bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in autonomic nervous function during the 4‐year follow‐up in middle‐aged diabetic and nondiabetic subjects initially free of coronary heart disease
Authors: MUSTONEN J. , UUSITUPA M. , MÄNTYSAARI M. , LÄNSIMIES E. , PYÖRÄLÄ K. , LAAKSO M.
Published: November 2003

Objectives. To study the changes in autonomic nervous function during the 4‐year follow‐up period in diabetic patients and to investigate factors predicting autonomic nervous dysfunction

Design. A 4‐year follow‐up study.

Setting. At baseline the study subjects without known cardiovascular disease were recruited from a large group of diabetic and nondiabetic subjects selected randomly from a baseline population.

Subjects. Middle‐aged control subjects (n=44), patients with insulin‐dependent diabetes mellitus (n=32) and patients with non‐insulin‐dependent diabetes mellitus (n=32) were studied at baseline and after the 4‐year follow‐up.

Interventions. Autonomic nervous function tests and exercise test at baseline and after the 4‐year follow‐up.

Results. At the baseline, heart rate variation during deep breathing was significantly lower in patients with insulin‐dependent diabetes (13.0±1.2 beats min−1; P<0.05) and in patients with non‐insulin‐dependent diabetes (12.9±1.5 beats min−1; P<0.05) than in control subjects (16.6±1.1 beats min−1). At baseline, autonomic nervous function score was significantly higher indicating disturbed autonomic nervous function in patients with insulin‐dependent diabetes (1.74±0.19; P<0.01) than in control subjects (1.24±0.14), but the difference was not significant between control subjects and patients with non‐insulin‐dependent diabetes (1.47±0.12). During the follow‐up, autonomic nervous function score increased in patients with noninsulin‐dependent diabetes to 2.00±0.21 (P<0.001, as compared to baseline), but did not change in patients with insulin‐dependent diabetes (1.77±0.18) or control subjects (1.22±0.12). In both diabetic groups, the deterioration of autonomic nervous function score during the 4‐year follow‐up was associated with poor glycaemic control at baseline. Clinical manifestation of coronary heart disease was found in three (7%) control subjects, 12 (37%; P<0.001) patients with insulin‐dependent diabetes and 11 (34%; P<0.01) patients with non‐insulin‐dependent diabetes mellitus at follow‐up examination. Autonomic nervous function was more abnormal in those insulin‐dependent diabetic patients with coronary heart disease than those without.

Conclusions. During the 4‐year follow‐up the impairment in autonomic nervous function occurred mainly in patients with noninsulin‐dependent diabetes. Poor glycaemic control appears to be an important determinant of the progression of autonomic nervous dysfunction in diabetes.
Paracetamol‐induced cholestatic and granulomatous liver injuries
Authors: LINDGREN A. , ALDENBORG F. , NORKRANS G. , OLAISON L. , OLSSON R.
Published: November 2003

Objective. To describe uncommon (previously unreported) types of adverse liver reactions to paracetamol

Design. In addition to describing patients, with uncommon types of liver reactions to paracetamol, admitted to our hospitals, we surveyed all the liver reactions to paracetamol reported to the Swedish Adverse Drug Reactions Advisory Committee from 1973 to 1993.

Setting. The Swedish population of 8 million inhabitants.

Measurement. Extensive medical evaluation.

Results. We found one case with a cholestatic liver reaction and one with granulomatous hepatitis. The reactions were probably idiosyncratic and took several months to disappear.

Conclusion. In addition to the well‐known dose‐related toxic liver damage paracetamol may rarely cause non‐dose‐related severe, prolonged cholestasis or granulomatous hepatitis with cirrhosis.
Interpretation of data from multiple trials: a critical review
Authors: CONN H. O.
Published: November 2003

The objective was to review the current status of meta‐analysis, the process of combining the results of similar investigations, which was described in 1976. It has evolved rapidly, and thousands have already been published. Meta‐analysis may be performed in a narrative form or in the preferred statistical format, which is more rigorous and demanding. The criteria for designing and performing a meta‐analysis of high quality are presented and discussed in detail, including selection criteria, quality, biases and heterogeneity, both of component studies and of results. Critical reviews of meta‐analyses are presented. Examples are given from my own field – hepatology – and from my own research experience. Although meta‐analysis is simple in concept, it is complex in execution. If performed with insufficient insight or attention to detail, it may give ambiguous or erroneous results.
Interrelationships between weight loss, body fat distribution and sex hormones in pre‐ and postmenopausal obese women
Authors: TURCATO E. , ZAMBONI M. , DE PERGOLA G. , ARMELLINI F. , ZIVELONGHI A. , BERGAMO‐ANDREIS I. A. , GIORGINO R. , BOSELLO O.
Published: November 2003

Objectives. Relationships between regional body fat distribution and sex hormones as well as changes in sex hormones after weight loss were evaluated.

Setting. All subjects were hospitalized in the Institute of Internal Medicine of the University of Verona.

Subjects. Twenty‐six premenopausal (age 33.7± 10.2 years) and 15 postmenopausal (age 57.9±5.9 years) obese women.

Interventions. Body weight, body‐mass index, waist and hip circumferences, visceral fat by computed tomography and sex hormones were evaluated before and after 4 weeks on a very low energy diet.

Results. Body‐mass index was higher in pre‐ than in postmenopausal women, although the difference was not significant. Total and free testosterone were significantly higher in the pre‐ than in the postmenopausal group (P<0.001). Significant negative correlations were found between age and total testosterone (r=−0.65; P<0.001), free testosterone (r=−0.54; P<0.001), androstenedione (r=−0.46; P<0.01) and urinary cortisol excretion (r=−0.50; P<0.01). A negative correlation was found between visceral fat and total testosterone (r=−0.41; P<0.01). After adjusting for age, the negative correlation between total testosterone and visceral fat encountered both in the subject group as a whole and in premenopausal women was no longer significant, whilst a significant negative association between visceral fat and sex hormone binding globulin (SHBG) (r=−0.56; P<0.001) was always found. When step‐down regression analysis was used to evaluate the joint effect of age, menopausal status, and anthropometric and metabolic variables on sex hormones, age was the most powerful independent variable for predicting total testosterone, free testosterone and androstenedione levels, whilst menopausal status was the most powerful predictor of FSH and LH levels. Changes in hormones after VLED were analysed separately in pre‐ and postmenopausal women. None of the hormones changed significantly after VLED in the postmenopausal group, except for FSH values. LH, free testosterone and urinary cortisol excretion values decreased significantly after VLED in the premenopausal group.

Conclusions. Our data show that age, to a greater extent than visceral fat, seems to be negatively associated with steroid sex hormones. Weight loss seems to be associated with changes in sex hormones only in premenopausal women.
Microcirculation: its significance in clinical and molecular medicine
Authors: FAGRELL B. , INTAGLIETTA M.
Published: November 2003

Microcirculation represents the smallest functional unit of the cardiovascular system, where the interaction between blood and tissue creates the environment necessary for cell function. Analysis of physiology and pathophysiology of this system gives a unique perspective to the disease process, and provides the link between clinical and molecular medicine. The present status and future directions of this medical and scientific frontier were assessed and projected by experts in the field at a meeting in Italy in 1995, and the conclusions are presented in this article.
Atrial natriuretic peptides predict mortality in the elderly
Authors: WALLÉN THOMAS , LANDAHL STEN , HEDNER THOMAS , HALL CHRISTIAN , SAITO YOSHIHIKO , NAKAO KAZUWA
Published: November 2003

Objectives. To ascertain whether atrial natriuretic peptides could be used as prospective and independent predictors of total mortality in an elderly population.

Design. Atrial natriuretic peptides, ANP(1–98) and ANP(99–126), were measured in 541 subjects from the 85‐year‐old population of Gothenburg, Sweden. Before the study cardiovascular disorders such as congestive heart failure, ischaemic heart disease, hypertension and atrial fibrillation were defined. Total mortality was recorded during the prospective 60‐month follow‐up period.

Setting. Individuals aged 85 years from the population of Gothenburg, Sweden, were visited once at home and made one visit to Vasa Hospital.

Main outcome measures. Sixty‐month mortality in relation to circulating concentrations of atrial natriuretic peptides.

Results. Circulating concentrations of ANP(1–98) and ANP(99–126) were significantly correlated with 60‐month mortality in the total study population (ANP(1–98), P<0.001; ANP(99–126), P<0.01). In subjects with cardiovascular disorders, 60‐month mortality was significantly correlated with increased concentrations of ANP(1–98) (P<0.01) and ANP(99–126) (P<0.05). In subjects with no defined cardiovascular disorder, 60‐month mortality was significantly correlated with increased ANP(1–98) concentrations (P<0.01).

Conclusions. In the elderly population, atrial peptides predict mortality in subjects with defined cardiovascular disorders as well as in the total population and may predict future cardiovascular disorder.
Prevalence of activated protein C resistance and analysis of clinical profile in thromboembolic patients. A Belgian prospective study
Authors: HAINAUT P. , AZERAD M.‐A. , LEHMANN F. , SCHLIT A.‐F. , ZECH F. , HEUSTERSPREUTE M. , PHILIPPE M. , COL C. , LAVENNE E. , MORIAU M.
Published: November 2003

Objectives. To assess the prevalence of activated protein C resistance (APC‐R) among healthy subjects and thromboembolic patients and to determine the clinical characteristics associated with APC‐R.

Design. A prospective study.

Setting. One academic medical centre.

Subjects. 91 health controls and 126 thromboembolic patients.

Measurements. Patients and control were genotyped for the factor V Leiden (VaQ506) mutation. The anticoagulant response of the patient's plasma to activated protein C was also determined.

Results. The frequency of APC‐R was 3.3% among healthy control subjects and 22% among thrombotic patients of whom 18% were heterozygous and 4% were homozygous. The mean age at the first thrombotic event and the severity of thrombotic disease including the proportion of proximal deep vein thrombosis and the frequency of lung embolism were identical among APC‐R positive and negative patients. A family history of thromboembolic disease was elicited more frequently in APC‐R positive than in APC‐R negative patients (57% vs. 22%, P<0.001). The recurrence rate was higher for APCR‐R positive patients (57% vs. 34%, P<0.05). The percentage of cases with a factor predisposing to thrombosis was very similar in APC‐R positive (57%) and negative (68%) patients.

Conclusions. A familial history of thromboembolic disease and recurrences are significantly more frequent among APC‐R positive than APC‐R negative patients.
Delayed hypersensitivity to flurbiprofen
Authors: ROMANO A. , PIETRANTONIO F.
Published: November 2003

Immune‐mediated reactions to nonsteroidal antiinflammatory drugs (NSAIDs) are unusual, and true allergy to the drug flurbiprofen has never been documented. We observed a patient who developed a maculopapular rash 48 h after beginning oral therapy with this drug, and 2 days later, angioedema and hypotension. Patch tests with flurbiprofen were positive 48 and 72 h after application. The clinical and allergologic features of this case strongly suggest type IV hypersensitivity. Patch testing with NSAIDs is useful for confirming clinical diagnoses of delayed hypersensitivity to these drugs.
Ethnic differences in the clinical and laboratory associations with retinopathy in adult onset diabetes: studies in patients of African, European and Indian origins
Authors: KALK W. J. , JOANNOU J. , NTSEPO S. , MAHOMED I. , MAHANLAL P. , BECKER P. J.
Published: November 2003

Objective. To evaluate the prevalence of diabetic retinopathy (DR) and its associations in adult onset diabetic patients of African, European and Indian origins.

Design. The prevalence of retinopathy was determined by 60° retinal photography in 507 consecutive out‐patients. Clinical and laboratory associations were evaluated.

Setting. Diabetes clinic in a large community hospital.

Main outcome measures. The associations between clinical and laboratory measurements with retinopathy.

Results. African patients (A) had shorter duration of diabetes (P < 0.001), higher HbA1 levels (P < 0.01) compared to those of Europeans (E) and Indian (I) extraction. A also had lower C‐peptide levels (median 0.57 nmol L−1; vs. E, 0.81 nmol L−1 and I, 0.93 nmol L−1) (P < 0.001). The prevalences of retinopathy at diagnosis (21–25%) and overall were similar (A 37%, E 41%, I 37%). Severe DR was more frequent in the Africans (52%, P < 0.0001) and Indians (41%, P = 0.03) compared to the Europeans (26%). In Africans DR was significantly associated only with duration of diabetes (P < 0.0001) and macro‐albuminuria (P = 0.01); in I it was also associated with systolic BP (P = 0.03); in E also with lower C‐peptide levels (P = 0.0002), worse glycaemic control and greater use of insulin (P < 0.0001). In patients with DR insulin was used less frequently in A (35%) than in E patients (62%) (P = 0.001).

Conclusions. In South Africa, the African population with adult onset diabetes has the highest prevalence of severe retinopathy, probably the result of very poor glycaemic control attributable to more severe insulinopenia and infrequent insulin treatment. Visual loss from diabetic retinopathy is likely to be considerable in Africans.
Moderate beer consumption and the blood coagulation in patients with coronary artery disease
Authors: GORINSTEIN S. , ZEMSER M. , LICHMAN I. , BEREBI A. , KLEIPFISH A. , LIBMAN I. , TRAKHTENBERG S. , CASPI A.
Published: November 2003

Objectives. To evaluate the influence of a short period of moderate beer consumption on the status of the thrombolitic activity in patients with coronary artery disease (CAD).

Subjects and design. From 28 patients with two‐ or three‐vessel CAD 22 were randomly assigned to an experimental group (EG) and six to control group (CG). Before and after completion of the study every one of the 28 patients was examined and a wide range of laboratory tests was performed.

Setting. A University Hospital in Israel.

Intervention. Of the EG, 22 patients consumed 330 mL of beer day−1 (20 g of alcohol) for the 30‐day period in addition to the usual antiatherosclerotic diet. Patients of the CG did not consume alcohol beverages.

Main outcome measures. Fibrinogen, prothrombin time (PT), coagulant activity of Factor VII (F VIIc) and Factor VII antigen (F VIIag), and plasminogen activator inhibitor (PAI) levels were studied.

Results. After the investigation a statistically significant decrease was found in F VIIc (P < 0.01) and F VIIag (P < 0.001) and to a lesser extent a decrease in the value of PAI. Fibrinogen and PT remained unchanged in the EG also.

Conclusions. Even a short period of moderate beer consumption results in a decrease in thrombogenic activity. The only sensitive tests were F VIIc and F VIIag. The decrease in thrombogenic activity may be the main cause of decreased mortality in patients with CAD who consume moderate quantities of alcoholic beverages.
4Pi‐confocal images with axial superresolution
Authors: Schrader M. , Hell S. W.
Published: November 2003

We present two‐photon excitation 4Pi‐confocal images of clustered fluorescence beads demonstrating three‐dimensional far‐field light microscopy with unprecedented resolution. For an excitation wavelength of 760 nm, the lateral and axial resolution amounts to 200 and 145 nm, respectively. The four‐fold improved axial resolution is achieved by engineering the point‐spread function through a suitable combination of aperture enlargement, two‐photon excitation, confocalization and three‐point deconvolution. In contrast to their confocal counterparts, 4Pi‐confocal images do not exhibit the typical axial elongation. The axial resolution in the 4Pi‐confocal images corresponds to about one‐fifth of the wavelength and surpasses the lateral resolution by 25%.
Mapping of ELNES on a nanometre scale by electron spectroscopic imaging
Authors: MAYER J. , PLITZKO J. M.
Published: November 2003

The amorphous interfacial layer between Si substrates and diamond films grown by plasma‐assisted chemical vapour deposition has been studied by electron spectroscopic imaging. The amorphous layer consists mainly of carbon, which can only be distinguished from the diamond film by analysis of the near‐edge structure (ELNES) of the carbon K edge. Series of electron spectroscopic images were acquired across the carbon K edge and were analysed in order to reveal the presence of the π*‐ and σ*‐excitations. After background removal from the corresponding images, phase maps for the distribution of sp2 and sp3 hybridized carbon can be obtained. From the whole series of images, electron energy‐loss spectra can be extracted for any given area in the images. The results show that the amorphous layer covers large areas along the interface and that regions with only 1–2 nm layer thickness can clearly be analysed. The results obtained with the electron spectroscopic imaging technique will be compared with results obtained on a field emission gun scanning transmission electron microscope.
A rapid‐flow perfusion chamber for high‐resolution microscopy
Authors: KAPLAN D. , BUNGAY P. , SULLIVAN J. , ZIMMERBERG J.
Published: November 2003

Perfusion chambers employing laminar flow have dead volumes and unstirred layers which limit the minimum time required to effect a change in the local chemical environment of the sample. We have fabricated and tested a chamber capable of developing turbulent flow at reasonable flow rates of aqueous solutions. Transition to turbulence occurred at ≈1 mL s−1. To minimize dead space, a dual‐exit cross‐flow pattern was employed. The chamber was designed to mount on optical microscope stages for visual sample observation supplemented by a variety of techniques, such as fluorescence, light scattering and electrochemical monitoring. As indicated by fluorescence from a fluorescein‐labelled protein film adherent to the chamber wall, local pH changes were produced within 200 ms. Use of the chamber is illustrated by measurements of stopped‐flow kinetics in both calcium‐triggered cortical granule exocytosis and influenza virus haemagglutinin‐mediated cell–cell fusion.
Comparison of slam‐freezing and high‐pressure freezing effects on the DNA cholesteric liquid crystalline structure
Authors: Leforestier A. , Richter K. , Livolant F. , Dubochet J.
Published: November 2003

Using in parallel electron microscopy of ultrathin frozen‐hydrated sections and freeze‐fracture replicas, we compare the ultrastructural consequences of two freezing techniques: slam‐freezing at liquid helium temperature and high‐pressure freezing, on a model system, the DNA cholesteric liquid crystalline phase. Both freezing techniques are able to vitrify DNA liquid crystalline solutions containing up to 85% water, but induce structural rearrangements of the molecular organization. The cholesteric structure is preserved by the slam‐freezing method despite the formation of periodic distortions induced by the mechanical compressive stress. In contrast, high‐pressure freezing does not preserve the structure of the liquid crystal: the long‐range cholesteric stratification disappears, and the local continuous twist between molecules is modified. These results show that vitrification, though necessary, may not be a sufficient token of preservation of the native state of hydrated materials. We discuss the possible origins of the molecular rearrangements that have time to occur in the specimens as a result of the low freezing rate permitted by the high‐pressure freezing process.
The design and implementation of a high‐fidelity Raman imaging microscope
Authors: GOLDSTEIN S. R. , KIDDER L. H. , HERNE T. M. , LEVIN I. W. , LEWIS E. N.
Published: November 2003

We describe a Raman imaging microscope that produces high‐fidelity, large format Raman images and Raman spectra from samples as small as 1 μm in size. Laser illumination is delivered to the object by means of an infinity corrected microscope objective, either by a galvanometer scanning system or a widefield fibre optic. Wavelength selection of Raman scattered emission is achieved by an acousto‐optic tunable filter (AOTF), which maintains image fidelity and provides either continuous or random wavelength selection. The collimated AOTF output is imaged first by a tube lens and then by a projection lens onto a cooled silicon CCD array. Instrument features, including factors that determine the system’s spatial and spectral resolution, and design considerations are discussed in detail. Images and spectra of test objects and samples that demonstrate the capability of this imaging spectrometer are presented. The potential of intrinsic chemical imaging is discussed in terms of its use in the analyses of a variety of chemical and biological samples.
Contrast effects using a two‐detector system in low‐voltage scanning electron microscopy
Authors: KÄSSENS M. , REIMER L.
Published: November 2003

A system of two opposite Everhart–Thornley detectors A and B has formerly been applied in conventional SEM for electron energies between 5 and 20 keV to separate material, topographic and other types of contrast by sum and difference signals. This technique can also be used successfully for low‐voltage scanning electron microscopy. The decreasing information depth with decreasing electron energy shows differences in the surface composition and contamination which cannot be observed beyond 5 keV. Also below 5 keV material and topographic contrast can be separated and increased by the A + B and A − B signals, respectively.
Maximum‐likelihood estimation for discrete Boolean models using linear samples
Authors: HANDLEY J. C. , DOUGHERTY E. R.
Published: November 2003

An observation of a Boolean model consists of a set of covered points. In the one‐dimensional discrete case, the likelihood function of an observation can be expressed via the lengths of sequences of covered points and points not covered, called black and white runlengths, respectively. The black and white runlengths are independent random variables whose respective distributions determine the one‐dimensional discrete Boolean model completely. Under certain conditions, a two‐dimensional discrete Boolean model induces a one‐dimensional discrete Boolean model, thereby allowing the likelihood function of a one‐dimensional observation to be expressed in terms of the parameters of the two‐dimensional model. This relationship enables maximum likelihood estimation to be performed on the two‐dimensional model using linear samples. Examples are given including an application involving micrographs of toner particles.
A precise light attenuation correction for confocal scanning microscopy with O(n4/3) computing time and O(n) memory requirements for n voxels
Authors: Margadant F. , leemann T. , Niederer p.
Published: November 2003

Some dyes and tissues observed by confocal fluorescence microscopy show remarkable opacity, caused by absorption and scattering within the scanned volume. The efficiency of the excitation and fluorescence process may vary by a factor of 10 from top to bottom regions even in not very extended scan volumes. Based on known attenuation properties of a specimen, the amount of damping is computed by integrating the attenuation along all light paths within the numerical aperture of the objective, Thus, to correct a single volume element (voxel), one has to take into account the damping within the whole extended conical volume between the lens and the focus, resulting in intolerable execution times. Common approaches reduce resolution or simplify the integration paths, thereby resulting in either a loss of fine resolution or showing a low resolution versus computation time ratio. This paper presents a more efficient reformulation of this spatial integration process without simplifying the physical background. This algorithm requires computing times slightly longer than those of programs using simplified physical and statistical approaches. However, the algorithm may be tuned to achieve a precision and stability comparable to exhaustive integration.
A simple cryo‐SEM method for delicate plant tissues
Authors: CRAIG S. , BEATON C. D.
Published: November 2003

A simple modification to the existing preparative procedures for examining delicate plant tissues and insect–plant interactions by cryo‐SEM is described. Samples are attached to a stub with colloidal graphite, introduced into the air lock, evacuated briefly, then transferred onto the cryo‐stage of the preparation chamber where full evacuation and slow cooling take place. After sputter coating with gold, the samples are transferred onto the microscope cold stage and examined at 15 kV.
A confocal fibre scanning microscope for magnetic domain imaging
Authors: Ping G. L. , See C. W. , Somekh M. G.
Published: November 2003

A compact confocal single‐mode optical fibre scanning microscope for imaging magnetic domain structures, based on the polar magneto‐optic Kerr effect, has been developed. The images obtained correspond to those obtained using single mirror scanning but this design offers a more compact structure and can be made more immune from system depolarization which makes two‐axis mirror scanning difficult to implement when very small changes in polarization need to be detected.
New direct observations of asphalts and asphalt binders by scanning electron microscopy and atomic force microscopy
Authors: LOEBER L. , SUTTON O. , MOREL J. , VALLETON J.‐M. , MULLER G.
Published: November 2003

Observations made using AFM and SEM have been combined in order to study the structure of asphalts. Fluorescence microscopy was used to aid in understanding the structural changes occurring when polymer is added to the asphalts.  With the atomic force microscope we are able to study the structure of the asphalts without any pre‐preparation. Despite very low resolution, our study reveal ed a network of asphaltene molecules with regard to asphalt gel. The same result is obtained by SEM observation but with a much better resolution. SEM observation, however, needs an adequate preparation method.  In the presence of polymer we observed a rearrangement of the initial asphaltene association which leads to the assumption that polymer can aggregate the asphaltene phase.
Elimination of the effects of stray light in measurements by total internal reflection aqueous fluorescence (TIRAF)
Authors: GINGELL D. , HEAVENS O.
Published: November 2003

Total internal reflection aqueous fluorescence has been shown to be capable of achieving spatial resolution in surface contours of about 1 nm. When used with highly structured objects, errors in measurements can arise from light scattered either by the object or within the body of the microscope. We describe how these errors can be eliminated when studying surface contours of human platelets.
MULTIVARIATE STATISTICAL ANALYSIS OF FEG‐STEM EDX SPECTRA
Authors: Titchmarsh J. M. , Dumbill S.
Published: November 2003

A multivariate statistical analysis method has been used to examine the information content of a series of EDX spectra acquired using a FEG‐STEM recorded across a segregated grain boundary in a ferritic steel. The spectra have been factored into a set of orthogonal information components (eigenspectra), each of which corresponds to an independent source of information. Two meaningful eigenspectra are identified: one is related to the segregation of P and Cr and the other to a combination of self‐absorption and coherent bremsstrahlung. Spectra are reconstructed from the meaningful eigenspectra to reduce noise and improve the sensitivity for detection of trace element segregation.
The effect of deformation on the lateral resolution of atomic force microscopy
Authors: YANG J. , MOU J. , YUAN J.‐Y. , SHAO Z.
Published: November 2003

A computer model based on the elastic properties of rubber is introduced for the evaluation of the lateral resolution in atomic force microscopy of deformable specimens. The computational results show that, if the full width at half‐height can be defined as the lateral resolution, it is continuously improved at greater probe forces, at the expense of a reduced molecular height. In fact, even for a probe that is bigger than the molecule, the real size of the molecule can be ‘recovered’ at about 25% compression. This result demonstrates that for a better lateral resolution, a greater probe force can be beneficial, provided that the molecule is not moved or damaged and the response remains elastic. Measurements on isolated low‐density lipoproteins (LDL) show that with 26% vertical compression, the lateral size measured in atomic force microscopy is only about 72% of the value predicted by a simple convolution, and is only slightly larger (≈ 13%) than the known size of LDL. Therefore, the results on LDL provide a direct support for the conclusions of the computational model.
Membrane deformation of living glial cells using atomic force microscopy
Authors: HAYDON P. G. , LARTIUS R. , PARPURA V. , MARCHESE‐RAGONA S. P.
Published: November 2003

Using atomic force microscopy (AFM) it has been possible to detect actin filaments that are beneath the cell membrane of living cells despite the fact that the AFM tip is applied to the surface of the cell. To determine whether the AFM tip actually penetrates or deforms the cell membrane we determined whether an intracellularly trapped fluorescent indicator was lost from cells during AFM. Using epi‐fluorescence illumination to monitor the presence of fluo‐3 in the cell, we found that AFM did not cause dye leakage from the cell. Further, force–distance curves indicated that standard tips did not penetrate the membrane while sharper SupertipsTM did. In addition, the physiology of cells was found to be unaffected by AFM with standard tips since volume regulatory signal transduction mechanisms were intact in such studies. Thus, traditional AFM tips deform the cell membrane in order to reveal the presence of subcellular structures.
Electron‐beam‐induced amorphization of ice III or IX obtained by high‐pressure freezing
Authors: SARTORI N. , BEDNAR J. , DUBOCHET J.
Published: November 2003

Cryo‐electron microscopy of vitrified specimens makes it possible to observe fully hydrated biological samples unimpaired by chemical fixation, staining and dehydration. High‐pressure freezing represents important progress since it allows a 10‐fold increase in the vitrification depth. High‐pressure freezing can also induce the formation of undesirable high‐pressure forms of ice. We show that ice III or IX is amorphized under the electron beam at a dose of about 2400 electronsnm−2 and that the resulting amorphous ice is similar to the vitreous water obtained by high‐pressure freezing.
High‐pressure freezing of tissue obtained by fine‐needle biopsy
Authors: HOHENBERG H. , TOBLER M. , MÜLLER M.
Published: November 2003

High‐pressure freezing (HPF) permits adequate cryoimmobilization (without detectable ice crystals after freeze‐substitution) of biological tissue up to a thickness of about 200 μm. Until now the preparation of tissue prior to freezing has been unsatisfactory: sizing of the tissue to the required dimensions takes minutes, during which structural alterations must occur. We demonstrate that the use of a fine‐needle biopsy technique minimizes tissue damage and guarantees sample dimensions close to the optimal thickness for HPF. The tissue cores can be cryoimmobilized within 40 s of excision.
Analysis of efficiency of two‐photon versus single‐photon absorption for fluorescence generation in biological objects
Authors: BRAKENHOFF G. J. , MÜLLER M. , GHAUHARALI R. I.
Published: November 2003

Starting from basic absorption cross‐section values and breakdown limitations on possible field strengths it is shown that the total exposure required for equivalent fluorescence generation by single‐photon absorption (SPA) is at least an order of magnitude lower than by two‐photon absorption (TPA). The difference is such that TPA may in fact in many cases offer no advantage over SPA with respect to the damage induced during fluorescence imaging of biological materials.
Multimodal microscopy by digital image processing
Authors: Glasbey C. A. , Martin N. J.
Published: November 2003

A matching algorithm is proposed for aligning microscope images obtained using different modalities, making use of cross‐correlations of outputs from Prewitt's edge filter. Brightfield, phase contrast and differential interference contrast microscope images of algal and bacterial cells from an experimental, high‐rate algal pond are used for illustration. The information content of multimodal images is explored using principal components analysis and colour displays, and an image which represents optical thickness is constructed digitally.
Application of confocal scanning laser microscopy for an automated nuclear grading of prostate lesions in three dimensions
Authors: BEIL M. , IRINOPOULOU T. , VASSY J. , RIGAUT J. P.
Published: November 2003

Confocal scanning laser microscopy provides the opportunity to obtain three‐dimensional (3‐D) images by piling up consecutive confocal planes. This technique was applied to capture 3‐D images from 100‐μm‐thick tissue blocks from prostate lesions (hyperplasia, dysplasia, adenocarcinomas). Automated methods were implemented to perform a nuclear grading of 3‐D cell nuclei from these specimens. Special attention was focused on the development of a new approach to 3‐D chromatin texture analysis. This method uses mathematical morphology operations to tessellate the chromatin into homogeneous domains. The nuclear features (volume, shape, texture) were subjected to a discriminant analysis. Using a set of five features, the classification of cell nuclei yielded an accuracy of 963%. The results indicate the potential of 3‐D imaging and analysis techniques for an automated nuclear grading of prostate lesions.
Sampling and transportation of food materials for freeze‐fracture from the industrial environment
Authors: Price J. C. , Brooker B. E.
Published: November 2003

A simple holder is described which enables the multiple sampling of food materials for freeze‐fracturing in an industrial environment. The holder allows frozen samples to be transferred safely to the laboratory under liquid nitrogen for freeze‐fracturing and examination by transmission electron microscopy. The technique has been successfully applied to sampling food from pilot plant and production lines under factory conditions.
New polyacrylamide gel‐based methods of sample preparation for optical microscopy: immobilization of DNA molecules for optical mapping
Authors: MOROZOV V. N. , MOROZOVA T. Y. , HIORT C. , SCHWARTZ D. C.
Published: November 2003

New methods have been developed for rapid immobilization of biological macromolecules and other microscopic objects from aqueous solution at gel/gel, gel/solid and gel/solution interfaces using thin polyacrylamide gels covalently bound to glass surfaces. When quickly spread over a dry gel, an aqueous sample loses most of its water and low‐molecular‐weight solutes due to migration of these components into the gel. All optically observable objects thus become concentrated at the gel surface and may be easily located by light microscopy. Based on this, a procedure for binding DNA at a positively charged gel/solution interface was developed. A mild immobilization of the DNA molecules was obtained, allowing ‘all in focus’ observations of DNA digestion by restriction endonucleases with an apparent rate close to that in solution.
Novel micromachined cantilever sensors for scanning near‐field optical microscopy
Authors: MÜNSTER S. , WERNER S. , MIHALCEA C. , SCHOLZ W. , OESTERSCHULZE E.
Published: November 2003

The reproducible micromachining of hollow metal tips on Si cantilevers and their applicability to scanning near‐field optical microscopy (SNOM) is described. This sensor is fabricated using semiconductor compatible technologies. A hollow metal pyramid is employed as an optical aperture sensor for SNOM and simultaneously as a force sensor for scanning force microscopy applications. Apertures down to 120 nm were realized. To confirm the feasibility of the sensor we present measurements on microstructured chromium films as well as on hot filament chemical vapour deposition grown (111) diamond membranes. The SNOM images show a resolution of about 100 nm, demonstrating the usefulness of these probes.
Recent applications of the new stereology have thrown fresh light on how the human placenta grows and develops its form
Authors: MAYHEW T. M.
Published: November 2003

The availability of design‐based stereological methods has made it possible to readdress certain key and contentious issues in placental growth and morphogenesis. Three particular questions are: (i) does the population of cytotrophoblast cells decline during gestation?, (ii) is placental growth biphasic or monophasic? and (iii) what are the consequences for intervillous porosity of the elaboration of terminal villi? These questions cannot be answered definitively without recourse to the new stereology. Applying the disector to estimate nuclear number and star volume to assess pore size, recent studies have helped to resolve these issues. Their findings are reviewed. Nuclei were counted in the trophoblastic epithelium, stroma and vascular endothelium of placental villi. It was found that growth is monophasic and proliferative. All types of nuclei increased in number throughout gestation and this included cytotrophoblast. Trophoblast grows by the continuous recruitment of new proliferative units of uniform mean volume. The so‐called ‘loss’ of cytotrophoblast cells is a misinterpretation of what is seen on microscopical sections and is attributable to disproportionate growth in villous surface area. Cells simply become more widely dispersed. Elaboration of finer terminal branches on villous trees leads to a decline in the star volumes of villi and intervillous pores. Some of the functional implications of these findings are discussed.
Unbiased stereological estimation of the number and volume of nuclei and nuclear size variability in invasive ductal breast carcinomas
Authors: ARTACHO‐PÉRULA E. , ROLDÁN‐VILLALOBOS R.
Published: November 2003

The application of design‐based stereological methods for estimating nuclear features quantitatively in invasive ductal breast cancer is described. Nuclear number, size and size variability are explored in relation to the tumour grade and patient prognosis. The study includes an examination of the efficiency in estimating different nuclear volumes, and two different estimators of the nuclear size variability are contrasted. Forty‐two invasive ductal breast carcinomas diagnosed and graded by two pathologists were used. Both 5‐μm and 25‐μm‐thick sections were obtained from paraffin blocks for stereological study. More undifferentiated tumours show significantly larger nuclei than low‐grade tumours. The estimates based on the disector method demonstrate a decrease in the number of tumour cell nuclei per unit volume of tissue from grades 1 to 2 and especially from grades 2 to 3. The univariate survival analysis shows a high prognostic value of the nuclear volume estimates. The study shows that an efficient sampling procedure was performed, particularly when estimating volume‐weighted mean nuclear volume using the point‐sampled intercepts method. This method is more efficient than estimation of the number‐weighted mean nuclear volume using the selector method; however, the latter provides paired estimates of volume‐ and number‐weighted mean nuclear volume, as well as an estimate of the coefficient of variation of nuclear volume in the number distribution of the same cells.
Comparison of two‐photon excitation laser scanning microscopy with UV‐confocal laser scanning microscopy in three‐dimensional calcium imaging using the fluorescence indicator Indo‐1
Authors: SAKO Y. , SEKIHATA A. , YANAGISAWA Y. , YAMAMOTO M. , SHIMADA Y. , OZAKI K. , KUSUMI A.
Published: November 2003

Two‐photon excitation laser scanning fluorescence microscopy (2p‐LSM) was compared with UV‐excitation confocal laser scanning fluorescence microscopy (UV‐CLSM) in terms of three‐dimensional (3‐D) calcium imaging of living cells in culture. Indo‐1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p‐LSM than in UV‐CLSM, 2p‐LSM exhibited several advantages over UV‐CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3‐D images were repeatedly acquired, the calcium concentration determined by UV‐CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo‐1 and autofluorescence, while the calcium concentration evaluated by 2p‐LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p‐LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two‐photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV‐CLSM. These results indicate that 2p‐LSM is preferable for repeated 3‐D reconstruction of calcium concentration in living cells. In UV‐CLSM, 0.18‐mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal‐to‐noise ratio, contrast and resolution than 0.09‐mW laser power with a 4.9‐φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3‐D images.
Hydrogen incorporation in Ti‐based metal–matrix composites fabricated by vacuum plasma spraying and vacuum hot pressing
Authors: LI J. H. , GRANT P. S. , JENKINS M. L. , CANTOR B.
Published: November 2003

In this paper we investigate the incorporation of hydrogen as a temporary alloying element during vacuum plasma spraying (VPS) of Ti‐based metal–matrix composites and show that it has probable beneficial effects for secondary processing by vacuum hot pressing (VHP).

The composites investigated included both commercial purity Ti and Ti–6Al–4V matrices, which were sprayed onto DRA Sigma SiC fibres. The as‐sprayed matrix microstructures were investigated by transmission electron microscopy and X‐ray diffractometry, and were found to consist in both cases of a fine mixture of (1) hexagonal α′‐Ti martensite plates, with an increased lattice parameter relative to the usual value, presumably due to dissolved hydrogen; and (2) an increased proportion of equiaxed b.c.c. β grains, probably stabilized by dissolved hydrogen. Both phases, but particularlyβ, contained needle‐shaped b.c.t. hydride precipitates.

It has been found that monotapes produced by VHP can be consolidated more easily by secondary VHP than those produced by electric arc spraying, leading to reduced adverse interfacial reactions between fibre and matrix. This improved processability is believed to be due to the combination of a greater proportion of the more ductile β phase and the very small grain sizes inherent in VPS. However, the dissolved hydrogen and the potentially embrittling hydride precipitates are removed during VHP. The presence of hydrogen after primary VPS may therefore have an overall beneficial effect.
On the interpretation of magnetic and electric fields imaged by low‐magnification off‐axis electron holography
Authors: FROST B. G. , JENKINS T. L.
Published: November 2003

The interpretation of magnetic and electric fields imaged by off‐axis electron holography is not always straightforward. We will demonstrate this by some examples of possible misinterpretations which can result from, for example, comparing a hologram with a second one acquired such that the reference wave travelled on the opposite side of the sample, the diffraction of the electron beam at the biprism fibre, not centring an area to the true centre of the sideband when reconstructing the image wave by Fourier optics, quantification of ‘magnetic’ and ‘electric’ phases, procedures to separate electric from magnetic phases and dislocations in the interference fringes of the hologram.
Localization of chemical elements and isotopes in the leaf of soybean (Glycine max) by secondary ion mass spectrometry microscopy: critical choice of sample preparation procedure
Authors: Grignon N. , Halpern S. , Jeusset J. , Briançon C. , Fragu P.
Published: November 2003

Secondary ion mass spectrometry (SIMS) is one of the few microscopical methods that potentially can detect and in situ localize the various isotopes of virtually all elements. Recent work with SIMS has demonstrated the possibility of imaging the distribution of various elements in plant cell and tissues. However, in these studies, the elements were incorporated in cell macromolecules or associated with structural polymers, precipitated or immobilized in dry seeds. The localization of mineral ions is of particular significance for the physiology of higher plants owing to their quantitative importance and the impact of their cellular distribution on metabolic regulation. Here we analyse the possibility of mapping different elements (K, Ca, Mg, P, S, 15N and 14N) present as soluble and/or bound forms in highly vacuolated leaf cells. Cryoprocedures to prepare samples for SIMS detection are described and discussed. The quality of the results is assessed at each step of the sample preparation and analysis. Various methodologies are used, including photonic and electronic microscopies, and the agreement of the observed ion distribution with current knowledge of ion compartmentalization in plant cells. The K/Ca emission ratio is proposed as an index of the degree of preservation of the natural ion distribution to critically evaluate the results and identify where artefacts are likely to occur.
Morphological studies of Cu(I)‐complexed bipyridine‐containing block copolymers by combined transmission electron microscopy techniques
Authors: Göldel A. , Schubert U. S. , Eisenbach C. D.
Published: November 2003

The design of molecular species which can form well‐defined macromolecular architectures by spontaneous, recognition‐directed self‐assembly of their components is a major goal in supramolecular chemistry. We synthesized block copolymers with polyether and bipyridine or oligo‐(bipyridine) containing segments where the latter could form double‐helical structures by metal complexation. Upon complexation with Cu(I) these systems showed a dramatic change in the thermal and mechanical properties from a waxy system to a thermoplastic elastomeric behaviour. This effect could be explained by the formation of a multiphase polymer system consisting of domains of bipyridine or oligo(bipyridine) Cu(I) complexes in a matrix of amorphous soft segments.

In the solution‐cast films both nitrogen and copper could be detected with electron energy‐loss spectroscopy. Element spectroscopic imaging pictures of the net nitrogen and copper element distribution were identical and revealed a microphase‐separated system with identical morphological structures. High‐resolution bright‐field images revealed fine structures (interference lines) which were in accordance with stacks of [Cu(I)(bpy)2] complexes. Electron diffraction patterns showed that the supramolecular order could be perfected through annealing.