bluuebunny/crossref_metadata_2025 huggingface dataset:
Parental age and coronary disease in the general male population
Authors: Rosengren A. , Thelle D. , Wilhelmsen L.
Published: March 2003

Abstract. Rosengren A, Thelle D, Wilhelmsen L (Sahlgrenska University Hospital, Östra; and Göteborg University, Göteborg, Sweden). Parental age and coronary disease in the general male population. J Intern Med 2002; 251: 258–267.

Objective. To investigate the role of achieved age in father or mother for long‐term prognosis in men with respect to fatal and nonfatal coronary events, and mortality from cardiovascular disease, cancer, and all causes.

Design. Prospective follow‐up study.

Setting. City of Göteborg, Sweden.

Subjects. A total of 6242 men aged 51–59 out of 7100 men who took part in the second screening of the Primary Prevention Study in 1974–77 and who had stated the age of both mother and father at death, or their current age, if they were still alive.

Main outcome measures. Fatal and nonfatal coronary events, and mortality from cardiovascular disease, cancer, and all causes during follow‐up until 1996.

Results. Of the men (n=2135) whose father had died before the age of 70, 8.4 per 1000 observation years died from coronary disease during follow‐up. Coronary mortality decreased continuously with increasing age in the father and in those whose father had achieved an age of 90 or more 3.2 per 1000 years died [hazard ratio (HR) after adjustment for coronary risk factors, socio‐economic status, and history of myocardial infarction in either parent, 0.41 (95% CI 0.23–0.73; P for trend <0.0001)]. This was reflected in reduced risk of mortality from any cause (P for trend after adjustment 0.003). No association with cancer death was found. With respect to hospitalization for myocardial infarction men whose father had survived to at least 90 had an adjusted HR of 0.60 (0.40–0.89) compared with men whose father died before 70 (P for trend 0.0006). The effect of achieved age in the mother was weaker and after adjustment trends were weakly significant for death from cardiovascular and all causes (P=0.01 and 0.03, respectively), but not for any other end‐point.

Conclusion. Paternal, but not maternal, longevity appears to protect against coronary disease, by mechanisms that are largely unknown.
A rapid increase in β‐cell function by multiple insulin injections in type 2 diabetic patients is not further enhanced by prolonging treatment
Authors: KÄRVESTEDT L. , ANDERSSON G. , EFENDIC S. , GRILL V.
Published: March 2003

Abstract. Kärvestedt L, Andersson G, Efendic S, Grill V (Stockholm's Sjukhem Foundation; and Karolinska Hospital, Stockholm, Sweden). A rapid increase in β‐cell function by multiple insulin injections in type 2 diabetic patients is not further enhanced by prolonging treatment.J Intern Med2002;251:307–316.

Objective. Intensive insulin treatment in type 2 diabetes can improve β‐cell function. It is not known which duration of treatment achieves maximal improvement. We addressed this question in type 2 diabetic patients who displayed features of `secondary failure'.

Research design and method. Ten patients were randomized to multiple insulin injection (MI) therapy for 9 weeks. Another 10 patients started with bedtime insulin (BTI) and continued their peroral medication. Following 9 weeks of treatment, patients on MI switched to BTI and glibenclamide.

Results. Three days of MI led to a decrease in fasting proinsulin/insulin ratio, 0.43 ± 0.20 vs. 0.29 ± 0.11,P=0.01 and an increase in glucagon‐stimulated C‐peptide over baseline, 0.77 ± 0.43 vs. 1.28 ± 0.44 nmol L–1,P 0.02. Nine weeks of MI treatment successively decreased fasting and nonfasting blood glucose in parallel with increasing insulin dosage. Initial improvements in secretion parameters were upheld but not further enhanced, the 9 week proinsulin/insulin ratio being 99 ± 23% and that of glucagon‐stimulated C‐peptide being 95 ± 24% of the values obtained after 3 days of treatment. Eight weeks after termination of MI there persisted a total weight gain that tended to be larger than after continuous peroral medication with BTI.

Conclusion. Improvement of insulin secretion by intensive insulin treatment is rapidly gained with no further effect obtained after a longer treatment period. This finding, as well as undesirable effects of MI on body weight, argues against prolonged MI treatment as a prelude to other therapeutic regimens in type 2 diabetic patients.
Antioxidant vitamins in the prevention of cardiovascular disease: a systematic review
Authors: ASPLUND K.
Published: March 2003

Abstract. Asplund K (Umeå and Stockholm, Sweden). Antioxidant vitamins in the prevention of cardiovascular disease: a systematic review (Review). J Intern Med 2002; 251: 372–392.

Do antioxidant vitamins, in regular food or as food supplements, protect against myocardial infarction and stroke? In this systematic literature review on the effects of antioxidant vitamins in the primary prevention of cardiovascular disorders, studies with ischaemic heart disease, stroke or combined cardiovascular events as end‐points have been included. Studies on the effects of antioxidant vitamins on intermediary end‐points (such as blood lipids and blood pressure) and as secondary prevention in patients with manifest cardiovascular disease are reviewed in a conventional manner.

In observational studies (case–control or cohort design), people with high intake of antioxidant vitamins by regular diet or as food supplements generally have a lower risk of myocardial infarction and stroke than people who are low‐consumers of antioxidant vitamins. The associations in observation studies have been shown for carotene, ascorbic acid as well as tocopherol. In randomized controlled trials, however, antioxidant vitamins as food supplements have no beneficial effects in the primary prevention of myocardial infarction and stroke. Serious adverse events have been reported. After an initial enthusiasm for antioxidants in the secondary prevention of cardiovascular disease, recent reports from of several large randomized trials have failed to show any beneficial effects.

Thus, the apparent beneficial results of high intake of antioxidant vitamins reported in observational studies have not been confirmed in large randomized trials. The discrepancy between different types of studies is probably explained by the fact that supplement use is a component in a cluster of healthy behaviour. Antioxidant vitamins as food supplements cannot be recommended in the primary or secondary prevention against cardiovascular disease.
A family with IgA nephropathy and hereditary lymphoedema praecox
Authors: USTA M. , DILEK K. , ERSOY A. , ALPER E. , ÖZBEK S. , ÖZDEMIR B. , FILIZ G. , YAVUZ M. , GÜLLÜLÜ M. , YURTKURAN M.
Published: March 2003

Abstract. Usta M, Dilek K, Ersoy A, Alper E, Özbek S, Özdemir B, Filiz G, Yavuz M, Güllülü M, Yurtkuran M (Uludağ University Medical School, Bursa, Turkey). A family with IgA nephropathy and hereditary lymphoedema praecox (Case Report). J Intern Med 2002; 251: 447–451.

Immunoglobulin A (IgA) nephropathy is the most common primary glomerulonephritis worldwide. The pathogenesis is still unknown and treatment has not yet been established. Rarely it can be associated with other disorders. Its association with hereditary lymphoedema is not reported before. We report four patients, a 60‐year‐old father, his two sons and his daughter, with hereditary lymphoedema. The family had nine members and in four of them lymphoedema was evident. The other members had neither lymphoedema nor IgA nephropathy. This is the first report of IgA nephropathy in association with hereditary lymphoedema.
Carotid artery intima‐media thickness and brachial artery flow‐mediated vasodilation in asymptomatic Japanese male subjects amongst apolipoprotein E phenotypes
Authors: Haraki T. , Takegoshi T. , Kitoh C. , Wakasugi T. , Saga T. , Hirai J.‐I. , Aoyama T. , Inazu A. , Mabuchi H.
Published: March 2003

Abstract. Haraki T, Takegoshi T, Kitoh C, Wakasugi T, Saga T, Hirai J‐I, Aoyama T, Inazu A, Mabuchi H (Fukui Prefectural Hospital, Fukui; Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan). Carotid artery intima‐media thickness and brachial artery flow‐ mediated vasodilation in asymptomatic Japanese male subjects amongst apolipoprotein E phenotypes. J Intern Med 2002; 252: 114–120.

Background. Epidemiological studies suggest that apolipoprotein E (apoE) polymorphism influences plasma lipoprotein levels and the development of cardiovascular disease.

Objective. To clarify the role of apoE polymorphism as a risk factor for early atherosclerosis.

Design. Using a high‐resolution ultrasound method, we investigated the association between apoE phenotypes, carotid intima‐media thickness (CCA‐IMT), and flow‐mediated dilation in the brachial artery (brachial‐FMD) in 96 healthy asymptomatic Japanese men (mean ± SD age, 50 ± 8 years).

Results. Serum cholesterol and LDL‐cholesterol levels in subjects with E3E4 were highest and those with E2E3 were lowest (P < 0.05 and P < 0.05, respectively). The CCA‐IMT in E3E4 subjects (0.76 ± 0.17 mm) was greater than that in E2E3 and E3E3 (0.61 ± 0.15 and 0.64 ± 0.14 mm, respectively; P < 0.01). In contrast, there was no difference between brachial‐FMD and apoE phenotypes (P=0.15). By univariate analysis, CCA‐IMT was positively correlated with age (r=0.51, P < 0.01), LDL‐chol/HDL‐chol ratio (r=0.37, P < 0.01), triglycerides (r=0.23, P < 0.05), and negatively correlated with HDL‐cholesterol (r=−0.31, P < 0.01). An association between CCA‐IMT and the presence of E4 allele was also found (P < 0.05). Logistic regression analysis revealed that the presence of E4 allele was a higher risk for increased IMT (relative risk of 4.4, 95% CI 1.5–12.5), even after adjustment for age, LDL‐cholesterol, blood pressure and other known risk factors. A negative correlation between brachial‐FMD and CCA‐IMT was also found in all subjects (r=−0.21, P < 0.05), being most apparent in the E3E4 subjects (r=−0.53, P < 0.02).

Conclusion. ApoE4 phenotype was independently associated with an increased risk of carotid atherosclerosis and elevated LDL‐cholesterol levels in asymptomatic middle‐aged Japanese men.
Determination of cardiac involvement in sarcoidosis by magnetic resonance imaging and Doppler echocardiography
Authors: Sköld C. M. , Larsen F. F. , Rasmussen E. , Pehrsson S. K. , Eklund A. G.
Published: March 2003

Abstract. Sköld CM, Larsen FF, Rasmussen E, Pehrsson SK, Eklund AG (Karolinska Hospital and Institutet, Stockholm, Sweden). Determination of cardiac involvement in sarcoidosis by magnetic resonance imaging and Doppler echocardiography. J Intern Med 2002; 252: 465–471.

Objectives. To elucidate whether cardiac magnetic resonance imaging (MRI) could be useful in disclosing structural changes in the myocardium in sarcoidosis patients and to relate echo‐Doppler derived indices of left ventricular function to electrocardiogram (ECG) findings.

Design. The MRI was performed in 18 consecutive patients with sarcoidosis. Left ventricular ejection fraction (LVEF), i.e. systolic function, was estimated echocardiographically by Simpson's two‐dimensional method (n = 16). Diastolic function was estimated by age‐corrected Doppler‐derived indices: isovolumetric relaxation time (IVRT), deceleration time (DT) and early filling/atrial contraction ratio (E/A ratio).

Results. Eleven patients had conduction defects or dysrhythmias (ECG+) whilst seven patients had a normal ECG (ECG–). In two patients, high signalling, contrast‐enhanced, isolated regions, suggestive of deposits, were seen in the left ventricular myocardium on MRI. Both these patients had abnormal ECGs and signs of systolic and/or diastolic dysfunction on echocardiography. LVEF was subnormal in seven of 10 of the ECG+ patients and in two of six of the ECG–. Signs of diastolic dysfunction were found in 59% and 56% of the measurements in the ECG+ and ECG– patients, respectively.

Conclusion. We conclude (i) that myocardial deposits on MRI in sarcoidosis patients have a high specificity for cardiac involvement but a rather low sensitivity; (ii) that a substantial proportion of sarcoidosis patients with abnormal ECGs have echocardiographic signs of systolic and/or diastolic dysfunction.
The cytokine network in sarcoidosis and its clinical relevance
Authors: Ziegenhagen M. W. , Müller‐Quernheim J.
Published: March 2003

Abstract. Ziegenhagen MW, Müller‐Quernheim J (University Hospital Freiburg, Freiburg, Germany). The cytokine network in sarcoidosis and its clinical relevance (Minisymposium). J Intern Med 2003; 253: 18–30.

In recent years, analysis of the cytokine network has substantially improved our knowledge of the immunopathogenesis of sarcoidosis. There is increasing evidence from clinical immunology that analysis of the cytokine network may be helpful for clinicians to assess the extent and activity of sarcoid inflammation. Genetic polymorphisms may contribute to interindividual differences in the regulation of cytokine release. Thus, disease phenotype‐associated haplotypes should exist and their analysis might disclose risk profiles of individual patients. Furthermore, serological assessment of cytokines or soluble cytokine receptors may become suitable parameters in clinical practice to detect an ongoing inflammation in chronic sarcoidosis.
Effects of glucocorticoids on leptin levels and eating behaviour in women
Authors: Uddén J. , Björntorp P. , Arner P. , Barkeling B. , Meurling L. , Rössner S.
Published: March 2003

Abstract.  Uddén J, Björntorp P, Arner P, Barkeling B, Meurling L, Rössner S (Karolinska Institute at Huddinge University Hospital, Stockholm, Sweden; and Sahlgren's Hospital, Gothenburg, Sweden). Effects of glucocorticoids on leptin levels and eating behaviour in women. J Intern Med 2003; 253: 225–231.

Objective.  Long‐term treatment with glucocorticoids induces weight gain and increased risk to develop obesity‐related metabolic complications. The underlying mechanisms are not fully understood. Glucocorticoid therapy has previously been associated with increased levels of circulating leptin. In this study the eating behaviour was therefore studied in relation to leptin levels before and after short‐term prednisolone treatment.

Design.  Within‐subject design.

Subjects.  Twelve healthy postmenopausal women with a mean body mass index (BMI) of 28.9 kg m−2 (±0.8 SEM) volunteered after recruitment by an advertisement in the local paper.

Interventions.  The subjects received 25 mg prednisolone daily for 7 days.

Main outcome measurements.  Fasting serum samples were obtained before, during and after treatment for determination of leptin and insulin, glucose and fractionated lipoproteins in plasma. The microstructure of the eating behaviour was registered with a universal eating monitor, VIKTOR. Appetite was estimated by visual analogue rating scales and food intake by a 48‐h recall.

Results.  Serum leptin increased after 2 and 7 days of glucocorticoid administration (P < 0.01), and the food intake measured by VIKTOR after 7 days of treatment (P < 0.05). No statistically significant changes were however, found in the 48‐h food‐ recall or in the subjective appetite registrations. Insulin levels were borderline elevated (P = 0.062) after treatment, but no significant changes of fasting glucose were seen. High density lipoprotein cholesterol (HDL) increased (P < 0.05), whilst low density lipoprotein cholesterol (LDL) decreased (P < 0.05).

Conclusion.  Food intake was elevated after glucocorticoid administration as observed with an objective, quantitative method, in spite of increased levels of circulating leptin.
Decreased muscle strength and contents of Mg and Na,K‐pumps in chronic alcoholics occur independently of liver cirrhosis
Authors: Aagaard N. K. , Andersen H. , Vilstrup H. , Clausen T. , Jakobsen J. , Dørup I.
Published: March 2003

Abstract. Aagaard NK, Andersen H, Vilstrup H, Clausen T, Jakobsen J, Dørup I (Aarhus University Hospital; and University of Aarhus, Aarhus, Denmark). Decreased muscle strength and contents of Mg and Na,K‐pumps in chronic alcoholics occur independently of liver cirrhosis. J Intern Med 2003; 253: 359–366.

Objectives.  To evaluate the influence of established liver cirrhosis on muscle strength and muscle contents of magnesium (Mg), potassium (K) and sodium, potassium pumps (Na,K‐pumps) in chronic alcoholic patients.

Design.  An open cross‐sectional study.

Setting and subjects.  Forty consecutive chronic alcoholics (18 with cirrhosis and 22 without cirrhosis) admitted to the Department of Hepatology, Aarhus University Hospital, Denmark, or to a collaborating alcoholism treatment centre, and 36 healthy control subjects.

Main outcome measures.  Evaluation of participant's subjective physical ability and measurement of maximum isokinetic muscle strength and muscle mass, as well as measurements of Mg, K and Na,K‐pumps in skeletal muscle.

Results.  Maximum isokinetic muscle strength and muscle mass were equally reduced in patients with and without cirrhosis (P < 0.01 all). In keeping with this, both groups of patients felt equally physically restricted. Muscle Mg was reduced to the same extent in the two groups of patients (by 12 and 9%, P < 0.001, both), whereas the muscle K content was only significantly lower in the cirrhotic patients (10%, P < 0.001). The muscle content of Na,K‐pumps was reduced by 14%, (P < 0.01) in the cirrhotic patients and by 8% (P < 0.05) in the noncirrhotic patients.

Conclusion.  Our alcoholic patients complained of physical disability, had reduced skeletal muscle mass, isokinetic muscle strength, content of muscle Mg and content of Na,K‐pumps. There was no difference between patients with and without cirrhosis. It appears that it is the heavy alcohol intake, and not the cirrhosis per se, that is responsible for the observed defects.
Helicobacter pylori: resurrection of the cancer link
Authors: Björkholm B. , Falk P. , Engstrand L. , Nyrén O.
Published: March 2003

Abstract.  Björkholm B, Falk P, Engstrand L, Nyrén O (Swedish Institute for Infectious Disease Control, Solna; AstraZeneca R & D, Mölndal; Karolinska Institute, Stockholm, Sweden). Helicobacter pylori: resurrection of the cancer link (Review). J Intern Med 2003; 253: 102–119.

Helicobacter pylori is one of the most common pathogenic bacterial infections, colonizing an estimated half of all humans. In a subset of individuals, the infection leads to serious gastroduodenal disease such as peptic ulcers and gastric adenocarcinoma. The factors contributing to skewing this, in most cases benign, relationship into disease development are largely unknown. However, factors emanating from the bacterium, host and the environment have been shown to affect the risk for disease, although no factor can be singled out to be most important. The known factors are associated with affecting the risk of disease, and are not absolute. Virulence of H. pylori is affected by the existence and regulation of certain genes present in the bacterial population in a stomach. The effects of H. pylori on gastric cancer development have been challenged and the risk associated with infection with virulent (i.e. Cag PAI positive) H. pylori has likely been underestimated.
Cryo‐electron diffraction as a tool to study local variations in the lipid organization of human stratum corneum
Authors: Pilgram  , Van Pelt  , Spies  , Bouwstra  , Koerten
Published: March 2003

The human skin provides the body with a barrier against transepidermal water loss and the penetration of harmful agents (e.g. microbes) from outside. This barrier function is produced mainly by the outermost, nonviable layer of the epidermis, the stratum corneum (SC). The SC consists of terminally differentiated corneocytes surrounded by a continuous intercellular lipid domain, which contains mostly ceramides, cholesterol and free fatty acids. Small‐ and wide‐angle X‐ray diffraction studies have elucidated the lamellar and lateral lipid organizations in these domains. However, these techniques require bulk quantities of SC, as a result of which local structure information on the lipids cannot be obtained. Insights to these local lipid arrangements are important when new transdermal drug delivery systems have to be developed. Therefore, the technique of electron diffraction arose as a tool to study the lateral packing of the lipids in the intercellular domains of SC, locally. In a previous study, the suitability of electron diffraction was demonstrated using a lipid model system that resembled the lipid composition of the SC. The spacings calculated from the electron diffraction patterns were in good agreement with the spacings revealed by wide‐angle X‐ray diffraction. The results presented here succeed this previous study. We improved the microscope settings and developed a new preparation method to study ex vivo human SC by cryo‐electron diffraction. The method is based on the conventional tape‐stripping method and offers the possibility to study depth‐related changes in the lipid organization of human SC. Diffraction patterns of both hexagonal and orthorhombic lipid lattices have been recorded with spacings that resembled those found in human SC by wide‐angle X‐ray diffraction. After lipid extraction, such diffraction patterns could no longer be detected in the samples.
On optimizing high‐pressure freezing: from heat transfer theory to a new microbiopsy device
Authors: Shimoni  , Müller
Published: March 2003

High‐pressure freezing (HPF) is currently the only method which enables adequate cryoimmobilization of biological samples thick enough to describe the bulk of the sample. In the current state of HPF instrumentation and preparation methods, the technique has not yet reached its full potential. While suspensions can be prepared easily for HPF, tissue preparation is restricted by the need to compromise between different requirements and difficulties. (i) In order to achieve optimal freezing quality, very thin samples are required. (ii) There is mechanical difficulty in cutting such thin samples without distorting the organization of the tissue. (iii) The cutting and the succeeding preparation steps of small samples require long handling times (minutes), which may result in physiological and hence structural alterations. Computerized heat transfer simulations are presented which confirm that the efficiency of heat extraction from cylindrical samples contained within thin‐walled metal tubes is higher than from standard flat discoid samples sandwiched between relatively thick aluminium platelets. Based on this fact, we developed a prototype of a new microbiopsy device which enables the quick excision of such cylinders of soft tissues. The device utilizes sharp gold needles of an inner diameter of 200 μm and wall thickness of 50 μm. The frozen sample contained in the soft gold needle permits all the manipulations needed for conventional cryo‐preparation techniques for electron microscopy (e.g. cryo‐sectioning, freeze‐fracturing, freeze‐substitution).
The coefficient of error of optical fractionator population size estimates: a computer simulation comparing three estimators
Authors: Glaser E. M. , Wilson P. D.
Published: March 2003

The optical fractionator is a design‐based two‐stage systematic sampling method that is used to estimate the number of cells in a specified region of an organ when the population is too large to count exhaustively. The fractionator counts the cells found in optical disectors that have been systematically sampled in serial sections. Heretofore, evaluations of optical fractionator performance have been made by performing tests on actual tissue sections, but it is difficult to evaluate the coefficient of error (CE), i.e. the precision of a population size estimate, by using biological tissue samples because they do not permit a comparison of an estimated CE with the true CE. However, computer simulation does permit making such comparisons while avoiding the observational biases inherent in working with biological tissue.

This study is the first instance in which computer simulation has been applied to population size estimation by the optical fractionator. We used computer simulation to evaluate the performance of three CE estimators. The estimated CEs were evaluated in tests of three types of non‐random cell population distribution and one random cell population distribution. The non‐random population distributions varied by differences in ‘intensity’, i.e. the expected cell counts per disector, according to both section and disector location within the section. Two distributions were sinusoidal and one was linearly increasing; in all three there was a six‐fold difference between the high and low intensities. The sinusoidal distributions produced either a peak or a depression of cell intensity at the centre of the simulated region. The linear cell intensity gradually increased from the beginning to the end of the region that contained the cells. The random population distribution had a constant intensity over the region. A ‘test condition’ was defined by its population distribution, the period between consecutive sampled sections and the spacing between consecutive sampled disectors. There were 1000 independently simulated cell populations for each test condition, and a ‘trial’ was conducted for each of these cell populations. In each trial we calculated the (unique) true CE of the population size estimate and the three CE estimates obtained by applying the Scheaffer–Mendenhall–Ott (SMO) and both Gundersen–Jensen (GJ) estimators. We compared the estimated CEs with the true CEs for each population distribution. We found that the CE estimates obtained by the SMO estimator were closer to the true CEs and had less scatter than those of the nugget‐modified GJ estimator. Both had small positive bias. The CE estimates obtained by the unmodified GJ estimator exhibited widely varying bias and large scatter. In all the population distributions we tested, the average true CE was very nearly proportional to 1/√Q¯T, where Q¯T is the average number of cells counted in the two‐stage systematic sample.
Exit wave reconstruction using a conventional transmission electron microscope
Authors: Steinecker A. , Mader W.
Published: March 2003

Exit wave reconstruction of a focus series of Ge in [110] using the PAM–MAL algorithm was performed on a conventional electron microscope. The simulated images using the reconstructed object wave match very well with those obtained experimentally. Amplitudes from the complex wave function were extracted by means of local Fourier transformation. Crystal thickness and tilt were determined locally by quantitative comparison of the reconstructed amplitudes with amplitudes from multislice calculations. Detailed analysis yields the quasicoherent imaging approach used in the PAM–MAL algorithm to produce the largest error in the analysis. For the Ge crystal specimen parameters were quantified to spatial frequencies of 5 nm−1. In the case of an object producing strong diffracted beams, the reconstruction may fail because the quasicoherent approximation will not describe correctly the nonlinear image formation.
Quantitative comparison of images and transforms
Authors: Saxton W. O.
Published: March 2003

Many different measures are currently used for quantifying the difference between pairs of images. Those in common use are surveyed, their interrelationships are made clear and their dependence on transform band‐width pointed out. Adaptations to image transforms, with complex data, are given. For comparing simulated with experimental images, the χ2 difference is recommended as allowing a simple prediction of the likelihood of obtaining at random a difference as great as that actually obtained. In particular, the χ2 difference in Fourier space is shown to be the only interpretable difference measure available in cases when the image noise is not normally distributed or when — as is normally the case — the noise in neighbouring image pixels is not independent.
Photogrammetric calibration of a stereo light microscope
Authors: DANUSER G.
Published: March 2003

Quantitative stereo light microscopy has been used in very rare cases to obtain geometric representations of microscopic objects. Although the instrument itself has limitations, most of them efficiently overcome by other 3D microscopes, it bears considerable advantages in observing and measuring dynamic scenes with multiple objects. The biggest asset of the stereo method is that the full 3D work space is imaged in one shot — a property which distinguishes stereo from all the scanning techniques in 3D microscopy. With the work presented in this paper, we contribute to making the stereo technique applicable to the numerous mensuration tasks in the microscopic domain, where its potential would be invaluable.

We report the photogrammetric calibration of a common main objective lens type stereo light microscope. Such a calibration is the initial step in ensuring accurate measurements with this instrument. First, we derive a mathematical formulation of the imaging function and discuss the estimation of the parameters involved. Then, three main problems of the practical implementation of the framework are addressed: the finding of a calibration standard, the automatic measuring of the many image coordinates required, and the stabilization of the parameter estimation. Finally, results of various calibration runs are presented and analysed under different aspects. Among these, the most important is the accuracy of the calibrated instrument in measuring 3D positions and positional relationships. With a Zeiss Stemi 11, Achromat 1.6 × we achieve accuracies of 1‰ laterally and 1–2% axially relative to the volume of the work space. On the highest magnification level this corresponds to 700 nm and 1.8 μm, respectively.
Non‐coating fixation techniques or redundancy of conductive coating, low kV FE‐SEM operation and combined SEM/TEM of biological tissues
Authors: Jongebloed  , Stokroos  , Van Der Want  , Kalicharan
Published: March 2003

Non‐coating fixation methods, in particular the tannic acid/arginine/osmium tetroxide procedure, are employed for a number of reasons on the guinea‐pig organ of Corti hair cell stereocilia glycocalyx and the imprints of the stereocilia at the bottom side of the tectorial membrane, and on the rat and cat intestinal epithelial microvilli glycocalyx and mucus‐producing goblet cells.

These methods are used firstly to confirm that non‐coating prepared specimens can be embedded for TEM observation at 60–100 kV without loss of detail information, and these images can be compared with cryo‐FE‐SEM images of the same structure/tissue. Secondly, they show that specimens treated according non‐coating techniques become optimally preserved and electrically conductive, so that no external conductive coating is required. In this way a comparison of images of subsequent fresh fracture faces is possible without a decrease in information on detail, which otherwise could happen after subsequent coating layers required after standard fixation. Thirdly, they show that non‐coating methods can be used quite well with low accelerating voltages because the osmium–tannic acid complex in the specimen surface produces a large number of backscattered and secondary electrons in the surface layer, showing in particular surface phenomena. Fourthly, they show that with an optimal non‐coating preservation, in combination with a well‐balanced pre‐fixation mixture, preparation artefacts due to extraction and even dehydration and drying are minimized. This is compared with images of the organ of Corti hair cells treated with a so‐called three‐aldehyde pre‐fixation mixture, which causes disrupted stereocilia to cling onto the bottom side of the tectorial membrane.
Three‐dimensional morphometry in scanning electron microscopy: a technique for accurate dimensional and angular measurements of microstructures using stereopaired digitized images and digital image analysis
Authors: Minnich  , Leeb  , Bernroider  , Lametschwandtner
Published: March 2003

A method for accurate dimensional and angular measurements of microstructures analysed in the scanning electron microscope is described. The method considers central and parallel projections and involves (a) digital image acquisition of stereopaired images from the scanning electron microscope's photodisplay, (b) generation of 3D‐image representations, (c) setting of measuring points in the digitized stereopaired images, (d) computation of exact space coordinates (x/y/z) from the corresponding point coordinates (xL/yL; xR/yR), (e) determination of distances and angles between consecutive corresponding points using vector equations, and (f) transfer of computed data into spreadsheets of the data analysis software using dynamic data exchange with simultaneous graphical display of the frequency distribution of variables.

Measurements performed on specimens with known dimensions (grid with 10 μm wide square meshes, polystyrene beads with 0.33 μm diameter) and angles (synthetic crystals of K(Al,Cr)[SO4], CuSO4.5H2O and NaCl) revealed a high accuracy in dimensional as well as angular measurements (total error 1 ± 0.5%).

In Monte Carlo experiments the overall error was found to depend strongly on the size of the measured structure relative to the size of the measurement field (field width).
Grain size distribution analysis in polycrystalline LiF thin films by mathematical morphology techniques on AFM images and X‐ray diffraction data
Authors: Cremona  , Mauricio  , Scavarda Do Carmo  , Prioli  , Nunes  , Zanette  , Caride  , Albuquerque
Published: March 2003

The influence of the deposition temperature on the grain size of polycrystalline lithium fluoride (LiF) thin films is studied using a mathematical morphology method. On atomic force microscopy images of the LiF surface, the grain sizes and shapes are determined by applying the watershed technique, together with a shape factor algorithm. Also, the domain size of the film structure, determined by an X‐ray diffraction data analysis, is compared and correlated with the mean grain size as a function of the deposition temperature. In both cases a linear increase with temperature and a very good agreement among the two structural parameters (grain and domain size) was found.
Bituminous emulsions and their characterization by atomic force microscopy
Authors: Loeber  , Alexandre  , Muller  , Triquigneaux  , Jolivet  , Malot
Published: March 2003

We present a new method for observing oil‐in‐water emulsions with a continuous water phase and a dispersed bitumen phase. The fine polydispersed bitumen micelles were adsorbed to an atomically smooth mica substrate and imaged in solution by atomic force microscopy in a liquid cell. The height of the adsorbed bitumen sheet in wet and dry states can be measured and the homogeneity of film formation by coalescence can be determined. Localization of surfactant onto and between bitumen micelles is also visualized.
Quantification of boundary segregation in the analytical electron microscope
Authors: Keast V. J. , Williams D. B.
Published: March 2003

When studying equilibrium grain‐boundary segregation using the small (~1 nm) electron probe of the scanning transmission electron microscope and X‐ray energy dispersive spectroscopy, the assumptions made about the size and shape of the beam–specimen interaction volume may introduce errors in quantification of up to a factor of five. Comparisons between experimental segregation profiles and different theoretical models have shown that a Gaussian model for the electron distribution will provide the best description for the interaction volume. Calculations of minimum detectable segregation levels have shown that optimum sensitivity is not achieved in the thinnest samples or even with the smallest probe sizes. In addition, operating at 300 keV (rather than 100 keV) will halve the minimum detectable segregation level, assuming all other experimental conditions are equal. Rastering the electron probe over a fixed area while acquiring spectra improves the accuracy of quantification but at the price of reducing sensitivity by at least a factor of three.
Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus
Authors: Fisher K. E. , Lowry D. S. , Roberson R. W.
Published: March 2003

We have used the vital fluorescent dye, FM4‐64, as a marker of membrane development during zoospore formation in living zoosporangia of Allomyces macrogynus. Membrane development was visualized and documented using standard epifluorescence and laser scanning confocal microscopy. Video‐enhanced light microscopy and transmission electron microscopy, using cryopreparation methods, were also employed in this study. In the first 10–12 min after the induction of zoospore formation, only the plasma membrane labelled with FM4‐64. During this time, nuclei were strictly located in the cortical cytoplasm with their associated centrosomes positioned immediately adjacent to the plasma membrane (Lowry & Roberson, 1997). Between 12 and 20 min post‐induction, increased fluorescence appeared along regions of the plasma membrane adjacent to the nuclei. From these sites, membranes (i.e. cleavage elements) extended laterally within the cortex and then, in conjunction with nuclear migration, rapidly elongated into the sporangial cytoplasm. By 25–35 min post‐induction, cleavage elements had ramified throughout the cytoplasm forming a complex, interconnected membranous network. Transmission electron microscopy revealed that cleavage elements were paired membrane sheets with a lumen consisting of an electron opaque, granular matrix. Cleavage elements developed into a highly ordered network by 35–40 min post‐induction, which fully delimited zoospore initials into polyhedral‐shaped cells. Zoospore discharge occurred between 40 and 50 min post‐induction. Our results have shown that cleavage elements undergo four stages of development during zoospore formation in A. macrogynus: (i) development of membrane initials, (ii) cortical extension, (iii) cytoplasmic elongation and ramification and (iv) zoospore initial delimitation.
A two‐channel four‐dimensional image recording and viewing system with automatic drift correction
Authors: Bürglin T. R.
Published: March 2003

Four‐dimensional image acquisition systems have been described to analyse various developmental processes, for example, the Caenorhabditis elegans cell lineage. A practical problem that is often encountered during recordings is mechanical slippage of the microscope stage, causing the sample to drift out of focus. Furthermore, with the advent of green fluorescent protein (GFP) as an in vivo marker, affordable two‐channel imaging systems are needed to correlate gene expression with changes through development. To overcome the mechanical drift a device‐independent, software‐only solution for the MacOS was devised that can compensate for Z‐axis drifts in sample position. The software also allows recording of 4D stacks in two channels. To correct for drift, a small reference object beside the main object to be recorded is kept in focus using a simple autofocus principle, and this automatic drift correction allows for effective 4D recordings. In addition to the Z‐axis drives and the shutters of the microscope, a video camera can be computer controlled to switch between two light levels. Second channel live GFP recordings are presently limited by the fact that the high intensity of the blue light heats and kills C. elegans embyros quickly. To view and annotate the stacks a MacOS viewing application was developed.
Near‐field fluorescence imaging of single molecules with a resolution in the range of 10 nm
Authors: Hosaka N. , Saiki T.
Published: March 2003

We demonstrate fluorescence imaging of single molecules, by near‐field scanning optical microscopy (NSOM), using the illumination‐collection mode of operation, with an aperture probe. Fluorescence images of single dye molecules were obtained with a spatial resolution of 15 nm, which is smaller than the diameter of the aperture (20 nm) of the probe employed. Such super‐resolution may be attributable to non‐radiative energy transfer from the molecules to the coated metal of the probe since the resolution obtained in the case of conventional NSOM is limited to 30–50 nm due to penetration of light into the metal.
Quantitative analyses of the biomineralization of different hard tissues
Authors: Arnold S. , Plate U. , Wiesmann H. P. , Stratmann U. , Kohl H. , Höhling H. J.
Published: March 2003

The primary crystallites of the different developing hard tissues have an apatite structure. However, they have crystal lattice distortions representing an intermediate state between amorphous and fully crystalline. We have applied energy‐filtering transmission electron microscopy in the selected area electron diffraction mode to analyse different stages of crystal formation in dentine, bone, enamel and inorganic apatite mineral. We have obtained quantitative information on the degree of crystal lattice distortion using the paracrystal theory of Hosemann and Bagchi.

We have found that the early formed crystallites of the hard tissues being analysed have a paracrystalline character comparable to biopolymers. However, with maturation, the lattice fluctuations of the crystallites of the hard tissues bone, enamel and dentine decrease to form a typical (para)crystalline character. Also the decrease of the organic proportion in the matrix corresponds to the decrease of the lattice fluctuation of the crystallites in the different hard tissues during maturation.
High resolution protein localization using soft X‐ray microscopy
Authors: Meyer‐Ilse W. , Hamamoto D. , Nair A. , Lelièvre S. A. , Denbeaux G. , Johnson L. , Pearson A. L. , Yager D. , Legros M. A. , Larabell C. A.
Published: March 2003

Soft X‐ray microscopes can be used to examine whole, hydrated cells up to 10 µm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X‐ray transmissive ‘water window’, where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X‐ray microscope at photon energies just below the oxygen edge (λ = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver‐enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X‐ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three‐dimensional information on the distribution of proteins in cells.
Combined application of electron backscatter diffraction and stereo‐photogrammetry in fractography studies
Authors: Davies P. A. , Randle V.
Published: March 2003

The main aim of this paper is to report on recent experimental developments that have succeeded in combining electron back‐scatter diffraction (EBSD) with stereo‐photogrammetry, compared with two other methods for study of fracture surfaces, namely visual fractography analysis in the scanning electron microscope (SEM) and EBSD directly from facets. These approaches will be illustrated with data relating to the cleavage plane orientation analysis in a ferritic and C‐Mn steel. It is demonstrated that the combined use of EBSD and stereo‐photogrammetry represents a significant advance in the methodology for facet crystallography analysis. The results of point counting from fractograph characterization determined that the proportions of intergranular fracture in C‐Mn and ferritic steels were 10.4% and 9.4%, respectively. The crystallographic orientation was determined directly from the fracture surface of a ferritic steel sample and produced an orientation distribution with a clear trend towards the {001} plane. A stereo‐photogrammetry technique was validated using the known geometry of a Vickers hardness indent. The technique was then successfully employed to measure the macroscopic orientation of individual cleavage facets in the same reference frame as the EBSD measurements. Correlating the results of these measurements indicated that the actual crystallographic orientation of every cleavage facet identified in the steel specimens is {001}.
Studies of cell division (mitosis and cytokinesis) by dynamic secondary ion mass spectrometry ion microscopy: LLC‐PK1 epithelial cells as a model for subcellular isotopic imaging
Authors: Chandra S.
Published: March 2003

The feasibility of the renal epithelial LLC‐PK1 cell line as a model for cell division studies with secondary ion mass spectrometry (SIMS) was tested. In this cell line, cells undergoing all stages of mitosis and cytokinesis remained firmly attached to the substrate and could be cryogenically prepared. Fractured freeze‐dried mitotic cells showed well‐preserved organelles as revealed by fluorescence imaging of rhodamine‐123 and C6‐NBD‐ceramide by confocal laser scanning microscopy. Secondary electron microscopy analysis of fractured freeze‐dried dividing cells revealed minimal surface topography that does not interfere in isotopic imaging of both positive (39K, 23Na, 24Mg, 40Ca, etc.) and negative (31P, 35Cl, etc.) secondaries with a CAMECA IMS‐3f ion microscope. Mitotic cells revealed well‐preserved intracellular ionic composition of even the most diffusible ions (total concentrations of 39K+ and 23Na+) as revealed by K : Na ratios of approximately 10. Structurally damaged mitotic cells could be identified by their reduced K : Na ratios and an excessive loading of calcium. Quantitative three‐dimensional SIMS analysis was required for studying subcellular calcium distribution in dividing cells. The LLC‐PK1 model also allowed SIMS studies of M‐phase arrested cells with mitosis‐arresting drugs (taxol, monastrol and nocodazole). This study opens new avenues of cell division research related to ion fluxes and chemical composition with SIMS.
Multiphoton versus confocal high resolution z‐sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors
Authors: Drummond D. R. , Carter N. , Cross R. A.
Published: March 2003

Multiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z‐axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.