bluuebunny/crossref_metadata_2025 huggingface dataset:
The relationship between educational level and mortality. The Reykjavík Study
Authors: Hardarson T. , Gardarsdóttir M. , Gudmundsson K. TH. , Thorgeirsson G. , Sigvaldason H. , Sigfússon N.
Published: March 2003

Abstract. Hardarson T, Gardarsdóttir M, Gudmundsson KTh, Thorgeirsson G, Sigvaldason H, Sigfússon N (National University Hospital, Reykjavík and University of Iceland; and Icelandic Heart Association, Reykjavík, Iceland). The relationship between educational level and mortality. The Reykjavík Study. J Intern Med 2001; 249: 495–502.

Objectives. Epidemiological studies have indicated an association between socioeconomic factors and health. It has not been clearly established whether this association is wholly or partly independent of classical risk factors. Our objective was to estimate the relationship between educational level and coronary artery disease (CAD), mortality and all‐cause mortality. The Reykjavík Study involving 18 912 participants followed‐up 4–30 years provides an ideal opportunity to address this question.

Design and subjects. The participants were aged 33–81 years and living in the Reykjavík area. They were divided into four groups according to education. The standard risk factors were assessed on entry and mortality, and cause of death registered during follow‐up. Multiple Cox regression analysis was applied to assess the relationship between age at examination, year of examination, educational level and mortality.

Results. The all‐cause mortality and CAD mortality was significantly related to education, even after adjustment for classical risk factors. For men, 14% (95% CI: 2–24) reduction was found in CAD mortality for those having high school education relative to elementary school. The figures for junior college and university education were 17% (95% CI: 1–31) and 38% (95% CI: 21–32), respectively. These figures were only slightly lower when major CAD risk factors were controlled for and still significant. Similar figures were found for all‐cause mortality. For women 34% (95% CI: 18–48) reduction was found in CAD mortality for high school education and 55% (95% CI: 22–74) for junior college, but too few had university education for reliable results. The figures were lower for all‐cause mortality, but significant. The figures were reduced when major CAD risk factors were controlled for, but still significant.

Conclusion. Education is a strong protective factor both for all‐cause and CAD mortality. Only a small part of this effect can be explained through conventional risk factors.
Rosiglitazone treatment of patients with extreme insulin resistance and diabetes mellitus due to insulin receptor mutations has no effects on glucose and lipid metabolism
Authors: Vestergaard H. , Lund S. , Pedersen O.
Published: March 2003

Abstract. Vestergaard H, Lund S, Pedersen O (Herlev Hospital, University of Copenhagen, Herlev; and Steno Diabetes Center, Gentofte; Denmark). Rosiglitazone treatment of patients with extreme insulin resistance and diabetes mellitus due to insulin receptor mutations has no effects on glucose and lipid metabolism. J Intern Med 2001; 250: 406–414.

Background. Rosiglitazone, a thiazolidinedione (TZD), increases insulin sensitivity by reducing levels of plasma NEFA, triglycerides (TG), glucose and serum insulin. Rosiglitazone treatment decreases insulin resistance in type 2 diabetic patients, but no data exist concerning rosiglitazone treatment of patients with syndromes of extreme insulin resistance.

Objectives. To evaluate whether hyperglycaemia in two lean patients with primary severe insulin resistance due to insulin receptor (IR) mutations and diabetes mellitus could be reduced by supplement of rosiglitazone for 180 days and secondary, to evaluate the effects on plasma NEFA, TG, Apo B, PAI‐1 and serum insulin.

Subjects. Both patients (brothers) have known mutations in the IR gene localized to the tyrosine kinase domain and a deletion of exon 17 in part of their IR mRNA. Prior to the study the HbA1C values were higher than 10% in both patients for more than 12 months during treatment with insulin and metformin.

Results. After 180 days of rosiglitazone supplement (8 mg day−1), no changes were observed in fasting plasma glucose and HbA1C. Incremental plasma glucose areas under the curves during a 75‐g oral glucose tolerance test (OGTT) were unchanged. Likewise, no improvements were seen in either first or second phase insulin secretion during a 0.3 g kg−1 intravenous glucose tolerance test (IVGTT). Fasting plasma VLDL and HDL cholesterol, TG and Apo B levels were unchanged, whereas a small increase was seen in total and LDL cholesterol levels. Fasting plasma NEFA increased by 51% in KC after 90 days of treatment, and after 180 days plasma NEFA was still 26% higher, when compared with pretreatment levels. In BC an initial 16% decrease was seen in plasma NEFA after 90 days of treatment. Plasma NEFA was increased 14% after 180 days of treatment, when compared with pretreatment levels, but 35% when compared with day 90. Plasma PAI‐1 decreased in both patients after 45 and 90 days of treatment but the decrease was only maintained in KC (47%).

Conclusions. Rosiglitazone treatment, in combination with insulin and metformin, of patients with severe primary insulin resistance due to IR mutations and diabetes mellitus, had no impact on the measured estimates of glucose and lipid metabolism. These findings may suggest that the effect of rosiglitazone on glucose and lipid metabolism are dependent on the presence of intact IR protein.
Serum free fatty acid pattern and risk of myocardial infarction: a case‐control study
Authors: YLI‐JAMA P. , MEYER H. E. , RINGSTAD J. , PEDERSEN J. I.
Published: March 2003

Abstract. Yli‐Jama P, Meyer HE, Ringstad J, Pedersen JI (Medical Faculty, University of Oslo; National Health Screening Services, Oslo; and Østfold Central Hospital, Norway). Serum free fatty acid pattern and risk of myocardial infarction: a case‐control study. J Intern Med 2002; 251: 19–28.

Objectives. To investigate the association between composition of serum free fatty acid (FFA) fraction and risk of a first myocardial infarction (MI).

Design. A case‐control design.

Setting. The patients were recruited from Ullevål Hospital in Oslo and Østfold Central Hospital in Fredrikstad and Sarpsborg, Norway.

Subjects. A total of 103 patients with first MI and 104 population controls, both men and postmenopausal women, age 45–75  years.

Results. The mean molar percentage content of docosahexaenoic (DHA), eicosapentaenoic (EPA), stearic and myristic acid in the serum FFA fraction was significantly lower in cases than in controls, whereas that of oleic and linoleic acid was higher in cases. Increased percentage content of total very long‐chain omega‐3 fatty acids (VLC n‐3) in serum FFA was associated with decreased risk of MI. Multivariate odds ratio (OR), adjusted for age, sex, waist‐hip ratio, smoking, family history of coronary heart disease (CHD) and years of education was 0.20 (95% CI 0.06–0.63) for the highest vs. lowest quartile. Also increased content of stearic acid was associated with decreased risk. Multivariate OR adjusted as above was 0.38 (95% CI 0.14–1.04) for the highest versus lowest quartile. After adjustment for oleic acid, however, the inverse linear trend was no longer significant.

Conclusions. The percentage content of VLC n‐3 as well as of stearic acid in serum FFA was inversely associated with risk of myocardial infarction. That of VLC n‐3 may reflect diet, but additionally these free fatty acids might in some way be related to the pathogenetic process and not only reflect their content in adipose tissue.
Costs for screening, intervention and hospital treatment generated by the Malmö Preventive Project: a large‐scale community screening programme
Authors: NORINDER A. , PERSSON U. , NILSSON P. , NILSSON J.‐Å. , HEDBLAD B. , BERGLUND G.
Published: March 2003

Abstract. Norinder A, Persson U, Nilsson P, Nilsson J‐Å, Hedblad B, Berglund G (Swedish Institute for Health Economics, Lund; and University Hospital, Malmö, Sweden). Costs for screening, intervention and hospital treatment generated by the Malmö Preventive Project: a large‐scale community screening programme. J Intern Med 2002; 251: 44–52.

Objectives. The purpose of this study was to estimate retrospectively the costs of health care resources used in the Malmö Preventive Project, Sweden and estimate the costs of in‐patient care that were avoided because of early intervention.

Setting and subjects. A large‐scale community intervention programme was conducted from 1974 to 1992 in Malmö, Sweden with the aim of reducing morbidity and mortality of cardiovascular diseases (CVD), alcohol related illnesses, and breast cancer. Between 1974 and 1992, 33 336 male and female subjects were screened for hypertension, hyperlipidaemia, type‐2 diabetes and alcohol abuse. Intervention programmes that included life‐style modifications, follow‐up visits with physicians and nurses and drug therapy were offered to about 25% of screened subjects.

Methods. Recruitment costs were generated through out the screening period. Intervention costs were estimated for 5 years after screening. Excess in‐patient care costs were estimated by subtracting hospital consumption for an unscreened, matched cohort from that of the screened cohort over follow‐up periods of 13–19 years. Intervention and excess in‐patient care costs were estimated until 1996.

Results. The net expenditures for recruitment and intervention was SEK253 million and saved costs for in‐patient care of SEK143 millions (1998 prices). Considering the opportunity cost of the resources used in the study, the net cost rises to about SEK200 millions.

Conclusion. The results suggest that only part of the intervention costs were offset by reduction in future morbidity health care costs. This is in line with results from prospective analyses of other primary prevention programmes.
Interleukin‐1 receptor antagonist is detectable in human carotid artery plaques and is related to triglyceride levels and Chlamydia pneumoniae IgA antibodies
Authors: GOTTSÄTER A. , FORSBLAD J. , MÄTZSCH T. , PERSSON K. , LJUNGCRANTZ I. , OHLSSON K. , LINDGÄRDE F.
Published: March 2003

Abstract. Gottsäter A, Forsblad J, Mätzsch T, Persson K, Ljungcrantz I, Ohlsson K, Lindgärde F (Malmö University Hospital, University of Lund, Malmö, Sweden). Interleukin‐1 receptor antagonist is detectable in human carotid artery plaques and is related to triglyceride levels and Chlamydia pneumoniae IgA‐antibodies. J Intern Med 2002; 251: 61–68.

Objectives. To investigate whether the interleukin‐1 receptor antagonist (Il‐1ra) and interleukin‐1β (Il‐1β) can be detected in human carotid artery tissue, and whether their presence is related to evidence of Chlamydia pneumoniae infection, risk factors for atherosclerosis, and clinical data.

Setting. Departments of Vascular Diseases and Surgical Pathophysiology, University Hospital, Malmö, Sweden.

Subjects. A total of 66 patients undergoing carotid endarterectomy (median age 74, range 53–89 years, 26 women). Il‐1β and Il‐1ra were studied in carotid artery plaques and in Il‐1ra in serum.

Results. Interleukin‐1 receptor antagonist was detected in mononuclear cells in plaques from 37/66 (56%) patients. Patients with Il‐1ra in plaques showed higher [2.04 (1.70–3.14) mmol L–1 vs. 1.69 (1.09–1.99) mmol L–1; P < 0.05] serum(s‐)triglyceride(tg) levels, and a higher frequency of IgA seropositivity for C. pneumoniae (76% vs. 52%; P < 0.05) than those without. S‐Il‐1ra levels correlated with s‐tg levels (r=0.38; P=0.047). There were no differences between patients with and without Il‐1ra in plaques concerning s‐Il‐1ra, blood(b‐)haemoglobin or leucocyte count, s‐cholesterol, b‐glucose, blood pressure, IgG seropositivity for C. pneumoniae, prevalence of neurological symptoms preceding operation, smoking, or diabetes mellitus. There were no differences in frequency of Il‐1ra in plaques or in s‐Il‐1ra levels between patients with symptomatic and asymptomatic stenosis, between smokers and nonsmokers, or between diabetic and nondiabetic patients. Il‐1β was not detected in plaques in the current study.

Conclusion. Interleukin‐1 receptor antagonist can be detected in human atherosclerotic carotid artery plaques, and is related to s‐triglyceride levels and IgA seropositivity for C. pneumoniae, but not to prevalence of neurological symptoms related to embolization.
Endothelial nitric oxide synthase gene Glu298Asp polymorphism and blood pressure, left ventricular mass and carotid artery atherosclerosis in a population‐based cohort
Authors: Karvonen J. , Kauma H. , Kervinen K. , Rantala M. , Ikäheimo M. , Päivänsalo M. , Savolainen M. J. , Kesäniemi Y. A.
Published: March 2003

Abstract. Karvonen J, Kauma H, Kervinen K, Rantala M, Ikäheimo M, Päivänsalo M, Savolainen MJ, Kesäniemi YA (University of Oulu, Oulu; Finland). Endothelial nitric oxide synthase gene Glu298Asp polymorphism and blood pressure, left ventricular mass and carotid artery atherosclerosis in a population‐based cohort. J Intern Med 2002; 251: 102–110.

Objective. Decreased production of endothelial nitric oxide (NO) is associated with different cardiovascular pathology. We studied the association between the Glu298Asp polymorphism of the NO producing gene, endothelial nitric oxide synthase (eNOS), and hypertension, left ventricular mass (LVM) and carotid artery intima‐media thickness (IMT) in a population‐based cohort of hypertensive and control subjects.

Design. Cross‐sectional case–control study.

Setting. District around Oulu University Hospital, Northern Finland.

Subjects and methods. The study population consisted of 600 middle‐aged hypertensive subjects (300 men and 300 women) and 600 controls (300 men and 300 women) living in the City of Oulu. The hypertensive subjects were randomly selected by age stratification from the Social Insurance Institute register for reimbursement of antihypertensive medication. For each hypertensive subject, an age‐ and sex‐matched control was randomly selected from the national health register. The overall participation rate was 87.8%. In the present study a total of 1024 subjects were screened. Echocardiographic examinations were performed by a trained cardiologist and carotid ultrasonographic examinations by a trained radiologist.

Results. The genotype distributions and allele frequencies between the hypertensive and control subjects and the relationship between the Glu298Asp variant and blood pressure, LVM and carotid artery IMT were determined. No differences in genotype distribution or allele frequencies were found between the hypertensive and control groups (the frequency of the Asp allele 0.299 vs. 0.288, respectively). Also, we could not find any association between the eNOS genotype and the measured cardiovascular complications.

Conclusions. The Glu298Asp variant of the eNOS gene does not seem to be a major risk factor for cardiovascular alterations in the general population.
Preventing atherothrombotic events in peripheral arterial disease: the use of antiplatelet therapy
Authors: Hiatt W. R.
Published: March 2003

Abstract.  Hiatt WR (University of Colorado Health Sciences Center and the Colorado Prevention Center, Denver, CO, USA). Preventing atherothrombotic events in peripheral arterial disease: the use of antiplatelet therapy (Review). J Intern Med 2002; 251: 193–206.

Patients with peripheral arterial disease (PAD) are at increased risk of generalized atherothrombotic events. Epidemiologic data shows a high rate of co‐prevalence of PAD and atherosclerosis in other vascular beds. Aggressive risk‐factor modification and antiplatelet therapy has become the cornerstone of treatment to prevent ischaemic events associated with PAD. Recent clinical trials have confirmed the clinical benefit of clopidogrel and ticlopidine in patients with PAD, agents that irreversibly inhibit the binding of adenosine diphosphate to its platelet receptor. In the clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE) trial, clopidogrel was associated with an overall risk reduction of 8.7% (compared with aspirin, P=0.043) in myocardial infarction (MI), ischaemic stroke and vascular death. These results demonstrated that long‐term administration of clopidogrel was effective in preventing ischaemic events in patients with atherosclerotic vascular disease including PAD. Aspirin and/or clopidogrel are the antiplatelet agents of choice for the reduction of atherothrombotic events in patients with PAD.
Treatment of sarcoidosis – from a basic science point of view
Authors: Moller D. R.
Published: March 2003

Moller DR (John Hopkins University School of Medicine, Baltimore, MD, USA). Treatment of sarcoidosis – from a basic science point of view (Minisymposium).J Intern Med2003;253:31–40.

Progress in our understanding of the scientific basis of granulomatous inflammation in sarcoidosis provides a framework for enlightened treatment decisions. Current evidence supports the concept that the pathogenesis of sarcoidosis involves a highly polarized T‐helper 1 (Th1) immune response to pathogenic tissue antigens. Conventional treatment is focused on attenuating granuloma formation with antimalarial drugs that inhibit antigen presentation or with nonspecific anti‐inflammatory agents such as glucocorticosteroids, methotrexate, or azathioprine. Anti‐tumour necrosis factor (TNF)‐α agents such as pentoxifylline, thalidomide, etanercept and remicade, have recently shown some successes in sarcoidosis. Designing future therapies depends on improved knowledge of the critical immunological processes operative in different stages of disease.
Contrast, resolution, pixelation, dynamic range and signal‐to‐noise ratio: fundamental limits to resolution in fluorescence light microscopy
Authors: Stelzer
Published: March 2003

In a perfect optical system numerical aperture and wavelength determine resolution. In a real optical system, however, the number of photons collected from a specimen determines the contrast and this limits the resolution. Contrast is affected by the number of picture elements per unit area, the number of photons and the aberrations present in every optical system. The concept of contrast vs. distance functions is used to compare the resolution achievable in confocal and wide‐field fluorescence microscopes and the effect of a further reduction of the observable volume. In conclusio: (a) real optical systems will never be able to achieve the theoretical resolution, (b) wide‐field fluorescence microscopy will often provide a better resolution than confocal fluorescence microscopy, (c) decreasing the observed volume does not necessarily increase the resolution and (d) using multiple fluorophores can improve the accuracy with which distances are measured. Some numbers for typical situations are provided.
Quantitative elemental X‐ray imaging of frozen‐hydrated biological samples
Authors: Marshall  , XU
Published: March 2003

It is shown that quantitative X‐ray imaging of planed, frozen‐hydrated, biological bulk samples that have not been etched is possible. X‐ray imaging represents a better alternative to static beam (selected area) analysis of fractured frozen‐hydrated samples. This procedure avoids the undesirable necessity of etching planed frozen‐hydrated samples to provide an interpretable electron image. Qualitative oxygen and carbon X‐ray images, which can be acquired in a short time, can be used for distinguishing morphological features and remove the requirement for electron images. In test samples of frozen‐hydrated albumin, containing salts, analyses by X‐ray images compared well with static beam (selected area) analyses from the same samples. An example of an analysis of frozen‐hydrated insect Malpighian tubules is given in which the response to ouabain treatment was analysed. In this example X‐ray imaging showed that ouabain resulted in a significant increase in cytoplasmic and luminal Na and a significant decrease in cytoplasmic and luminal K. X‐ray imaging also showed that there was a significant increase in cellular water content. The presence of a potassium gradient in soybean root nodules was also demonstrated. The use of standard deviation images for processing low count images increases analytical precision but results in underestimates of the true concentrations.
Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution
Authors: Erk  , Nicolas  , Caroff  , Lepault
Published: March 2003

Freezing of bulk biological objects was investigated by X‐ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High‐pressure freezing induces the formation of different ices (hexagonal, cubic and a high‐pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high‐pressure freezing. Cryo‐sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high‐pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high‐pressure frozen yeast cells survive successive high‐pressure freezing and thawing steps.
Atomic force microscopy imaging of fragments from the Martian meteorite ALH84001
Authors: Steele  , Goddard  , Beech  , Tapper  , Stapleton  , Smith
Published: March 2003

A combination of scanning electron microscopy (SEM) and environmental scanning electron microscopy (ESEM) techniques, as well as atomic force microscopy (AFM) methods has been used to study fragments of the Martian meteorite ALH84001. Images of the same areas on the meteorite were obtained prior to and following gold/palladium coating by mapping the surface of the fragment using ESEM coupled with energy‐dispersive X‐ray analysis. Viewing of the fragments demonstrated the presence of structures, previously described as nanofossils by McKay et al. (Search for past life on Mars — possible relic biogenic activity in martian meteorite ALH84001. Science, 1996, pp. 924–930) of NASA who used SEM imaging of gold‐coated meteorite samples. Careful imaging of the fragments revealed that the observed structures were not an artefact introduced by the coating procedure.
Quantitative analysis of valence electron energy‐loss spectra of aluminium nitride
Authors: Dorneich  , French  , Müllejans  , Loughin  , Rühle
Published: March 2003

The optical properties and electronic structure of aluminium nitride are determined using valence electron energy‐loss spectroscopy in a dedicated scanning transmission electron microscope. Quantitative analysis of the experimental valence electron energy‐loss spectra to determine the electronic structure encompasses single scattering deconvolution of the valence electron energy‐loss spectra to calculate the energy‐loss function, Kramers–Kronig analysis of the energy‐loss function to reveal the complex dielectric function, transformation of the dielectric function into the optical interband transition strength via optical property relations and finally critical‐point analysis of the interband transition strength. The influence of both experimental and analytical parameters on the final result was studied systematically to define and improve the understanding of the methods. To check the reliability of this technique the interband transition strength determined was compared with results of vacuum ultraviolet spectroscopy. Good agreement was found if sample preparation was taken into account. The preparation of the specimen for the transmission electron microscopy has an effect on the electronic structure. Quantitative analysis of valence electron energy‐loss spectroscopy, using the methods presented, is an important and capable method to determine the electronic structure of materials and it has the benefit of high spatial resolution.
High‐pressure freezing for immunocytochemistry
Authors: Monaghan  , Perusinghe  , Müller
Published: March 2003

Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross‐linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample‐stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well‐frozen material obtainable to ≈ 15 μm or less. In contrast, high‐pressure freezing has been demonstrated to provide ice‐crystal‐artefact‐free freezing of samples up to 200 μm in thickness. There have been few reports of high‐pressure freezing for immunocytochemical studies and there is no consensus on the choice of post‐freezing sample preparation. A range of freeze‐substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin‐embedding, polymerization and immunocytochemical labelling. Freeze‐substitution in acetone containing 2% osmium tetroxide followed by epoxy‐resin embedding at room temperature gave optimum morphology. Freeze‐substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl‐embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze‐substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice‐crystal damage in sections of embedded material. Thus a slow warm‐up to freeze‐substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze‐substitution media from immunocytochemical labelling experiments.
Real time two‐photon absorption microscopy using multi point excitation
Authors: Buist  , Müller  , Squier  , Brakenhoff
Published: March 2003

In this communication we present the development of a real time two‐photon absorption microscope, based on parallel excitation with many foci. This pattern of foci is created by a two‐dimensional microlens array. The fluorescence is detected by direct, non descanned detection on a CCD camera. Due to the parallel nature of both excitation and detection it is possible to speed up image acquisition significantly. This makes the instrument especially suitable for studying living specimens and/or real time processes. The optical design of the instrument is discussed and an imaging example is given. We specifically address the relation between the axial sectioning capability and the distance between the illumination foci at the sample.
Accurate structure determinations of very small (4–20 nm) areas, using refinement of dynamic electron diffraction data
Authors: Zandbergen H. W. , Jansen J.
Published: March 2003

It is shown that accurate structure refinements are possible from electron diffraction data obtained using areas 4–20 nm in diameter and 4–20 nm thick. To obtain accurate atomic positions it is essential to take the dynamic diffraction fully into account, for which new software was developed. Examples (La3Ni2B2N3, Ba2Ca3Cu5O10+δ and Ce5Cu19P12) are given of the structure refinements of known and unknown structures with R‐values in the range 2–6%. The procedure reported in this paper opens the way to structure determination of particles in many materials of industrial and scientific interest which cannot be solved by conventional structure determination (e.g. because of small size, heavily twinned materials or small fractions in polyphase materials).
Element specific imaging with high lateral resolution: an experimental study on layer structures
Authors: FREITAG  , MADER
Published: March 2003

Planar defects and individual layers in ceramic material are chemically imaged by high resolution energy‐filtering TEM using a post‐column imaging electron energy filter. Objects are barium layers in the cuprate superconductor NdBa2Cu3O7−δ (isostructual to YBa2Cu3O7−δ) as well as planar defects and precipitates of β‐WB in tungsten‐ and chromium‐doped TiB2. The barium layers with a spacing of 0.42 nm in the cuprate are resolved in jump‐ratio images using the Ba_N edge. In the boride system the β‐WB precipitates with thickness of 0.8 nm can be chemically imaged in elemental maps of B_K, Ti_L ,Cr_L and W_M. The B as well as the Ti map show a decrease in intensity at the precipitates, whereas in the W map an increase in intensity is observed. The boron‐deficient layers with a spacing of 0.38 nm in the β‐WB precipitate can be resolved in boron jump‐ratio images. Additionally, defects containing single boron‐deficient layers are chemically imaged. Hence structures in the dimension of interatomic distances can be imaged with respect to their elemental constituents. Although high resolution electron spectroscopic images contain strong interference contrast from elastic scattering, after normalization or background subtraction the element specific images are dominated by chemical contrast.
Quantitative metallography by electron backscattered diffraction
Authors: HUMPHREYS F. J.
Published: March 2003

Although electron backscattered diffraction (EBSD) in the scanning electron microscope is used mainly to investigate the relationship between local textures and microstructures, the technique has now developed to the stage where it requires serious consideration as a tool for routine quantitative characterization of microstructures. This paper examines the application of EBSD to the characterization of phase distributions, grain and subgrain structures and also textures. Comparisons are made with the standard methods of quantitative metallography and it is shown that in many cases EBSD can produce more accurate and detailed measurements than the standard methods and that the data may sometimes be obtained more rapidly. The factors which currently limit the use of EBSD for quantitative microstructural characterization, including the speed of data acquisition and the angular and spatial resolutions, are discussed, and future developments are considered.
Source point calibration from an arbitrary electron backscattering pattern
Authors: KRIEGER LASSEN N. C.
Published: March 2003

Precise knowledge of the position of the source point is a requirement if electron backscattering patterns (EBSPs) are to be used for crystal orientation measurements or other types of measurements which demand a geometrical analysis of the patterns. Today, possibly the most popular method for locating the source point is a computational technique which uses the positions of a number of indexed Kikuchi bands for calculating the coordinates of the point. A serious limitation of this calibration technique is, however, that the localized bands must first be indexed, which is difficult if the location of the source point is not known with reasonable precision.

This paper describes a new technique which determines the location of the source point from the positions of a number of bands in an arbitrary EBSP. Besides the positions of the Kikuchi bands, the only information which is required by this new calibration procedure is the same crystallographic information which is used for normal indexing of EBSPs. The procedure is shown to work successfully with patterns from a simple cubic crystal, as well as with patterns from an orthorhombic BiSCCO superconductor. In the former case, four bands are shown to be sufficient to ensure a unique determination of the source point, whereas five bands are required in the latter case. Once the bands have been localized, the time required for calculating the source point position is of the order of 1 min on a standard PC.
A procedure to prepare cultured cells in suspension for electron probe X‐ray microanalysis: application to scanning and transmission electron microscopy
Authors: Fernández‐Segura  , Cañizares  , Cubero  , Campos  , Warley
Published: March 2003

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X‐ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g, washed to remove the extracellular medium, cryofixed and freeze‐dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m
m ammonium acetate, 300 m
m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X‐ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.
Unexpected property of trehalose as observed by cryo‐electron microscopy
Authors: De Carlo  , Adrian  , Kälin  , Mayer  , Dubochet
Published: March 2003

Trehalose is an agent useful in maintaining the integrity of many biological systems submitted to various stresses. It is also presumed to improve specimen preparation for electron microscopy and to reduce beam damage. Here we study the effect of trehalose on the preparation and observation by cryo‐electron microscopy of thin vitrified films of biological suspensions. We observe that trehalose, as compared to sucrose, can indeed reduce electron beam damage to biological particles, as determined from the dose necessary for the onset of bubbling. Surprisingly, we also find that the contrast of biological particles is higher in a vitrified solution of trehalose than in one of sucrose. This effect can be explained if the water evaporation during the specimen preparation is less in the presence of trehalose than with sucrose, but we do not yet understand the underlying reasons since the evaporation properties of both sugars are similar at a macroscopic level. We conclude that trehalose is truly a remarkable substance and that more investigation is needed in order to fully understand its properties, and that the addition of ca. 3–5% trehalose to biological suspensions is a simple and useful method to reduce commonly arising drying artefacts and water evaporation in the thin film vitrification method.
Biological ultrastructure as revealed by high resolution cryo‐SEM of block faces after cryo‐sectioning
Authors: Walther  , Müller
Published: March 2003

Ultrastructural information was obtained by imaging the block face of high‐pressure‐frozen cryo‐sectioned biological samples in a high‐resolution cryo‐SEM. Cryo‐sectioning leads to a well‐defined flat artificial surface in contrast to cryo‐fracturing. Typical artefacts of cryo‐sections such as compression and crevasses were not visible on the block face. The ultrastructural features known from resin sections and from freeze‐fractures could also be found on the block faces. The cytoplasms show particles of different size which most likely represent proteins. The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging.

High quality cryo‐sections are only obtained from vitrified material. Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state.
Wavelets as chromatin texture descriptors for the automated identification of neoplastic nuclei
Authors: Van De Wouwer  , Weyn  , Scheunders  , Jacob  , Van Marck  , Van Dyck
Published: March 2003

Chromatin distribution reflects the organization of the DNA of a nucleus and contains important cellular diagnostic and prognostic information. Feulgen staining of breast tissue enables the chromatin distribution of the nucleus to be visualized in the form of texture. Describing texture in an objective and quantitative way by means of a set of texture parameters, combined with the study of the relationship of such parameters to the pathobiological cell properties, is useful both for reduction of the subjectivity inherently coupled to visual observation and for more accurate prognosis or diagnosis.

We have presented an automated classification scheme for the diagnosis and grading of invasive breast cancer. The input to this scheme was a digitized microscopical image, from which nuclei were segmented. Chromatin texture was described using a set of textural parameters that include first‐ and second‐order statistics of the image grey levels. The more recently developed wavelet energy parameters were also included in our study. Classification was performed by a Knn‐classifier, which is a versatile multivariate statistical classification technique.

We investigated the role of the tissue preparation technique and found that parameters derived from cytospins were better texture descriptors than those from sections. A 100% correct classification was achieved in a patient diagnosis experiment and 82% in a nuclear grading experiment.
Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation
Authors: Noyan  , Kahveci  , Cavusoglu  , Minbay  , Sunay  , Sirmali
Published: March 2003

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16–24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium–formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.
Effect of electron beam parameters on simulated CBED patterns from edge‐on grain boundaries
Authors: Bokel  , Tichelaar  , Schapink
Published: March 2003

Convergent beam electron diffraction (CBED) at vertical grain boundaries (parallel to the electron beam) can be applied to determine the symmetry of bicrystals. It can also be used to investigate the structure of the boundary region itself when subnanometre probe sizes are employed. In this paper it is shown that (sub)nanometre‐probe CBED patterns are largely influenced by the electron‐beam geometry. In particular, simulations of coherent CBED patterns based on the multislice algorithm show that the CBED pattern of an edge‐on interface depends on the defocus distance between the probe position and the specimen midplane, the probe size and the beam‐convergence angle. The pattern symmetry may be lower than the theoretically predicted symmetry in case of large spherical aberration. This effect increases with smaller accelerating voltages. An increase in the beam‐convergence angle also increases the possibility of a non‐optimum symmetry due to spherical aberration of a coherent probe. Thus, for the determination of an interface structure using subnanometre (coherent) probes, the imaging conditions play an important role.
Immunocytochemical strategies for electron microscopy: choice or compromise
Authors: Skepper J. N.
Published: March 2003

This review attempts to describe the major immunocytochemical strategies that are in current use for transmission electron microscopy. Emphasis is placed on suggesting the most appropriate application of each technique that is described. The review is not intended to identify the microscopist's ‘cure all’ technique for immunocytochemistry but rather to describe what techniques are available and what are their relative merits. Expensive equipment and technically complicated methods are not always necessary to collect robust data. I hope to present a review that will enable its readers to select the most profitable technique (or compromise) available, to reflect both their resources and their ability to address their biological questions with vigour.
Confocal microscopy of FM4‐64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae
Authors: Fischer‐Parton S. , Parton R. M. , Hickey P. C. , Dijksterhuis J. , Atkinson H. A. , Read N. D.
Published: March 2003

Confocal microscopy of amphiphilic styryl dyes has been used to investigate endocytosis and vesicle trafficking in living fungal hyphae. Hyphae were treated with FM4‐64, FM1‐43 or TMA‐DPH, three of the most commonly used membrane‐selective dyes reported as markers of endocytosis. All three dyes were rapidly internalized within hyphae. FM4‐64 was found best for imaging the dynamic changes in size, morphology and position of the apical vesicle cluster within growing hyphal tips because of its staining pattern, greater photostability and low cytotoxicity. FM4‐64 was taken up into both the apical and subapical compartments of living hyphae in a time‐dependent manner. The pattern of stain distribution was broadly similar in a range of fungal species tested (Aspergillus nidulans, Botrytis cinerea, Magnaporthe grisea, Neurospora crassa, Phycomyces blakesleeanus, Puccinia graminis, Rhizoctonia solani, Sclerotinia sclerotiorum and Trichoderma viride). With time, FM4‐64 was internalized from the plasma membrane appearing in structures corresponding to putative endosomes, the apical vesicle cluster, the vacuolar membrane and mitochondria. These observations are consistent with dye internalization by endocytosis. A speculative model of the vesicle trafficking network within growing hyphae is presented.
Temperature‐controlled microscopy for imaging living cells: apparatus, thermal analysis and temperature dependency of embryonic elongation in Caenorhabditis elegans
Authors: Rabin Y. , Podbilewicz B.
Published: March 2003

A new experimental apparatus for temperature‐controlled microscopy has been developed for the study of the temperature dependency of developmental processes in the nematode Caenorhabditis elegans. However, the application of this apparatus is rather general and can be used for a wide range of temperatures between − 10 and 90 °C. The new apparatus is easy to use, inexpensive, simple to construct, and is designed for precise temperature control of oil‐immersion microscopy using epifluorescence. Thermal analysis of the experimental apparatus shows the effects of each of its components, as well as the effects of uncertainty in temperature measurements. Finally, results of this study indicate that: (i) embryos incubated and imaged at temperatures of 8 °C and below do not elongate; (ii) the initial elongation rate is strongly temperature‐dependent between 9 and 25 °C.
A new method for three‐dimensional skeleton graph analysis of porous media: application to trabecular bone microarchitecture
Authors: Pothuaud L. , Porion P. , Lespessailles E. , Benhamou C. L. , Levitz P.
Published: March 2003

This paper introduces a new three‐dimensional analysis of complex disordered porous media. Skeleton graph analysis is described and applied to trabecular bone images obtained by high resolution magnetic resonance imaging. This technique was developed bearing in mind topological considerations. The correspondence between vertices and branches of the skeleton graph and trabeculae is used in order to get local information on trabecular bone microarchitecture. In addition to real topological parameters, local structural information about trabeculae, such as length and volume distributions, are obtained. This method is applied to two sets of samples: six osteoporosis and six osteoarthritis bone samples. We demonstrate that skeleton graph analysis is a powerful technique to describe trabecular bone microarchitecture.
A simplified application of systematic field sampling and low‐cost video recording set‐up for viewing disector pairs – exemplified in the rat cochlear nucleus
Authors: Korkmaz A. , Çiftçi N. , Boşnak M. , Agˇar E.
Published: March 2003

A description of a simple and efficient way of systematic field sampling along with a low‐cost set‐up for simultaneous viewing of physical disector pairs at the light microscopic level is presented. So far the use of a programmable motorized stage for the former purpose and two projection microscopes for the latter have proved to be the most efficient and fastest solutions, but the relatively high expense of purchasing such equipment constitutes a major impediment to the widespread use of such stereological methods. We describe practical applications of two new cost‐effective alternative approaches derived from existing ones. These approaches are used in estimating the total number of neurones in the cochlear nucleus of the rat and involve the systematic selection of section fields at a magnification significantly lower than the magnification used for counting and a video recording set‐up for comparing section pairs. Apart from providing a cheap way for the implementation of the physical disector, the main feature of the application, which may also encourage its use, is that it provides an efficient and simple design for systematic area sampling without requiring any specialized equipment. The same is true for optical disector designs, where the procedure is simplified even further.
Near‐field optical probing of two‐dimensional photonic crystals: theory and experiment
Authors: Mulin D. , Girard C. , Colas Des Francs G. , Spajer M. , Courjon D.
Published: March 2003

We report new experimental results, and their theoretical analysis, on the mechanisms that control light transfer between two integrated waveguides connected by two‐dimensional matrices of dielectric pillars. The optical properties of the system are analysed from the well‐established formalism of classical field susceptibilities (Green dyadic functions). We apply this scheme to investigate the optical properties of two‐dimensional arrays connected to two integrated waveguides. Comparisons with current experimental work based on near‐field optical probing are provided together with a spectral analysis of the phenomenon.