bluuebunny/crossref_metadata_2025 huggingface dataset:
Fitness, fatness and activity as predictors of bone mineral density in older persons
Authors: Stewart K. J. , Deregis J. R. , Turner K. L. , Bacher A. C. , Sung J. , Hees P. S. , Tayback M. , Ouyang P.
Published: March 2003

Abstract. Stewart KJ, DeRegis JR, Turner KL, Bacher AC, Sung J, Hees PS, Tayback M, Ouyang P (Johns Hopkins Bayview Medical Center, Johns Hopkins University, School of Medicine, Baltimore, MD, USA). Fitness, fatness, and activity as predictors of bone mineral density in older persons. J Intern Med 2002; 252: 381–388.

Objectives. To determine relationships of bone mineral density (BMD) with fitness, physical activity, and body composition and fat distribution.

Design. Cross‐sectional.

Setting. General Clinical Research Center, Johns Hopkins Bayview Medical Center, Baltimore, Maryland.

Subjects. Men (n = 38) and women (n = 46), aged 55–75 years with high normal blood pressure or mild hypertension but otherwise healthy.

Methods. Aerobic fitness (oxygen uptake) on a treadmill, muscle strength by one‐repetition maximum, activity by questionnaire, abdominal obesity by magnetic resonance imaging; anthropometrics, and body composition by dual energy X‐ray absorptiometry (DXA) which measured total fat and lean mass, and BMD for the total skeleton, lumbar spine (L1–L4) and total hip.

Results. Aerobic fitness did not correlate with BMD. Using multivariate analysis to ascertain independent contributions to the variance in BMD, in women, with adjustment for hormone replacement therapy (HRT), total skeleton BMD was independently related to muscle strength and abdominal total fat; total hip BMD to body weight; lumbar spine BMD to abdominal total fat. HRT also influenced BMD in the lumbar spine. In men, lumbar spine BMD was independently related to abdominal total fat physical activity and total hip BMD related to lower body strength. P < 0.05 for all of these correlations.

Conclusions. Abdominal obesity and muscle strength emerge as predominant correlates of BMD in older persons with stronger relationships seen in women. Body weight and HRT also explained portions of the variance in BMD in women. Whether abdominal obesity is simply a marker for general obesity or has independent protective effects on bone is yet to be determined.
An integrated approach to the study of living cells by atomic force microscopy
Authors: Nagao  , Dvorak
Published: March 2003

We describe a technique for studying living cells with the atomic force microscope (AFM) in tapping mode using a thermostated, controlled‐environment culture system. We also describe the integration of the AFM with bright field, epifluorescence and surface interference microscopy, achieving the highest level of integration for the AFM thus far described. We succeeded in the continuous, long‐term imaging of relatively flat but very fragile cytoplasmic regions of COS cells at a lateral resolution of about 70 nm and a vertical resolution of about 3 nm. In addition, we demonstrate the applicability of our technology for continuous force volume imaging of cultured vertebrate cells.

The hybrid instrument we describe can be used to collect simultaneously a diverse variety of physical, chemical and morphological data on living vertebrate cells. The integration of light microscopy with AFM and steady‐state culture methods for vertebrate cells represents a new approach for studies in cell biology and physiology.
Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two‐photon fluorescence microscopy
Authors: Schrader  , Hofmann  , Hell
Published: March 2003

Monomolecular films of polymerized dimethyl‐bis[pentacosadiinoic‐oxyethyl] ammonium bromide (EDIPAB) provide one‐ and two‐photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3‐D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi‐confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB‐layers can be used as substrates for the cultivation of cells.
Physical and chemical changes in polystyrene during electron irradiation using EELS in the TEM: contribution of the dielectric function
Authors: Varlot  , Martin  , Quet
Published: March 2003

We have performed electron energy‐loss spectroscopy analysis of polystyrene in the analytical transmission electron microscope in order to evaluate the possibility of obtaining both chemical and dielectric information on the polymer at the submicrometre scale. Irradiation has also been studied, particularly the influence of the specimen temperature on the kinetics of degradation.

The main results show that polystyrene is resistant to electron beam with a critical dose of about 104 C m−2 at 127 K. Spectra could be acquired with doses less than this critical dose. We were thus able to propose an identification of the different chemical bonds of carbon (including the C–H bond), in agreement with previous X‐ray absorption near‐edge structure experiments. The chemical changes in polystyrene due to severe irradiation damage are also visible in the carbon K‐edge near‐edge structure.

At the same time, we calculated the dielectric function from the low‐loss part of the spectra. Interestingly, this part of the spectrum is the most sensitive to irradiation, since great changes can be seen during exposure.

Lastly, we propose a degradation process, in agreement with all these results.
Picosecond pulsed two‐photon imaging with repetition rates of 200 and 400 MHz
Authors: Bewersdorf  , Hell
Published: March 2003

We show the viability of high‐resolution two‐photon fluorescence imaging of fixed and live cells by exciting the fluorophores with a train of near‐infrared pulses with duration in the picosecond range. This is exemplified with a compact, diode‐pumped Nd:YVO4 laser, emitting trains of 7‐ps pulses at a wavelength of 1064 nm, with a repetition rate of 200 MHz at two separate outputs. Incoherent combination of the outputs enabled two‐photon excitation with a repetition rate of 400 MHz. For a numerical aperture of 1.4 (oil), we used an average illumination power of up to 20–40 mW at the sample. The pulses were coupled into a beam scanning microscope, either directly or through a single mode glass fibre. Compared with standard femtosecond titanium–sapphire excitation conditions, our experiments were performed with a 2.5 or 5 times higher repetition rate, 30–70 times longer pulses and 10–35 times lower pulse peak intensity. The experiments indicate the possibility of significantly relaxing the temporal pulse width constraints for a series of applications.
A comparison of image restoration approaches applied to three‐dimensional confocal and wide‐field fluorescence microscopy
Authors: VERVEER P. J. , GEMKOW M. J. , JOVIN T. M.
Published: March 2003

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide‐field data. In general, restored confocal data are superior to restored wide‐field data, but given sufficient higher signal level for the wide‐field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide‐field data is presented.
Energy filtering transmission electron microscopy using the new JEM‐2010FEF
Authors: TOMOKIYO  , MATSUMURA  , MANABE
Published: March 2003

The new JEM‐2010FEF electron microscope provides useful techniques based on energy filtering as an omega‐type energy filter is integrated into a thermal field‐emission 200 kV transmission electron microscope. For example, the zero‐loss imaging improves the contrast of high resolution lattice images as well as images of precipitates or lattice defects in alloys. The acquisition time for elemental mapping with core‐loss electrons is one order in magnitude shorter than with energy‐dispersive X‐ray spectroscopy. The removal of inelastically scattered electrons enables us to observe weak lines in convergent‐beam electron diffraction patterns from a thicker specimen with a probe size 1–2 nm in diameter. A combination of the field emission gun and sensitive recording media such as an imaging plate and a slow‐scan CCD camera makes the energy filtering more powerful.
Stereological analysis and modelling of gradient structures
Authors: Hahn  , Micheletti  , Pohlink  , Stoyan  , Wendrock
Published: March 2003

Gradient structures are inhomogeneous along a particular gradient direction but homogeneous perpendicular to that direction. Consequently, structural parameters such as volume fraction or surface area density are local characteristics which depend on the ‘vertical’ coordinate with respect to the ‘vertical’ gradient axis.

Analogously, models for gradient structures have model parameters depending on the vertical coordinate z. For example, a Voronoi tessellation with a gradient is generated by a gradient point process with a local intensity which is a function of z. Similarly, a gradient germ grain model is obtained from a gradient point process where the grain size distribution may also depend on z. For a gradient Boolean model, local volume fraction VV(z) and local surface area density SV(z) can be calculated from the model parameters.

Stereological methods for gradient structures are based on vertical sections parallel to the gradient direction. Estimation of VV(z), SV(z) and local length density LV(z) is done by lineal analysis using horizontal test lines with vertical coordinate z. Similarly, lineal analysis is used to estimate local mean cell volume of gradient tessellations. For the estimation of local particle number density and size in the spirit of the Wicksell problem the use of kernel methods and distributional assumptions is required.
Surface imaging in near‐field optical microscopy by using the fluorescence decay rate: a theoretical study
Authors: Parent G. , Van Labeke D. , Barchiesi D.
Published: March 2003

In this paper, we study the fluorescence decay rate of a molecule above a corrugated interface, and particularly the variations of the decay rate as a function of the lateral position of the molecule. As a first step, one has to determine the field diffracted by a corrugated interface when the incident field is the field emitted by a dipole. For this purpose, we have used a perturbative Rayleigh method, and we show that the decay rate variations can be connected to the surface profile via a transfer function. Some numerical calculations of this transfer function and of decay rate variation images are presented for dielectric and metallic samples. The visibility of the theoretical images is up to 20% and, moreover, resolution of the images is good enough to use the fluorescence lifetime of molecules as signal in a life‐time scanning near‐field optical microscope. The technical problems are discussed briefly.
Electron backscatter diffraction of grain and subgrain structures — resolution considerations
Authors: HUMPHREYS F. J. , HUANG Y. , BROUGH I. , HARRIS C.
Published: March 2003

Characterization of microstructures containing small grains or low‐angle grain boundaries by electron backscattered diffraction (EBSD) is limited by the spatial and angular resolution limits of the technique. It was found that the best effective spatial resolution (60 nm) for aluminium alloys in a tungsten‐filament scanning electron microscope (SEM) was obtained for an intermediate probe current which provided a compromise between pattern quality and specimen interaction volume. The same specimens and EBSD equipment when used with a field‐emission gun SEM showed an improvement in spatial resolution by a factor of 2–3. For characterizing low‐angle boundary microstructures, the precision of determining relative orientations is a limiting factor. It was found that the orientation noise was directly related to the probe current and this was interpreted in terms of the effect of probe current on the quality of the diffraction patterns.
Membrane ion channels as physiological targets for local Ca2+ signalling
Authors: Gordienko  , Zholos  , Bolton
Published: March 2003

Ionized calcium plays a central role as a second messenger in a number of physiologically important processes determining smooth muscle function. To regulate a wide range of cellular activities the mechanisms of subcellular calcium signalling should be very diverse. Recent progress in development of visible light‐excitable fluorescent dyes with high affinity for Ca2+ (such as oregon green 488 BAPTA indicators, fluo‐3 and fura red) and confocal laser scanning microscopy provides an opportunity for direct visualization of subcellular Ca2+ signalling and reveals that many cell function are regulated by the microenvironment within small regions of the cytoplasm (‘local control’ concept). Here confocal imaging is used to measure and locate changes in [Ca2+]i on a subcellular level in response to receptor stimulation in visceral myocytes. We show that stimulation of muscarinic receptors in ileal myocytes with carbachol leading to activation of inositol 1,4,5‐trisphosphate receptors (IP3Rs) accelerates the frequency of spontaneous calcium sparks (discharged via ryanodine receptors, RyRs) and gives rise to periodic propagating Ca2+ waves oscillating with a frequency similar to that of carbachol‐activated cationic current oscillations. Furthermore, by combining the whole‐cell patch clamp technique with simultaneous confocal imaging of [Ca2+]i in voltage‐clamped vascular myocytes we demonstrate that calcium sparks may lead to the opening of either Ca2+‐activated Cl− channels or Ca2+‐activated K+ channels, and the discharge of a spontaneous transient inward current (STIC) or a spontaneous transient outward current (STOC), respectively.
Extending the dynamic range of fibre length and fibre aspect ratios by automated image analysis
Authors: Davidson  , Clarke
Published: March 2003

An automatic image analysis system has been developed, that is capable of scanning a large sample area (70 mm × 70 mm) using transmitted light microscopy. The analysis, based on transparent algorithms, successfully discriminates between overlapping and touching fibres, so that sample preparation time is minimized. The system utilizes overlapping image frames to reconstruct partial fibre images and impart unique fibre length information in the range micrometres to millimetres. The software has been made adaptable to enable the accurate measurement of fibre lengths for fibres that curve over many millimetres. Data are presented illustrating the accurate measurement of fibre lengths over a large range of fibre aspect ratios.
Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: II. Tomography
Authors: Wang  , Jacobsen  , Maser  , Osanna
Published: March 2003

Using a cryo scanning transmission X‐ray microscope ( Maser, et al. (2000
) Soft X‐ray microscopy with a cryo scanning transmission X‐ray microscope: I. Instrumentation, imaging and spectroscopy. J. Microsc. 197, 68–79), we have obtained tomographic data‐sets of frozen hydrated mouse 3T3 fibroblasts. The ice thickess was several micrometres throughout the reconstruction volume, precluding cryo electron tomography. Projections were acquired within the depth of focus of the focusing optics, and the three‐dimensional reconstruction was obtained using an algebraic reconstruction technique. In this first demonstration, 100 nm lateral and 250 nm longitudinal resolution was obtained in images of unlabelled cells, with potential for substantial further gains in resolution. Future efforts towards tomography of spectroscopically highlighted subcellular components in whole cells are discussed.
Long freeze‐drying times are not necessary during the preparation of thin sections for X‐ray microanalysis
Authors: Warley  , Skepper
Published: March 2003

The temperature profile that occurs when a brass block warms up in a vacuum evaporation unit was determined. Freshly drawn human blood was concentrated by centrifugation, and the pellet was cryofixed and cryosectioned. The cryosections were subject to different freeze‐drying protocols, using a freeze‐drier with a temperature‐controlled stage, to determine the effect of freeze‐drying time on element distribution. Spectra were collected by spot analyses at various distances across the interface between red cells and plasma. Concentrations of sodium were high and variable outside the cell and low in the cell interior, with potassium showing the reverse distribution. The number of counts under the iron peak closely followed the potassium distribution. The concentration of sodium was higher than expected at 40 nm inside the cell membrane. This was attributed to the formation of ice crystals at the interface between the cells and plasma during cryofixation and the use of a wide probe size.
Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance
Authors: Carlsson K. , Liljeborg A. , Andersson R. M. , Brismar H.
Published: March 2003

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH‐dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime‐dependent phase shift in the fluorescent signal is detected by a lock‐in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL‐2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8‐bit digital image. However, the pixel‐to‐pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.
Development of an X‐ray photoemission electron microscopy system with multi‐probes, and its application to surface imaging at static and dynamic states
Authors: Takakusagi S. , Kato M. , Sakai Y. , Fukui K. , Asakura K. , Iwasawa Y.
Published: March 2003

We have developed a new X‐ray photoemission electron microscopy system combined with low energy electron microscopy, photoemission electron microscopy, mirror electron microscopy (MEM), secondary electron emission microscopy (SEEM) and Auger electron emission microscopy, which provides multi‐angle information on the distribution and change of element, chemical state, structure, etc. at solid surfaces under the working conditions such as high temperature and gas atmosphere. The performance of each microscopical method was examined and typical images are presented. The dynamic behaviour of fabricated surfaces has been imaged in real time by SEEM and MEM.
Structural and morphological anomalies in magnetosomes: possible biogenic origin for magnetite in ALH84001
Authors: Taylor A. P. , Barry J. C. , Webb R. I.
Published: March 2003

We report biogenic magnetite whiskers, with axial ratios of 6 : 1, elongated in the [1 1 1], [1 1 2] and [1 0 0] directions, resembling the magnetite whiskers detected in the Martian meteorite ALH84001 by Bradley et al., and interpreted by those authors as evidence of vapour‐phase (abiogenic) growth. Magnetosomal whiskers with extended defects consistent with screw dislocations and magnetosomes resembling flattened twinned platelets, as well as other twinning phenomena and other structural defects, are also reported here. Magnetosomes with teardrop‐shaped, cuboidal, irregular and jagged structures similar to those detected in ALH84001 by McKay et al., coprecipitation of magnetite possibly with amorphous calcium carbonate, coprecipitation of magnetite possibly with amorphous silica, the incorporation of titanium in volutin inclusions and disoriented arrays of magnetosomes are also described. These observations demonstrate that the structures of the magnetite particles in ALH84001, their spatial arrangement and coprecipitation with carbonates and proximity to silicates are consistent with being biogenic.

Electron‐beam‐induced flash‐melting of magnetosomes produced numerous screw dislocations in the {1 1 1}, {1 0 0} and {1 1 0} lattice planes and induced fusion of platelets. From this, the lack of screw dislocations reported in the magnetite particles in ALH84001 (McKay et al., and Bradley et al.) indicates that they have a low‐temperature origin.
Near‐field optical addressing of single molecules in coplanar geometry: a theoretical study
Authors: Colas Des Francs G. , Girard C. , Weeber J.‐C. , Dereux A.
Published: March 2003

Photonic transfer through elongated optical structures of submicrometre section microfabricated at the surface of dielectric or semiconductor samples can be enhanced by an appropriate structuring of the local refraction index. We show from computerized simulations that both the light localization and the spectroscopic properties of such structures can be used to selectively excite, in coplanar geometry, individuals molecules located in the near‐field.
Mesoscopic structures and dynamics of merocyanine J‐aggregate studied by time‐resolved fluorescence SNOM
Authors: Miura A. , Yanagawa Y. , Tamai N.
Published: March 2003

Time‐resolved fluorescence SNOM is used to probe the mesoscopic structure and dynamics of long‐chain merocyanine (C18MC) J‐aggregates on glass plates prepared by spin coating, casting, and casting of water‐soluble polymer films. A globular structure with an average diameter of ∼ 1 µm and a height of ∼ 50 nm was attributed to the J‐aggregate of C18MC in the spin‐coating film. In polymer films, the bandwidth of the absorption of J‐aggregate is much narrower in polyvinyl alcohol (PVA, ∼ 20 nm) than that in polyvinyl sulphate (PVS, ∼ 60 nm). We have demonstrated that the large bandwidth of the spectrum is due to the inhomogeneous distribution of the J‐aggregate. The fluorescence image of the J‐aggregate in PVA film was rather uniform, whereas non‐uniform distribution of the fluorescence was observed in PVS film. The fluorescence of C18MC J‐aggregate in a small domain of PVA film was a single exponential decay with a lifetime as short as 19 ps, which was shorter than that in PVS film with a two‐exponential decay (average lifetime of ∼ 25 ps). The fluorescence lifetime of the J‐aggregate and its single exponential behaviour are considered to be indicators of the uniform distribution of the J‐aggregate. The non‐uniform distribution of the J‐aggregate in PVS film was interpreted in terms of electrostatic interaction between PVS and merocyanine.
In situ high resolution electron microscopy/electron energy loss spectroscopy observation of wetting of a Si surface by molten Al
Authors: Tsukimoto S. , Arai S. , Konno M. , Kamino T. , Sasaki K. , Saka H.
Published: March 2003

Electron energy loss spectroscopy was used to observe the segregation of Al on a Si surface above the melting point of Al. A mixture of Al and Si particles was heated above the melting point of Al in a vacuum of 1 × 10−5 Pa. The Si surface, which initially had been covered with an amorphous oxide layer before heating, became clean and atomically facetted when the Al melted. It was shown that the Si surface was segregated with Al.
Estimating the number of complex particles using the ConnEulor principle
Authors: Kroustrup J. P. , Gundersen H. J. G.
Published: March 2003

An unbiased counting rule for the number of topologically simple objects of any shape, size and distribution in 3D space is a pertinent problem in stereology. Combining the disector principle with the object's 3D Euler number makes possible number estimation, which until now has been obtainable only by exhaustive serial sections. The disector is a set of two sections where the object's profiles in one section are compared with its profiles on the neighbouring section, and the number of new 2D topological events is recorded. In a disector of known volume the sum of topological events is a direct estimate of the disector contribution to the total Euler number, which forms the basis for an ultimate number estimator in 3D, the ConnEulor. The method is illustrated by an electron microscopic study of the number of mitochondria in the exocrine cells of the pancreas.
Frequency‐domain fluorescence microscopy with the LED as a light source
Authors: Herman P. , Maliwal B. P. , Lin H.‐J. , Lakowicz J. R.
Published: March 2003

We describe a frequency‐domain lifetime fluorometer based on a microscope and a modulated light‐emitting diode (LED) excitation source (370/460 nm), which operates in the frequency range 120 Hz–250 MHz. We collected multifrequency phase and modulation fluorescence responses from cellular areas as small as 10–15 µm in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore, Coum‐Eu, with a millisecond lifetime, and Ru(bpy)2phe‐C12, with microsecond lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 14 and SYTO 16 probes were measured as well. We demonstrate that a simple LED excitation source can, for many applications, successfully replace complex and expensive laser systems, which have been used for cellular frequency‐domain lifetime measurements. As the LEDs are very stable with low noise, it will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths covering almost the entire visible spectrum it is easy to assemble a system for the fluorophore of choice. The ability to select an excitation source for a given fluorophore and low price make such an excitation source even more practical.
Microscopical investigations on the epicuticle of mammalian keratin fibres
Authors: Swift J. A. , Smith J. R.
Published: March 2003

The existence of a thin chemically resistant layer, the epicuticle, close to the surfaces of all undamaged mammalian keratin fibres has been known since 1916. The identification of such a specific structure within the fibre cuticle has remained elusive. Now, through transmission electron microscope investigations of stained transverse sections of hairs from various animal species, the epicuticle has been tentatively identified as a sharply defined and continuous layer approximately 13 nm thick covering the entire outwardly facing intracellular surface of every cuticle cell. The staining behaviour of the epicuticle leads one to suppose that it is rich in cystine and that thioester‐bound lipids might be present within its bulk. With the atomic force microscope it was established that the undamaged outer surface of all mammalian keratin fibres, even including those from the monotremes, were longitudinally striated. The lateral spacing of the striations was always in the range 0.29–0.39 µm. Striations only occurred on the freely exposed outer surfaces of the original undamaged fibres; evidently arising by some, as yet undefined, interaction in the follicle with the cuticle of the inner root sheath. By stripping off fatty acids known to be covalently attached to the fibre's outer surface, the striations were shown to reflect a corresponding irregularity of the epicuticle's surface.
Tissue shrinkage and unbiased stereological estimation of particle number and size*
Authors: Dorph‐Petersen K.‐A. , Nyengaard J. R. , Gundersen H. J. G.
Published: March 2003

This paper is a review of the stereological problems related to the unbiased estimation of particle number and size when tissue deformation is present. The deformation may occur during the histological processing of the tissue. It is especially noted that the widely used optical disector may be biased by dimensional changes in the z‐axis, i.e. the direction perpendicular to the section plane. This is often the case when frozen sections or vibratome sections are used for the stereological measurements. The present paper introduces new estimators to be used in optical fractionator and optical disector designs; the first is, as usual, the simplest and most robust. Finally, it is stated that when tissue deformation only occurs in the z‐direction, unbiased estimation of particle size with several estimators is possible.
Near real time in vivo fibre optic confocal microscopy: sub‐cellular structure resolved
Authors: Sung K. B. , Liang C. , Descour M. , Collier T. , Follen M. , Malpica A. , Richards‐Kortum R.
Published: March 2003

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 µm and axial resolution is 6 µm. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s−1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.
Pre‐screening for antigen detectability in cells: a TEM‐based solid phase digital immunogold detection method utilizing ultra low volumes of reagents
Authors: Kaur R. , Dikshit K. L. , Raje M.
Published: March 2003

Rapid and sensitive pre‐screening for the presence of antigens in cell samples and confirmation of reactivity of antibodies, before proceeding with electron microscopy, is highly desirable. Most of the methods developed for this purpose are generally not very efficient and suitable for dealing with very small volumes of sample and reagents. In this work we present a simple, sensitive and rapid solid phase transmission electron microscope (TEM) based method for the detection of picogram (pg) levels of soluble antigens using as little as 10 µL of reagents. Protein was adsorbed onto grids coated with polystyrene films to form the solid phase. The presence of antigen was detected using immunogold labelling. Gold particles adhering to the film were visualized and counted in a TEM providing a digital signal. This method was 100‐fold more sensitive than dot blot in detection of rabbit IgG. We have demonstrated the utility of this technique by screening for Vitreoscilla haemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold labelling transmission electron microscopy of cell sections.
Nonlinear bio‐photonic crystal effects revealed with multimodal nonlinear microscopy
Authors: Chu S.‐W. , Chen I.‐H. , Liu T.‐M. , Sun C.‐K. , Lee S.‐P. , Lin B.‐L. , Cheng P.‐C. , Kuo M.‐X. , Lin D.‐J. , Liu H.‐L.
Published: March 2003

Highly optically active nonlinear bio‐photonic crystalline and semicrystalline structures in living cells were studied by a novel multimodal nonlinear microscopy. Numerous biological structures, including stacked membranes and aligned protein structures are highly organized on a nanoscale and have been found to exhibit strong optical activities through second‐harmonic generation (SHG) interactions, behaving similarly to man‐made nonlinear photonic crystals. The microscopic technology used in this study is based on a combination of different imaging modes including SHG, third‐harmonic generation, and multiphoton‐induced fluorescence. With no energy release during harmonic generation processes, the nonlinear‐photonic‐crystal‐like SHG activity is useful for investigating the dynamics of structure–function relationships at subcellular levels and is ideal for studying living cells, as minimal or no preparation is required.
X‐ray tomographic imaging of Ti/SiC composites
Authors: Mcdonald S. A. , Preuss M. , Maire E. , Buffiere J.‐Y. , Mummery P. M. , Withers P. J.
Published: March 2003

In this paper, high‐resolution tomographic synchrotron X‐ray imaging is applied to study the occurrence and evolution of damage in Ti‐6Al‐4V/SCS6 SiC fibre composite materials. Three composite morphologies of increasing complexity have been studied, namely single fibre, single‐ply and multi‐ply composites. The single fibre composite was strained to full fibre fragmentation and the progressive introduction of damage monitored. For the single‐ply composite, damage was introduced deliberately by laser drilling to establish the effect of damaged fibres on their neighbours, whereas for the multi‐ply composite the morphology of a fibre bridging fatigue crack was studied. In addition to traditional mode I fibre fractures, subsequent fibre wedge cracks were observed presumably nucleating from damage introduced into the fibre surface by the first fracture event. In addition to these crack morphologies, spiral defects were observed for the single ply during failure. Finally, for the multi‐ply composite, the matrix crack front showed a number of characteristic features, including advancement in fibre‐free regions, crack bifurcation near fibres and different crack plane heights either side of a fibre.
Estrogen Down‐Regulates mRNA Encoding the Exocytotic Protein SNAP‐25 in the Rat Pituitary Gland
Authors: Jacobsson Gunilla , Razani Haleh , Ogren Sven‐Ove , Meister Björn
Published: March 2003

Exocytosis is dependent on specific proteins that are located at the secretory granule membrane, in the cytoplasm or at the plasma membrane. The mRNA expression of synaptosomal‐associated protein of 25 kDa (SNAP‐25) isoforms SNAP‐25a and SNAP‐25b, vesicle associated membrane protein (VAMP) 2, mammalian homologue of unc‐18 (munc‐18) and Hrs‐2 was studied in the pituitary of ovariectomized rats after subcutaneous insertion of capsules containing estrogen or placebo using in situ hybridization. Estrogen treatment (0.25 mg estradiol) significantly decreased SNAP‐25a (32%; 10%) and SNAP‐25b (25%; 22%) mRNA levels in the anterior and intermediate lobes, respectively, whereas VAMP‐2, munc‐18 and Hrs‐2 mRNA levels remained unchanged. The results suggest that estrogen selectively regulates SNAP‐25 transcription in the pituitary gland, but leaves VAMP‐2, munc‐18 and Hrs‐2 mRNA levels unaffected.
Different Developmental Patterns of Melanocortin MC3 and MC4 Receptor mRNA: Predominance of Mc4 in Fetal Rat Nervous System
Authors: Kistler‐Heer  , Lauber  , Lichtensteiger
Published: March 2003

Melanocortins are thought to be involved in neuronal development and regeneration. Proopiomelanocortin (POMC), the precursor of α‐melanocyte stimulating hormone (α‐MSH), γ‐MSH, ACTH, and β‐endorphin, becomes detectable in rat hypothalamic neurons from gestational day (E) 12.5. We recently described stage‐ and region‐specific ontogenetic patterns of binding sites for the α‐MSH analogue [125I]‐Nle4,
d‐Phe7‐α‐MSH ([125I]‐NDP), with the first localizations in epithalamus and sympathetic chain at E13. [125I]‐NDP binds to all known melanocortin receptors, including MC3‐R and MC4‐R, the predominant melanocortin receptors in nervous system. To identify the receptor type expressed during ontogeny, the developmental pattern of MC3‐R and MC4‐R mRNA was investigated by in situ hybridization in fetuses and offspring of time‐pregnant Long Evans rats between E14 and postnatal day (P) 27. MC4‐R mRNA was found to be the predominant species during the entire fetal period. It was localized in all fetal areas exhibiting distinct [125I]‐NDP binding, starting with sympathetic ganglia and epithalamus (E14), and including sensory trigeminal nuclei (E16), dorsal motor nucleus of vagus (E16) and cranial nerve ganglia, inferior olive (E18) and cerebellum (E18), striatal regions (E16), and entorhinal cortex (E22). In contrast, MC3‐R mRNA was detectable only in the postnatal period, with a fast increase in expression in the ventromedial and arcuate nuclei. The early presence of MC4‐R mRNA in central and peripheral nervous system and transient regional peaks of mRNA expression, often concomitant with periods of neural network formation, suggest a role of this receptor type in early ontogeny. The MC3 receptor may be involved in analogous processes during postnatal development.
Immunocytochemical Demonstration of CB1 Cannabinoid Receptors in the Anterior Lobe of the Pituitary Gland
Authors: Wenger  , Fernández‐Ruiz  , Ramos
Published: March 2003

Both exogenous and endogenous cannabinoids can influence hormone secretion from the anterior pituitary gland. A large body of information proves that the primary target of these effects is the neuroendocrine hypothalamus. However, recent studies using cannabinoid (CB) receptor autoradiography, messenger RNA in‐situ hybridization and in‐vitro analysis, indicate direct effects of cannabinoids at the level of the anterior pituitary gland itself. In the present paper, the immunocytochemical distribution of CB in the adult rat anterior pituitary was studied using specific polyclonal antibodies against CB1 (central) and CB2 (peripheral) receptors. Due to its resolution, this method allowed identification of individual anterior pituitary cells possessing cannabinoid receptors. The specific hormone immunoreactive cells with receptor‐like immunoreactivity were compared on adjacent sections. CB1‐like immunoreactivity (CB1ir) was found in the lactotroph cells as well as in luteinizing hormone (LH) secreting gonadotrophs. The CB1ir positive material present in the cytoplasm of these cells was less homogeneous than the hormone immunorective material, and it was also seen at the periphery of the cells, presumably on the cell membrane. No CB1ir was found in growth hormone (GH) secreting cells and it was hardly seen in the corticotrophs. No CB1ir was detected in the posterior pituitary. CB2ir was not observed in any part of the pituitary gland. The results support the view that the site of action of cannabinoids on neuroendocrine regulatory mechanisms may be both at pituitary and hypothalamic levels. We suggest that at least the direct effect of cannabinoids on the regulation of LH and prolactin secretion is mediated via CB1 cannabinoid receptors in the anterior pituitary.
Oxytocin and Vasopressin Neurones in Principal and Accessory Hypothalamic Magnocellular Structures Express Fos‐Immunoreactivity in Response to Acute Glucose Deprivation
Authors: Briski  , Brandt
Published: March 2003

Neurohypophyseal secretion of oxytocin and vasopressin is elevated in response to decreased systemic glucose availability. In these studies, dual‐label immunocytochemistry was used to identify hypothalamic neuropeptidergic magnocellular neurones that are transcriptionally activated in response to glucose substrate imbalance. Two h after i.p. injection of the glucose antimetabolite, 2‐deoxy‐ d‐glucose (2DG), or the vehicle, saline, groups of adult male rats were anaesthetized by i.p. injection with sodium pentobarbital and killed by transcardial perfusion. Sections (25 μm) through anterior and tuberal levels of the hypothalamus were processed for nuclear Fos‐ and cytoplasmic neuropeptide immunoreactivity (−ir). A high proportion of oxytocin‐ir neurones in the supraoptic, paraventricular, and adjunct structures, including the anterior commissural, periventricular magnocellular, posterior perifornical, recurrent supraoptic, medial forebrain, and circular nuclei, were colabelled for nuclear Fos‐ir following administration of 2DG. Large numbers of vasopressin neurones in the supraoptic, circular, posterior perifornical, and medial forebrain nuclei, and posterior magnocellular division and posterior subnucleus of the paraventricular nucleus were also immunostained for Fos in rats injected with the antimetabolite. These results show that decreased glucose metabolism is a stimulus for activation of the Fos stimulus‐transcription cascade within oxytocin‐and vasopressin‐immunopositive neurones in several hypothalamic loci, findings that reflect activation of the Fos‐stimulus transcription cascade within large proportions of these cell populations during this metabolic challenge. These data suggest that both peripheral hormonal and central modulatory functions of these neuropeptidergic neurones may be influenced by cellular glucose availability.
Distribution of Aromatase mRNA in the Ram Hypothalamus: An In Situ Hybridization Study
Authors: Roselli  , Stormshak  , Resko
Published: March 2003

The regional distribution of neurones expressing aromatase mRNA in the ram hypothalamus was examined by in situ hybridization using 33P‐labelled cRNA probes. The highest amounts of hybridization signal were observed in the central part of the medial preoptic nucleus and posterior medial part of the bed nucleus of the stria terminalis. Moderate amounts of hybridization signal were observed in the anteroventral periventricular preoptic nucleus, medial preoptic nucleus and a broad band extending between the medial preoptic nucleus and bed nucleus of the stria terminalis. Low levels of hybridization signal were observed in the organum vasculosum of the lamina terminalis, anterior part of the medial preoptic nucleus, and central part of the ventromedial nucleus of the hypothalamus. The presence of aromatase mRNA within neurones of the steroid‐sensitive hypothalamic circuit supports a role for aromatization in the mechanism of testosterone action on reproductive function in male sheep. The distribution of aromatase mRNA in the ovine hypothalamus was similar to that described for other vertebrate species, suggesting a high degree of functional conservation across species.
Expression of the Genes Encoding the Vasopressin‐Activated Calcium‐Mobilizing Receptor and the Dual Angiotensin II/Vasopressin Receptor in the Rat Central Nervous System
Authors: Hurbin  , Orcel  , Ferraz  , Moos  , Rabié
Published: March 2003

The distributions of two newly discovered receptors, the vasopressin‐activated calcium‐mobilizing receptor (VACM‐1) and the dual angiotensin II/vasopressin receptor (AII/AVP), in the central nervous system (CNS) of the rat were determined using reverse transcriptase‐polymerase chain reaction and in situ hybridization. The sequence of the rat VACM‐1 cDNA was determined and found very homologous to the rabbit and human sequences. Both VACM‐1 and AII/AVP receptor genes were widely expressed in the brain, but differed according to the cell type studied. Glial cells were very faintly labelled. The epithelial cells of the choroid plexuses, the ependymal cells and the pia mater were all labelled. Both genes were most active in neurones throughout the CNS. VACM‐1 and AII/AVP receptors were detected in neurones previously shown to possess V1a and V1b vasopressin receptors, and/or the AT1 and AT2 angiotensin II receptors in many brain areas. This was the case for the magnocellular neurones of the supraoptic and paraventricular nuclei of the hypothalamus. We suggest that the VACM‐1 and AII/AVP receptors may account for the V2‐like responses to vasopressin by these neurones which lack a genuine V2 vasopressin receptor.
Regulation of Central Corticosteroid Receptors Following Short‐Term Activation of Serotonin Transmission by 5‐Hydroxy‐ L‐Tryptophan or Fluoxetine
Authors: Sémont A. , Fache M.‐P. , Héry F. , Faudon M. , Youssouf F. , Héry M.
Published: March 2003

Alterations of the hypothalamic‐pituitary‐adrenal (HPA) axis function characterized by a decreased negative feedback capacity are often associated with affective disorders and are corrected by treatment with antidepressant drugs. To gain a better understanding of the effects of the antidepressant drug fluoxetine, a specific serotonin (5‐HT) reuptake inhibitor, on central corticosteroid receptors, the effects of short‐term activation of serotonin transmission on central corticosteroid receptor expression were analysed in adrenalectomized (ADX) rats either supplemented or not with corticosterone. Serotonin transmission was stimulated either by a single injection of the 5‐HT precursor, 5‐hydroxy‐ l‐tryptophan (5‐HTP), or by a 2‐day treatment with fluoxetine. In ADX rats, administration of 5‐HTP decreased hippocampal mineralocorticoid (MR) and glucocorticoid (GR) receptor numbers 24 h later, while their respective mRNAs were unchanged and these effects of 5‐HTP were mediated by 5‐HT2 receptors. In the hypothalamus, GR mRNAs and binding sites decreased 3 h and 24 h after 5‐HTP, respectively. By contrast, fluoxetine treatment increased hippocampal MR and GR mRNAs and MR binding sites while GR number remained unchanged. In ADX rats supplemented with corticosterone, 5‐HTP and fluoxetine treatment had the same effects on corticosteroid receptors compared to those observed in non supplemented ADX rats: 5‐HTP decreased hippocampal MR and GR and hypothalamic GR while fluoxetine treatment increased hippocampal MR. These results show that short‐term stimulation of 5‐HT transmission by 5‐HTP decreases hippocampal and hypothalamic corticosteroid receptor numbers through a corticosterone‐independent mechanism. It is hypothesized that the delayed maximal increase in extracellular 5‐HT contents after fluoxetine treatment, due to negative feedback regulations induced by the activation of 5‐HT1A and 5‐HT1B autoreceptors, is not the primary cause for the delayed normalization of corticosteroid receptor numbers that regulates the HPA axis functioning.
Trafficking of the Vasopressin and Oxytocin Prohormone Through the Regulated Secretory Pathway
Authors: De Bree F. M.
Published: March 2003

The trafficking of prohormones and of regulated secretory proteins in general has been studied extensively in the last decades of the last century. Prohormone trafficking starts with correct folding and subsequently efficient sorting into the secretory granule of the regulated secretory pathway. The vasopressin/oxytocin prohormone is particularly interesting for studying protein trafficking, because the physicochemical properties and three‐dimensional structure have been largely elucidated. In the case of pro‐vasopressin and pro‐oxytocin, folding and sorting depend completely on both intramolecular and intermolecular interactions. Proper folding is guided by the hormone–neurophysin association and the sorting event relies on the aggregative properties of the neurophysin domain in the prohormone, as well as a specific sorting signal, which is revealed when the aggregative property of the neurophysin domain is deleted. A comprehensive mechanism for trafficking of the vasopressin/oxytocin prohormone from the endoplasmic reticulum to the secretory granule is proposed.