bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous, non-interfering collection of optimal fluorescent and backscattered light signals on the MRC 500/600
Authors: Pawley JB , Amos WB , Dixon A , Brelje TC
Published: June 2020

One of the most important attributes of the confocal LM is that it can be used for imaging living cells. Unfortunately, cells that contain fluorescent dyes are far less tolerant of the intense illumination characteristic of microscopical examination than unstained cells. As a result, it early became evident that every effort should be made to extract as much information as possible from every photon striking the specimen. This implies not only utilizing detectors with high quantum efficiency but also detecting the light which is back-scattered by (BSL), or passing through, the specimen in addition to any fluorescent signal. Both transmitted light and BSL carry information about the optical properties of the specimen and it is possible to detect this information without compromising fluorescent signal collection.
Three-dimensional image analysis and visualization in confocal light microscopy
Authors: Samarabandu J. K. , Acharya R. , Pareddy D. R. , Cheng P. C.
Published: June 2020

In the study of cell organization in a maize meristem, direct viewing of confocal optical sections in 3D (by means of 3D projection of the volumetric data set, Figure 1) becomes very difficult and confusing because of the large number of nucleus involved. Numerical description of the cellular organization (e.g. position, size and orientation of each structure) and computer graphic presentation are some of the solutions to effectively study the structure of such a complex system. An attempt at data-reduction by means of manually contouring cell nucleus in 3D was reported (Summers et al., 1990). Apart from being labour intensive, this 3D digitization technique suffers from the inaccuracies of manual 3D tracing related to the depth perception of the operator. However, it does demonstrate that reducing stack of confocal images to a 3D graphic representation helps to visualize and analyze complex tissues (Figure 2). This procedure also significantly reduce computational burden in an interactive operation.
Confocal study of microaggregates in 2.5 billion year-old sediments
Authors: Fan G.Y. , Mojzsist S. , Arrheniust G.
Published: June 2020

Light microscopy conducted on samples of Archean sediments reveals microaggregates (Fig. 1) which are suggestive of a biotic origin. These aggregates, typically 15 μm wide and 50 μm long, are thought to be the mineral remains of colonies of microorganisms that lived 2.5 billion years ago. Confocal microscopy is used to study the structures of these microaggregates in 3-D.

Samples under study are from the lowest section drill core taken from the Dales Gorge Member of the Brockman Iron-Formation (Hamersley Basin) in Western Australia. These sediments are well preserved and escaped metamorphic conditions typically experienced by older rocks of this type. Two types of samples were prepared for study under the microscope: thin sections (~40 μm) for transmitted light microscopy to study the general rock texture and to locate the features of interest, and thick sections (3 mm) for confocal microscopy to determine the 3-D structure of the aggregates in their undisturbed state.
Confocal microscopy of cytoskeletal organization in amphibian oocytes and eggs
Authors: Gard David L. , Roeder Amy D. , Schroeder Marianne M.
Published: June 2020

Amphibian oocytes, eggs, and early embryos have been increasingly used as models for studying the regulation of microtubule assembly, the regulation and mechanics of spindle assembly, and the generation of cellular and embryonic axes. However, little information has been available regarding the organization of cytoskeletal elements in vivo during oogenesis. In large part, this results from the unique challenges posed by immunofluorescence microscopy of these large cells (up to 2 mm in diameter). Preservation and staining of labile cytoskeletal elements is often difficult in large cells, due to the limited penetration of fixative and antibodies or other dyes. In addition, the accumulation of yolk during oogenesis renders larger oocytes, eggs, and early embryos opaque. Finally, cell size also often limits the magnification and resolution possible, since the details of cytoskeletal organization in large oocytes and eggs often are obscured by fluorescence from outside of the focal plane when objectives with high numerical aperture are used.
Image compression and data integrity in confocal microscopy
Authors: Avinash Gopal B.
Published: June 2020

In confocal microscopy, one method of managing large data is to store the data in a compressed form using image compression algorithms. These algorithms can be either lossless or lossy. Lossless algorithms compress images without losing any information with modest compression ratios (memory for the original / memory for the compressed) which are usually between 1 and 2 for typical confocal 2-D images. However, lossy algorithms can provide higher compression ratios (3 to 8) at the expense of information content in the images. The main purpose of this study is to empirically demonstrate the use of lossy compression techniques to images obtained from a confocal microscope while retaining the qualitative and quantitative image integrity under certain criteria.

A fluorescent pollen specimen was imaged using ODYSSEY, a real-time laser scanning confocal microscope from NORAN Instruments, Inc. The images (128 by 128) consisted of a single frame (scanned in 33ms), a 4-frame average, a 64-frame average and an edge-preserving smoothed image of the single frame.
Gum arabic helps prepare better lacey polymer films for specimen support in electron microscopy
Authors: Sheth Shailesh R. , Bellare Jayesh R.
Published: June 2020

Specimen support and astigmatism correction in Electron Microscopy are at least two areas in which lacey polymer films find extensive applications. Although their preparation has been studied for a very long time, present techniques still suffer from incomplete release of the film from its substrate and presence of a large number of pseudo holes in the film. Our method ensures complete removal of the entire lacey film from the substrate and fewer pseudo holes by pre-treating the substrate with Gum Arabic, which acts as a film release agent.

The method is based on the classical condensation technique for preparing lacey films which is essentially deposition of minute water or ice droplets on the substrate and laying the polymer film over it, so that micro holes are formed corresponding to the droplets. A microscope glass slide (the substrate) is immersed in 2.0% (w/v) aq. CTAB (cetyl trimethyl ammonium bromide)-0.22% (w/v) aq.
X-ray detection performance of a 300KV field-emission Analytical Electron Microscope
Authors: Lyman C. E. , Goldstein J. I. , Williams D.B. , Ackland D.W. , von Harrach S. , Nicholls A. W. , Statham P.J.
Published: June 2020

A major goal of analytical electron micrsocopy (AEM) is to detect small amounts of an element in a given matrix at high spatial resolution. While there is a tradeoff between low detection limit and high spatial resolution, a field emission electron gun allows detection of small amounts of an element at sub-lOnm spatial resolution. The minimum mass fraction of one element measured in another is proportional to [(P/B)·P]-1/2 where the peak-to-background ratio P/B and the peak intensity P both must be high to detect the smallest amount of an element. Thus, the x-ray detection performance of an analytical electron microscope may be characterized in terms of standardized measurements of peak-to-background, x-ray intensity, the level of spurious x-rays (hole count), and x-ray detector performance in terms of energy resolution and peak shape.

This paper provides measurements of these parameters from Lehigh’s VG Microscopes HB-603 field emission AEM. This AEM was designed to provide the best x-ray detection possible.
From light to electron microscopic analysis of neurons intracellularly injected in fixed slices with “miniruby”, a fluorescent biocyttn compound
Authors: Liu Wei-lin , Shipley Michael T.
Published: June 2020

Intracellular labeling of neurons in fixed slices is a very useful method for studying morphological structures of neurons both at light and electron microscopic levels. Recently, biocytin has been widely used for intracellular labeling in living slices because this molecule is highly soluble, has high electrophoretic mobility and has high affinity for avidin. However, biocytin cannot be used in fixed slices because in fixed slices membrane potential cannot be used to signify that a cell is impaled. Thus, in fixed slices it is necessary to inject cells with a fluorescent compound so that impalement and filling can be visualized under fluorescent microscope. We have developed a fluorescent biocytin compound, “Miniruby” (MR), dextran-tetramethylrhodamine-biocytin. previously, we showed that mis molecule provides excellent intracellular labels in fixed slices at the light microscopic level. Here, we demonstrate MR can also be visualized at the electron microscopic level.

Fixed slices (200-400 ¼m) of adult rat olfactory bulb, piriform cortex and periaqeductal gray were used.
Comparative studies of Alaria and Diplostomum metacercariae using SEM
Authors: Ujiie M. , Heckmann R. A. , Gardner J. S.
Published: June 2020

Two species of metacercariae belonging to the genera Alaria and Diplostomum were obtained from lizards in Mexico and fish in Peru. Alaria was found in the lungs of Scleroporus grammicus (Mesquito lizard) and Diplostomum was in the intestines of Orestias agasii (Carachi). To study the taxonomy and morphology of these two parasites, their integuments were compared using SEM.

The parasites were fixed in 10% formalin, transferred into 2.5% glutaraldehyde and kept in 70% ethanol to be sent to Brigham Young University. Specimens were dehydrated to 100% ETOH and critical point dried. Samples were mounted on stubs using double-stick tape and were coated with approximately 30 nm of gold. The two species were compared using SEM.

The integuments of the two species of metacercariae exhibited major differences. In order to achieve maximum absorption, modification of the surface area of primitive parasites is more extensive than in subsequent species. Alaria was uniformly covered with microvilli - characteristic seen in many of the trematodes ‐ ‐ including its anterior sucker region (Fig. 1, 3).
Lysine effect on ruthenium red and alcian blue preservation of the staphylococci glycocalyx
Authors: Fassel Theresa A. , Sanger James R. , Edmiston Charles E.
Published: June 2020

The gram-positive coagulase-negative staphylococci are opportunistic organisms important in latent infection associated with prosthetic biomaterials. Their highly charged polysaccharide glycocalyx, or slime, aids in proliferation of bacteria on surfaces after an initial adhesion event, possibly at the time of surgical insertion. The cationic reagents, ruthenium red (RR) and alcian blue (AB), in en bloc procedures have aided visualization of the polysaccharide glycocalyx material in TEM or SEM applications. Further, enhancement of RR preservation or staining of slime by inclusion of lysine in a prefixation stage has been observed. The effect of lysine with and without RR and AB on preservation/staining of the staphylococci glycocalyx is investigated further in this study.

Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis RP62 (RP62) and Staphylococcus hominis SP2 (SP2) were cultured in trypticase soy broth for 18 hrs at 35°C, following recovery from frozen storage and plating on blood agar plates for 24 hrs. Cells with 75mM lysine in the prefixation were incubated for 20 minutes with 2.5% glutaraldehyde (GA), 2.5% GA and 0.075% RR, or 2.5% GA and 0.075% AB, respectively.
Microscopy ofTaxomyces andreanae, a new taxon isolated fromTaxus
Authors: Strobel Gary A. , Standing M. D. , Hess W. M.
Published: June 2020

A newly described fungus,Taxomyces andreanaeStrobel, Stierle, & Hess. has also been isolated from (Taxus brevifoliaNutt.), The growth pattern of the fungus was determined by growing it on various plant species, plant parts and agar cultures. The morphological characteristics appeared to be more diverse when the fungus was grown on twigs of yew wood. Therefore, the purpose of these investigations was to use electron microscopy to more carefully observe the nature of fungal growth and bulbil formation ofT.andreanaeon twigs of yew wood.

Small pieces of sterile plant tissues were used to grow the fungus. For microscopy studies, procedures outlined by Upadhyay et al. were used, which consisted of fixation and dehydration followed by critical point drying and sputter coating for SEM, and embedment in Spurr resin for TEM. All SEM photos were taken at 10 kV. Fungal colonies had sparse to very dense concentrations of hyphal cells (Fig. 1) which, in some areas of cultures, produced hyphal cells which averaged from 1.25 μm to larger cells which averaged 3.75 μm in diameter.
Drug-induced morphological alterations in liver
Authors: Mohamad Moustafa , Drake Richard L. , Cardell Robert R.
Published: June 2020

Chronic administration of phenobarbital, an anticonvulsant drug, or rifampicin, an antituberculous drug, alters hepatocyte ultrastructure by inducing the proliferation of smooth endoplasmic reticulum (SER) and changes in glycogen content. While these generalized effects are important, the lobular location of hepatocytes must be considered since pericentral and periportal hepatocytes differ morphologically and most hepatotoxic compounds affect specific zones of the liver. We treated adult male rats with phenobarbital or rifampicin for 5 days to evaluate the effect of each drug on hepatic glycogen levels, glycogen distribution across the liver lobule, and hepatocyte ultrastructure, especially the SER. Our results show a decrease in hepatic glycogen levels as determined biochemically in both treatment groups. Semithin sections of liver specifically stained for glycogen (Figs. 1-3) show that in treated animals glycogen in pericentral and periportal hepatocytes is reduced (Figs. 2,3). Ultrastructurally, periportal hepatocytes typically have clumped glycogen (Fig. 4); however, in treated animals periportal hepatocytes that show the greatest reduction of glycogen contain extensive SER (Fig. 5).
Histochemical and immunocytochemical ultrastructural localization of melanocyte specific proteins
Authors: Boissy Ying L. , Orlow Seth J. , Boissy Raymond E.
Published: June 2020

Melanin synthesis in melanocytes is localized to melanosomes, where enzymatic and structural gene products must be targeted. Some gene products are translocated through different pathways (i.e., tyrosinase travels through the Golgi apparatus during processing, whereas a putative matrix glycoprotein does not). To visualize trafficking routes, we used post-embedding immunocytochemistry to compliment DOPA-histochemistry. We report on the localization of tyrosinase, matrix glycoprotein, tyrosinase-related protein 1 (TRP-1) and lysosome-associated membrane protein-1 (LAMP-1).

DOPA histochemistry: Cultured melanoma cells were fixed in half-strength Karnovsky’s, incubated in 0.1 % 1-DOPA 2X for 2.5 h and embedded in Eponate 12. For immunocytochemistry, cells were fixed with 4% paraformaldehyde, 1% glutaraldehyde, and 0.2% picric acid with 0.5 mM CaCl2(pH 7.4) for 20 m at RT, quenched in 50 mM NH4Cl for 20 m at 0°C, washed in 0.1 M maleate buffer (pH 6.5), and treated with 2% uranyl acetate (pH 4.0) for 30 m at 0°C.
Cytoarchitecture of cultured olfactory neuroepithelial spheres: The role of olfactory sustentacular cells in cavity formation
Authors: Pixley Sarah K. , Miller Marian
Published: June 2020

When newborn rat olfactory mucosal cells were dissaggregated and plated as single cells onto astrocyte bed layers, abundant generation and maturation of olfactory receptor neurons was observed. Both immature and mature neurons were found in large multicellular spheres after 15 days in culture,. In sections of the spheres, almost all had central cavities, and the neurons were located almost exclusively in the external 1-2 cell layers. We show here that the spheres also contain olfactory sustentacular cells, neuronal supporting cells similar to retinal Muller cells. Sustentacular cell presence was shown by immunostaining cultured cells with a monoclonal antibody, SUS, which has previously been shown to recognize only sustentacular cells and a subset of the Bowman's gland cells. Fig. 1 shows that cultured cellular spheres contained numerous SUSpositive, roughly bipolar cells. These cells appear to be located internally rather than on the external surface. Sustentacular cell presence was further confirmed by immunostaining with a monoclonal antibody, 1F4, developed in this laboratory.
An SEM evaluation of normal, hypermineralized, and demineralized human dentin and its interaction with dentin bonding systems
Authors: Perdigao Jorge
Published: June 2020

The mechanism of bonding to enamel has been described as tag-like resin extensions into the etched enamel structure. However, the mechanism by which resins bond to dentin has not been fully elucidated. Bonding to dentin represents a greater challenge than bonding to enamel due to the organic composition of dentin, the presence of fluid-filled tubules, variations in intrinsic composition, and the presence of the odontoblastic process in dentinal tubules. Bonding to mineralized dentin surfaces is more difficult than bonding to normal dentin. The purpose of this study was to evaluate the morphology of three different dentin substrates and their interaction with four different dentin bonding systems (DBS).

One-hundred twenty caries-free human molar teeth stored in an thymol solution were randomly assigned into three groups (n = 40), according to the type of surface treatment. After removing the occlusal dentin, forty teeth were suspended in 600 ml of a mineralizing solution for two weeks.
Low-voltage SEM of urinary stones
Authors: Rez Peter , Reilly Michail
Published: June 2020

Urinary stones are predominantly composed of calcium oxalate and calcium apatite while a minority of stones are formed from uric acid. The oxalate stones occur with greatest frequency, mainly in the form of the monohydrate (whellewite) with some dihydrate (wedellite) present. Despite many years of extensive research, the mechanisms for kidney stone nucleation and growth are still unknown. In particular the urine from recurrent stone formers, and the urine from those who do not form stones, does not show large differences in concentration of either calcium or oxalate ions. Recent work has emphasised the role of possible “inhibitors” or “promoters” in the growth of urinary stones though the quest for these substances remains elusive. Much effort has been devoted to the study of crystallization of calcium oxalates in an attempt to understand nucleation, aggregation and growth as a function of pH and the presence of possible promoters or inhibitors.

Many analytical techniques have been applied to urinary stone analysis with X-ray diffraction, wet chemistry and infrared spectroscopy being the most successful for routine characterisation.
Ultrastructural diagnosis of cryptosporidium in a patient with acquired immunodeficiency syndrome (AIDS)
Authors: Ramirez E. , Ceseñas B. L. A. , Segovia F.
Published: June 2020

Cryptosporidium sp is an intestinal protozoan that is well known as parasite of different animals,but recently it has been recognized as a cause of human disease, princippally since apparition of the acquired immunodeficiency syndrome (AIDS). In this kind of patients it produces severe diarrhea (loss of a stool volume up to 17 liters per day), this condition may be a major factor leading to death.

Information about Cryptosporidiosis and AIDS incidence in developing countries is limited. In this paper we report the ultrastructural study of an intestinal biopsy from an hemophilic man,infected wit HIV through a blood transfussion that presented severe diarrhea.

Jejunal biopsy specimen was obtained and prepared for electron microscope study. The specimen was fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer pH 7.2;postfixed in 1% osmium tetroxide in 0.2M phosphate buffer pH 7.2;dehydrated in ethanol and embedded in epoxy resin (Medcast). Thin sections obtained in a LKB ultratome V with a diamond knife, stained with uranyl acetate and lead citrate, were observed in a Zeiss EM109 electron microscope.
The number and size of rat tubular cells in diabetic growth, regression, and regrowth using the optical disector
Authors: Nyengaard Jens Randel , Flyvbjerg Allan , Rasch Ruth
Published: June 2020

Diabetic renal growth, regression and regrowth was studied using stereological methods on perfusion-fixed rat kidneys. A razor blade tissue slicer was used for cutting the entire kidney into 2-mm-thick slices and every second slice was sampled systematically random. Every sampled slice from each kidney was embedded in a single capsule using glycolmethacrylate. From every plastic block one 35-μm-thick section and one 2-μm-thick section was cut and stained with PAS. The volume of kidney cortex was estimated on the thin sections using the Cavalieri principle. Total volume of proximal and distal tubules (V(tub)) were estimated by point-counting on the thin sections at a magnification of 332x using a projection microscope. Using an optical disector (Fig. 1a-d) on the thick sections, the numerical densities (NV) of proximal and distal tubular cells were estimated. The total number of tubular cells: N(total) = NV*V(tub). The mean volume of a tubular cell (
(tub)) including basement membrane and lumen was calculated as:
(tub)=V(tub)/N(total). Lumen of the tubular cell was excluded by point-counting using the thin sections and the microscope mentioned in Fig. 1.
A method for direct measurement of specimen noise in HREM images
Authors: Paciornik Sidnei , Kilaas Roar , Dahmen Ulrich , O'Keefe Michael Adrian
Published: June 2020

High resolution electron microscopy (HREM) is a primary tool for studying the atomic structure of defects in crystals. However, the quantitative analysis of defect structures is often seriously limited by specimen noise due to contamination or oxide layers on the surfaces of a thin foil.

For simple monatomic structures such as fcc or bcc metals observed in directions where the crystal projects into well-separated atomic columns, HREM image interpretation is relatively simple: under weak phase object, Scherzer imaging conditions, each atomic column is imaged as a black dot. Variations in intensity and position of individual image dots can be due to variations in composition or location of atomic columns. Unfortunately, both types of variation may also arise from random noise superimposed on the periodic image due to an amorphous oxide or contamination film on the surfaces of the thin foil. For example, image simulations have shown that a layer of amorphous oxide (random noise) on the surfaces of a thin foil of perfect crystalline Si can lead to significant shifts in image intensities and centroid positions for individual atomic columns.
The consolidation of microscopy technology into a university research support facility: Reflections on the 1980's and projections for the 1990's at the university of iowa
Authors: Moore Kenneth C.
Published: June 2020

The University of Iowa Central Electron Microscopy Research Facility(CEMRF) was established in 1981 to support all faculty, staff and students needing this technology. Initially the CEMRF was operated with one TEM, one SEM, three staff members and supported about 30 projects a year. During the past twelve years, the facility has replaced all instrumentation pre-dating 1981, and now includes 2 TEM's, 2 SEM's, 2 EDS systems, cryo-transfer specimen holders for both TEM and SEM, 2 parafin microtomes, 4 ultamicrotomes including cryoultramicrotomy, a Laser Scanning Confocal microscope, a research grade light microscope, an Ion Mill, film and print processing equipment, a rapid cryo-freezer, freeze substitution apparatus, a freeze-fracture/etching system, vacuum evaporators, sputter coaters, a plasma asher, and is currently evaluating scanning probe microscopes for acquisition. The facility presently consists of 10 staff members and supports over 150 projects annually from 44 departments in 5 Colleges and 10 industrial laboratories. One of the unique strengths of the CEMRF is that both Biomedical and Physical scientists use the facility.
Imaging biological samples with the atomic-force microscope
Authors: Henderson Eric , Jondle Daniel , Marsh Thomas , Shaiu Wen-Ling , Niu Luming , Vesenka James , Stanley Elis , Haydon Philip
Published: June 2020

The application of atomic force microscopy (AFM) to biological investigation is attractive for a number of reasons. Foremost among these is the ability of the AFM to image samples, even living cells, under near native conditions and at resolution equal to, or exceeding, that possible by the best light microscopes. Moreover, the ability of the AFM to manipulate samples it images provides a novel and far reaching application of this technology.

We have been studying a number of biological samples by AFM. These include conventional and non-conventional nucleic acid structures, ribosomes, neural cells and synapses, cellular organelles (chloroplasts and nuclei), among others. Each of these projects has its own set of associated difficulties and each reveals information about the uses and limits of the AFM in biology. Fig. 1 shows AFM images of various biological samples. In the case of nucleic acids, which have been extensively studied in a number of labs by AFM the problems of signal/noise sample deposition have been overcome in air and organic solvents.
Non-contact atomic-force microscopy for soft surfaces
Authors: Howland R. S. , Oot D. F. , Nowroozi-Esfahani R. , Maclay G. J. , Hesketh P. J.
Published: June 2020

The atomic force microscope (AFM) was invented in the mid-1980s, in response to strong interest in the high resolution, real-space surface imaging capabilities of the scanning tunneling microscope (STM). The AFM provides one real benefit that the STM cannot: it is able to image insulating surfaces. As a result, the AFM can operate on a wider variety of samples; it also can image samples in air, where many conductors oxidize rapidly, and in solution. Essentially no surface preparation is necessary. Historically, however, even the AFM has had limitations. Until recently, the contact forces exerted by the AFM tip on the sample surface meant that AFM was limited to surfaces of substantial rigidity. Noncontact AFM removes that barrier, opening up the possibility of AFM imaging of very soft surfaces, or of surfaces that cannot be contaminated by contact with the tip.

An AFM uses a piezoelectric transducer to scan the sample beneath a sharp probe.
The effect of probe-tip geometry on force microscopy images
Authors: Thornton J. T. , Velu E. M. T. , Mooney C. B. , Russell P. E.
Published: June 2020

The atomic force microscope (AFM) has been used to characterize a variety of surfaces to study the influence of the instrumental parameters and sample topography on the interpretation of the images and the data obtained. The effect of the tip shape on the resulting image resolution and artifacts has been studied. Test samples of varied topography have been used to establish imaging parameter optimization for specific sample topographies.

Chromium thin films of different thickness, deposited uniformly by rf diode sputtering, have been selected as test samples and imaged with a variety of tip shapes. These films were developed as underlayer films for magnetic recording media used in computer hard disks. The range of tips include commercial silicon nitride pyramidal tips, focused ion beam (FIB) sharpened silicon nitride pyramidal tips, and electron beam contamination induced tips (microtips) varying in growth time (i.e. length) from 10 to 40 seconds (approximately 0.40 to 0.65 μm).
Alignment without using markers in electron tomographic reconstruction
Authors: Liu Weiping , Agard David A. , Sedat John
Published: June 2020

In the electron tomographic reconstruction process the mutual alignment between projections of different view angles is a crucial step. Thus far the only alignment method routinely used is based on fiducial markers: gold particles are distributed on the specimen, and the relationship between the specimen and the digital projection coordinate systems is determined from least-square fitting the measured positions of colloidal gold beads on the projections. The inherent drawbacks with this method are that i) it is a tedious trial-and-error process to obtain the right amount of evenly distributed beads in the area of interest on the specimen and ii) the beads might obstruct the structural analysis since they are essentially opaque objects under the electron microscope (EM). The problems can become more acute if ice-embedded specimens are to be studied. The other two approaches to the alignment are the common line / moments method which requires that the object has clear boundaries in its projections and the cross-correlation method which assumes that the object has a flat structure.
Analysis of cryo-electron micrographs of HbS fibers
Authors: Lewis Michael R. , Gross Leon J. , Josephs Robert
Published: June 2020

We are studying the structure of deoxy-sickle hemoglobin (HbS) fibers by cryo-electron microscopy (cryo-EM). Cryo-EM affords potentially higher resolution than negative staining in part because the electron scattering by unstained protein includes internal structure which may be poorly depicted by an envelope of negative stain. This advantage, however, is partially compromised by a low signal to noise ratio (snr) and low image contrast. Our cryo-EM images were recorded with specimen doses of 7 e-/Å2 at 100 keV, ca. 100 nm defocus, and 39,000 magnification on Kodak SO-163 film.

Helical particles usually have a constant pitch. Thus the rotation about the axis is coupled to translation along the length of the particle. HbS fibers, however, have a variable pitch which ranges from 6° to 12° rotation per unit cell length (63 Å). Consequently, the rotation of HbS fibers is partially decoupled from translation and the twist of each unit cell and its relative angular rotation about fiber the axis cannot be determined by particle symmetry.
Symmetry-selected electron energy loss scattering
Authors: Batson P.E.
Published: June 2020

The differential scattering cross section for EELS has long been described within the Born approximation:

where v is the incident electron velocity, | ϕ0 > and | ϕn > are the initial and final one electron specimen states, and the sum is over the specimen electrons. This expression is suited to plane wave scattering where we have one well-defined momentum transfer given by q. We obtain equation by expanding the swift electron coulomb interaction in a Fourier series and picking one momentum transfer. This is not very useful for scattering in the STEM, because we must sum coherently over many plane wave states to make a small probe. This produces many cross terms in equation above, some of which may exactly cancel. Thus, the plane wave formulation can obscure a simple result by separating the scattering in a non-physical way.
Detection limits for Analytical Electron Microscopy with Electron Energy Loss Spectrometry and energy-dispersive x-ray spectrometry
Authors: Newbury Dale E. , Leapman Richard D.
Published: June 2020

The measurement of trace level constituents, arbitrarily defined for this study as concentration levels below 1 atom percent, has always been considered problematic for analytical electron microscopy (AEM) with energy dispersive x-ray spectrometry (EDS) and electron energy loss spectrometry (EELS). In a landmark study of various microanalysis techniques, Wittry evaluated the influence of various instrumental factors (source brightness, detection efficiency, accumulation time) and physical factors (cross section, peak-to-background) upon detection limits. Although the ionization cross section, fluorescence yield, and collection efficiency favor EELS over EDS, the peak-to-background ratio of EELS spectra is much lower than that of EDS spectra, leading Wittry to suggest that the limit of detection should be 0.1 percent for EDS and 1 percent for EELS for practical measurement conditions. Recent developments in parallel detection EELS (PEELS) indicate that a re-evaluation of the situation for trace constituent determination is needed for those elements characterized by "white line" resonance structures at the ionization edge.
Compositional mapping of biological material with the scanning ion microprobe
Authors: Levi-Setti Riccardo , Chabala Jan M. , Smolik Sarah
Published: June 2020

The use of ion probes for microscopy and imaging microanalysis by secondary ion mass spectrometry (SIMS) is a destructive observational technique that can provide information inaccessible to the electron microscope. The damage to the sample caused by the ion-induced sputter erosion of its surface layers is exploited to identify and map the chemical and isotopic composition of the removed layers. Particularly good image resolution (20-70 nm) can be attained with heavy-ion focused probes, as with the University of Chicago 40 keV Ga+ Scanning Ion Microprobe (UC SIM). The application of this instrument to biological research involving mineralized and soft tissue has been evaluated on several occasions, together with the requirements of specimen preparation for SIMS analysis. In a review recently presented, studies of mineralized tissues (bone, teeth) were discussed. We concentrate here on recent directions of our SIMS studies of soft biological material.

The sensitivity of SIMS to isotopes of any mass paves the way for the profitable use of tagged, tracer molecules in a host of biological applications as a preferable alternative to the use of radioisotopes and autoradiography.
Scanning farfield and scanning nearfield probe microscopy of catalase platelets
Authors: Knapp Helmut F. , Schabert Frank A. , Engel Andreas
Published: June 2020

High resolution (sub nm) and direct height information obtained through scanning probe microscopy (SPM) (such as from the STM and AFM) establishes SPM as a valuable tool for the investigation of biological samples. Unfortunately SPM allows only regions of a few μm to be scanned at high speed. For biological applications this is a disadvantage, because most samples will be only of the μm size and scattered across the much larger substrate. To make SPM investigation of biological samples less tedious, we combined a STM (or alternatively an AFM) with an inverted confocal scanning laser microscope (CSLM), with a lateral and axial resolution of 200 nm and 400 nm, respectively. This enables to locate areas of interest on the substrate and position the tip of the STM. STM tips used are electro-chemically etched from Au to routinely attain tip radii in the 5 nm range. Catalase platelets were chosen as a sample, because of their established two different lattice repeats (17.5 nm and 6.8 nm).
Direct phase determination from EM images
Authors: Hovmöller Sven , Zou Xiaodong , Eriksson Lars , Svensson Gunnar
Published: June 2020

EM images contain phase information, in contrast to X-ray and electron diffraction patterns. The phases are extracted from the Fourier transform (FT) of the EM image. However, there has been doubts as to what exactly this phase information is. Are the EM phases the same as those of the crystallographic structure factors F(hkl) as defined in x ray crystallography? Or are the phases so distorted by dynamical scattering that very sophisticated procedures are needed for phase retrieval, as has been suggested.

We have investigated this question, by comparing phases obtained from EM-images with those of the x ray structure factors from two accurately refined barium niobium oxide crystals.

EM images of Ba4Nb14O23 (orthorhombic Cmmm, a=20.782, b=12.448, c=4.148 Å) and Ba3Nb16O23 (orthorhombic Cmmm, a=20.930, b=12.478, c=4.162 Å) were taken with a Philips CM 30 300kV and a JEOL 200CX 200kV electron microscope. Thin areas near the edge were analyzed by our newly developed image processing system, CRISP.
Dislocation contrast in high-angle hollow-cone dark-field TEM
Authors: WANG Z. L.
Published: June 2020

Dislocations were imaged using the high-angle hollow-cone dark-field transmission electron microscopy (HADF-TEM) technique. Experiments were performed using a Philips CM30 TEM at 300 kV Dislocations and grain boundaries show bright contrast in HADF-TEM images and there are no contrast reversals with thickness or defocus. The dislocation contrast shows no dramatic variation when the average semi-conical angle θ was increased from 40 to 110 mrad, but does show strong dependence on the diffracting conditions that are set up for the corresponding on-axis bright-field (BF) TEM image (Fig. 1). Under the "one-beam" (random orientation without strong diffraction) reflection condition (Figs, 1a and 1a'), the visibility of the dislocations is poor in either the BF-TEM or the HADF-TEM image. Under the two-beam diffracting condition (Figs, 1b and 1b') both BF-TEM and HADF-TEM images show optimized contrast. The features appearing in the HADF-TEM images have a good one-to-one correspondence with the features shown in the corresponding BF-TEM images; the dislocation contrast disappears in the HADF-TEM images if the condition g·b = 0 is satisfied in the BF-TEM images, where b is the Burgers vector (Fig 2).
The atomic-force microscope and its future in biology
Authors: Revel Jean-Paul
Published: June 2020

The last few years have been marked by a series of remarkable developments in microscopy. Perhaps the most amazing of these is the growth of microscopies which use devices where the place of the lens has been taken by probes, which record information about the sample and display it in a spatial from the point of view of the context. From the point of view of the biologist one of the most promising of these microscopies without lenses is the scanned force microscope, aka atomic force microscope.

This instrument was invented by Binnig, Quate and Gerber and is a close relative of the scanning tunneling microscope. Today's AFMs consist of a cantilever which bears a sharp point at its end. Often this is a silicon nitride pyramid, but there are many variations, the object of which is to make the tip sharper. A laser beam is directed at the back of the cantilever and is reflected into a split, or quadrant photodiode.
Preparation of thin-film-metal/6H-SiC TEM specimens by RPR ion milling
Authors: Bow J.S. , Shaapur F. , Kim M.J. , Carpenter R.W.
Published: June 2020

HRTEM has proven to be the best method to study the interface structure of heterogeneous materials at the atomic level. One of the critical steps in these experiments is preparation of sufficiently thin cross section TEM (XTEM) specimens with different materials on both sides of the interface in good condition for HRTEM, especially when metal/ceramic materials are of interest. For the material system of a thin metal film on 6H-SiC substrate, the conventional ion milling method did not produce satisfactory XTEM specimens, because of the large difference in ion milling rates between metals and SiC. Most of the metal was ion milled way before a thin area of SiC was obtained. Two very important parameters, the thickness of the metal thin film normal to the interface and the atomic bonding structure across the interface were usually lost. Fig.l shows an example. The original thickness of the metal was 100 nm, and most of that has been removed. An amorphous layer of 1 to 1.5 nm thickness apparently due to ion milling formed at the metal/SiC interface.
Structural study of deactivation of gallosilicate catalysts
Authors: Choi-Feng C. , Hall J. B. , Huggins B. J. , Li D. , Kaduk J. A. , Green L. M. , Alexander B. D. , Reagan W. J.
Published: June 2020

Gallosilicate molecular sieves with an MFI structure are very promising catalysts for upgrading light olefins and paraffins to aromatics. Gallosilicate catalysts are quite stable at low reaction temperature, however, when subjected to high temperature, gallosilicate catalysts deactivate rapidly. The activity and selectivity of these catalysts are greatly influenced by both framework and non-framework gallium. Although the framework gallium imparts acidity to the sieve, the nature of the non-framework gallium is unclear. In the present study, combined techniques of analytical electron microscopy (AEM), high resolution transmission electron microscopy (HREM) and X-ray diffraction (XRD) have been applied to study the fate of framework and non-framework gallium in progressively deactivated gallosilicate molecular sieve catalysts. To study this progressive process, fresh gallosilicate molecular sieve and the gallosilicate catalysts (with 40% Cab-O-Sil matrix) subjected to steaming treatments at different temperatures and durations were characterized.

The microstructure of fresh gallosilicate catalysts consists of highly crystalline molecular sieve which is uniformly distributed in the amorphous supporting matrix (Fig. 1).
Characterization of colloidal materials by cryo-TEM
Authors: Minter J. R.
Published: June 2020

Many researchers develop products and processes that depend upon colloidal materials. Rheological and other important material parameters are influenced by the morphology of the colloid. The photography industry uses many such colloids including silver halide sols, photographic coupler sols and emulsions surfactants, and gelatin solutions and gels. High-resolution direct electron images of these systems in their native states permit photographic scientists to understand how changes in formulation produce changes in microstructure and, ultimately, to understand the photographic performance of the system.

We prepare thin biconcave liquid films spanning the holes in a perforated carbon film supported by an electron microscope grid (see Fig 1). To prevent changes in composition of the colloid caused by evaporation, the films are prepared in an environmental chamber and are propelled through a trap door into a container of liquid ethane. The specimen is cooled at a rate sufficient to prevent crystallization of water. When maintained below -150 °C, these specimens are stable and compatible with the high vacuuary of the electron microscope.
Chemical twinning in the Ti4C2S2-TiS system
Authors: Hua Mingjian , Garcia C. I. , DeArdo A. J.
Published: June 2020

Titanium carbosulfide Ti4C2S2 (H phase, P63/mmc, a=0.321 nm and c= 1.120) are TiS (Rm, a=0.3417 nm and c = 2.65) are frequently found to coexist within composite particle in ultra-low carbon interstitial-free (IF) steels. This more suggest that a direct transformation from TiS to Ti4C2S2 may be a major stabilization of C at higher temperatures in these steels. The deduced orient between Ti4C2S2 and TiS is in agreement with a reported crystal structure mode transformation mechanism between these two phases appears to be strongly related structures.

At the as-cast condition, most of the S- and C-rich particles in the studied IF step as pure M4C2S2 (M=Ti, Nb). After a re-heating treatment at 1300°C, M4C2S2 discharge and a new phase were observed. This new phase can be indexed as a trigonated with a=0.33 nm and c=5.3 nm (Figure 1). After a second re-heating treatment appeared at the center layer of the multi-phase particles (Figure 2). It should be noted that (i) some planar defects were observed inside the new phase and Ti4C2S2 carbon and 2a).
Hollow-cone analysis of intercalated particles in layered H(Ca2Nan-3NbnO3n+1) materials
Authors: Treacy M. M. J. , Bisher M. E. , Jacobson A. J.
Published: June 2020

The series of compounds H(Ca2Nan-3NbnO3n+1) form tetragonal perovskite-related layer structures that are strongly bound in two dimensions, but more weakly bound in the third. These materials can be delaminated by intercalating basic surfactant molecules into the layers, which spontaneously exfoliate the structure to form stable dispersions in a polar solvent. On drying a diluted droplet of this dispersion on an amorphous carbon film supported on a TEM specimen grid, all sheets are observed to lie flat on the carbon, with the thin tetragonal c-axis parallel to the optic axis. Typical sheet dimensions are 1 μm × 1 μm × 1.4 nm. Layered materials prepared in this way make fascinating specimens for transmission electron microscopy because the thickness, composition and orientation are known. Depending on the dispersion density, sheets will occasionally overlap with parallel c axes, but with random relationship between the a axes.

Layers of exfoliated H(Ca2Nb3O10) material were examined in a Hitachi H9000NAR microscope equipped with a high angle electronic hollow cone unit.
Scanning electron microscopy 1928-1965
Authors: McMullan D.
Published: June 2020

The beginning of the general use of the scanning electron microscope (SEM) can be accurately dated to 1965 when the Cambridge Instrument Company in the U.K. marketed their Stereoscan 1 SEM (to be followed about 6 months later by JEOL in Japan). But development had been in progress intermittently over the previous 30 years in Germany, the U.S.A., France, and the U.K. where in 1948 work was begun in Charles Oatley’s department at the Cambridge University Engineering Department which led directly to the Stereoscan. The purpose of this paper is to trace the development of the SEM and to show that some of the ideas pul forward by the early workers were well ahead of their time and only became technologically practicable much later.

The First proposal for the application of scanning to microscopy was made in 1928 by Synge, an Irish scientific dilettante, who conceived the near-field scanning optical microscope but did not attempt to build one.
Observation of supported catalyst particles by high-resolution SEM
Authors: Yao Ming-Hui , Smith David J. , Datye Abhaya K.
Published: June 2020

Supported metal particle catalysts have found wide application in the chemical industry and for environmental control. Many microscopy techniques have been employed to study these catalysts. Although scanning electron microscopy(SEM) is not commonly used, its ability to provide surface topological information as well as the ease of sample preparation and observation are positive attractions for catalysis research. Due to limited SEM resolution in the past, as well as severe charging of the insulating catalyst supports, very few studies of catalysts using SEM appear to have been reported. Recent development of in-lens specimen immersion systems and cold field emission sources of high brightness have made it possible to image specimen surfaces with secondary electrons at subnanometer resolution. Using a high resolution Hitachi S-5000 in-lens field emission SEM, we have studied Pt particles supported on TiO2 and CeO2. The purpose of this study was to evaluate relative merits and demerits of high resolution SEM for catalysis research, in particular with respect to TEM and STEM techniques.
Universal ESEM
Authors: Danilatos G.D.
Published: June 2020

The environmental scanning electron microscope (ESEM) has evolved as the natural extension of the scanning electron microscope (SEM), both historically and technologically. ESEM allows the introduction of a gaseous environment in the specimen chamber, whereas SEM operates in vacuum. One of the detection systems in ESEM, namely, the gaseous detection device (GDD) is based on the presence of gas as a detection medium. This might be interpreted as a necessary condition for the ESEM to remain operational and, hence, one might have to change instruments for operation at low or high vacuum. Initially, we may maintain the presence of a conventional secondary electron (E-T) detector in a "stand-by" position to switch on when the vacuum becomes satisfactory for its operation. However, the "rough" or "low vacuum" range of pressure may still be considered as inaccessible by both the GDD and the E-T detector, because the former has presumably very small gain and the latter still breaks down.