba Norio , Nakamura Shin‐Ichi , Kino Isamu , Kanaya Koichi
Published: February 2005

A method is described that allows a three‐dimensional object to be reconstructed from micrographs of serial sections by means of computer graphics. The reconstructed object, which can be rotated three‐dimensionally, is displayed on a colour visual display unit, and the object is shaded in order to provide an illusion of a three‐dimensional structure. Moreover, the technique makes it possible to observe an inner structure as seen through an outer one.
Characterization of chemically defined neurons and their cellular relationships by combined immunocytochemistry and radioautographic localization of transmitter uptake sites
Authors: Bosler Olivier , Beaudet Alain , Pickel Virginia M.
Published: February 2005

Radioautography and immunocytochemistry may be combined at the light and electron microscopic levels for simultaneously localizing uptake sites for exogenous transmitter molecules [such as (3H)monoamines or (3H)amino acids] and endogenous transmitter‐related antigens (classical transmitters and their synthesizing enzymes as well as neuropeptides) in the central nervous system. Silver grain accumulations indicative of transmitter uptake sites are readily distinguishable from immunocytochemical labels of the peroxidase‐antiperoxidase (PAP), avitin‐biotin, or colloidal gold methods. The combination of uptake radioautography and immunocytochemistry may be applied to the investigation of (1) the chemical identity of (3H) transmitter‐accumulating elements, (2) the coexistence of different neurotransmitters within the same neurons, and (3) the cellular basis of interactions between certain neurotransmitters, in particular monoamines, GABA, and neuropeptides. This article describes and evaluates the method and reviews the available experimental data derived from its application.
The use of scanning electron microscope autoradiography to localize the blueberry shoestring virus in its aphid vector
Authors: Klomparens Karen L. , Petersen Maureen A. , Ramsdell Donald C. , Chaney William G.
Published: February 2005

Scanning electron microscopy autoradiography (SEM‐AR) in conjunction with light microscope autoradiography (LM‐AR) was used to follow the movement of 125I‐labeled blueberry shoestring virus (BBSSV) through its aphid vector Illinoia pepperi with varying acquisition access periods (AAP). At 6 hr AAP, the virus had reached the stomach; at 12 hr AAP, it had reached the anterior of the intestines, and after 48 hr, AAP was present throughout the aphid. SEM‐AR using backscatter electron detection proved very useful because the sample bulk resulted in a shortened exposure time compared to LM sections, correlation of the autoradiograms could be made with fine structure, preparing large numbers of samples was easy, and examining whole longitudinal sections of aphids at low magnification with a clearly visible marker was possible. Limitations were mostly attributed to sample preparation and, in some cases, were easily remedied.
Freeze‐Fracture of physiologically identified neuromuscular junctions from single frog muscle fibers
Authors: Ko Chien‐Ping , Propst Jon W.
Published: February 2005

We developed a technique for freeze‐fracturing single physiologically identified neuromuscular junctions. This technique permits direct comparison of quantal content with morphological variables such as active zone length per unit terminal length for the same cell. The technique was developed to elucidate the structural basis for variability in transmitter release at the neuromuscular junction. The procedures were as follows: (1) record quantal content by conventional intracellular recordings; (2) mark cells for identification by fluorescent dye injection; (3) fix and stain endplate cholinesterase; (4) glycerinate and remove single fibers from the muscle; (5) draw endplate morphology; (6) freeze‐fracture single muscle fibers; (7) examine in a transmission electron microscope; and (8) photograph and measure nerve terminal membrane ultrastructure. We found that approximately 15% of freeze‐fractured single muscle fibers exhibited nerve terminal active zones. To demonstrate the usefulness of this technique, physiological and morphological information from an identified junction is presented. Freeze‐fracture of identified cells has several advantages over thin sections, which cannot accurately show such things as active zone length, spacing, or intramembrane particles. This technique also has applications to the study of active zone ultrastructure in situations where neurotransmitter release is known to differ from normal levels. In addition, direct correlations between membrane structure and function can be studied in other preparations by this method.
A modified goodman's technique for obtaining rocking‐beam type patterns
Authors: Kang Zhenchuan
Published: February 2005

A new method was developed for convergent beam electron diffraction (CBED) and large angle convergent beam electron diffraction (LACBED) in the JEM‐100CXII. This method is obtained in the imaging mode using the defocus objective lens and by re‐setting condenser‐2. A multi‐dark field CBED pattern was achieved in two ways.