The TRIXS end-station for femtosecond time-resolved resonant inelastic x-ray scattering experiments at the soft x-ray free-electron laser FLASH.

We present the experimental end-station TRIXS dedicated to time-resolved soft x-ray resonant inelastic x-ray scattering (RIXS) experiments on solid samples at the free-electron laser FLASH. Using monochromatized ultrashort femtosecond XUV/soft x-ray photon pulses in combination with a synchronized optical laser in a pump-probe scheme, the TRIXS setup allows measuring sub-picosecond time-resolved high-resolution RIXS spectra in the energy range from 35 eV to 210 eV, thus spanning the M-edge (M1 and M2,3) absorption resonances of 3d transition metals and N4,5-edges of rare earth elements. A Kirkpatrick-Baez refocusing mirror system at the first branch of the plane grating monochromator beamline (PG1) provides a focus of (6 × 6) µm2 (FWHM) at the sample. The RIXS spectrometer reaches an energy resolution of 35-160 meV over the entire spectral range. The optical laser system based on a chirped pulse optical parametric amplifier provides approximately 100 fs (FWHM) long photon pulses at the fundamental wavelength of 800 nm and a fluence of 120 mJ/cm2 at a sample for optical pump-XUV probe measurements. Furthermore, optical frequency conversion enables experiments at 400 nm or 267 nm with a fluence of 80 and 30 mJ/cm2, respectively. Some of the first (pump-probe) RIXS spectra measured with this setup are shown. The measured time resolution for time-resolved RIXS measurements has been characterized as 287 fs (FWHM) for the used energy resolution.

In situ focus characterization by ablation technique to enable optics alignment at an XUV FEL source.

In situ focus characterization is demonstrated by working at an extreme ultraviolet (XUV) free-electron laser source using ablation technique. Design of the instrument reported here allows reaching a few micrometres resolution along with keeping the ultrahigh vacuum conditions and ensures high-contrast visibility of ablative imprints on optically transparent samples, e.g., PMMA. This enables on-line monitoring of the beam profile changes and thus makes possible in situ alignment of the XUV focusing optics. A good agreement between focal characterizations retrieved from in situ inspection of ablative imprints contours and from well-established accurate ex situ analysis with Nomarski microscope has been observed for a typical micro-focus experiment.

Immunoregulatory properties of IL-13 in patients with inflammatory bowel disease; comparison with IL-4 and IL-10.

Activated monocytes with increased expression of proinflammatory cytokines play a major role in inflammatory bowel disease (IBD). Immunoregulatory cytokines such as IL-4 and IL-10 can effectively suppress the proinflammatory response of activated monocytes. IL-13 is a recently described antiinflammatory agent in vitro. The aim of our study was to determine the in vitro immunosuppressive capacity of IL-13, IL-4 and IL-10 in patients with IBD. Peripheral blood monocytes were isolated from 27 patients with ulcerative colitis (UC), 27 patients with Crohn's disease (CD) and 16 healthy controls. Cells were stimulated with pokeweed mitogen (PWM) after treatment with IL-13, IL-4 and IL-10, and secretion of IL-1beta, tumour necrosis factor-alpha (TNF-alpha) and IL-6 was assessed using sandwich ELISA systems. Peripheral blood monocytes secreted significantly increased amounts of TNF-alpha and IL-6 under stimulation with PWM in patients with CD, while UC patients showed significantly elevated levels of IL-1beta. The antiinflammatory cytokines IL-13, IL-4 and IL-10 were all capable of inhibiting monocyte secretion of IL-1beta in a dose-dependent manner. With regard to IL-13 and IL-4, there was no significant suppression of TNF-alpha and IL-6 in patients with active IBD. By contrast, IL-10 was able to down-regulate all proinflammatory cytokines in active IBD as well as in controls. Proinflammatory cytokines from patients with inactive IBD could be significantly down-regulated by all three immunoregulatory cytokines. The inhibitory effect of IL-13 on TNF-alpha and IL-6 production in differentiated macrophages was diminished in IBD patients, as well as in controls. In disease controls we also observed a reduced inhibition of TNF-alpha and IL-6 after treatment with IL-13. In conclusion, the antiinflammatory activity of IL-13 is partially reduced in patients with active IBD. The hyporesponsiveness of activated and differentiated monocytes to IL-13 and IL-4 does not seem to be a disease-specific phenomenon.

Vasomotor control: functional hyperemia and beyond.

Historically, functional hyperemia has been viewed largely as an interaction between a parenchymal cell and its associated microvasculature. Locally released metabolites have been thought to produce relaxation of the smooth muscle and a vasodilation that increases blood flow in proportion to metabolic need. This symposium report presents evidence from a variety of disciplines and a number of different types of biological preparations that demonstrates that functional hyperemia is a complex process involving several classes of microvessels including capillaries, arterioles, and small arteries. These vessels do not function independently but are coordinated by a complex set of interrelations involving at least three different modes of interaction between parenchymal cells and the various segments of the vascular bed. These are local metabolic effects, propagated effects extending over long segments of the vasculature, and flow-dependent vasodilation induced by local changes in blood flow. In addition to these acute responses to metabolic demand it appears that tissues may be capable of more long-term structural alterations of the arterial and arteriolar network in response to sustained changes in the relationship between supply and demand. The vascular bed appears to be able to adapt either by increasing the maximal anatomic diameter of the large arteries or by inserting new arterioles into the parenchyma. Thus, classical functional hyperemia appears to be but one manifestation of a multifaceted process leading to highly coordinated responses of many vascular elements, resulting finally in vascular patterns that are optimized to meet parenchymal cell demands.

Are endothelial cells rich in filaments involved in the phenomenon of electrically induced stoppages of flow in frog capillaries?

The aim of this study was to determine the frequency of occurrence of the phenomenon in skin and muscle capillaries in both young and mature frogs and to examine the ultrastructure of endothelial cells found in these capillaries. Using microelectrode techniques, trains of pulses of durations up to 90 s, amplitudes -0.2 to -2.0 V, frequencies 10 to 64 Hz and pulse widths 5 to 50 msec were applied to microscopically visualized capillaries situated either in skin (knee region) or at the surface of a sartorius muscle in metamorphosed, intermediately aged or mature frogs. Reproducible flow stoppages induced by these trains of pulses occurred in 2.3%, 1.4% and 6.7% of all tested muscle capillaries and in 11.1%, 82.5% and 68.6% of all skin capillaries in the three age groups, respectively. Examinations of electron micrographs of very thin sections of both skin and muscle capillaries revealed that only 13 out of 47 (27.7%) and 4 out of 45 (8.9%) endothelial cells in these two tissues contained filament bundles. In light of these ultrastructural findings, the dramatically high occurrence of the phenomenon of electrically induced stoppages of flow in skin capillaries (in both intermediately aged and mature frogs) cannot be explained in terms of the hypothesis involving endothelial cells rich in filaments. It is concluded that caution must be exercised when interpreting this phenomenon in terms of the several microvascular responses which the electrical stimulation can elicit.

Occurrence of the "capillary contractility" phenomenon and its significance in the distribution of microvascular flow in frog skeletal muscle.

The phenomenon of "capillary contractility" was first described more than 100 years ago. Recent reports on this phenomenon in the frog mesentery have been based on the observation of dramatic reductions of luminal diameter and blood flow during electrical stimulation of the capillary wall. The aim of this study was to establish the presence and the occurrence of this phenomenon in the frog sartorius muscle and to evaluate its contribution to the distribution of flow. Eight 1.3 X 1.7-mm areas of the muscle surface in eight anesthetized frogs were visualized by means of a microscope and also video-recorded during stimulation experiments. Microelectrodes and semitransparent surface electrodes introduced constant currents (0.1 to 1.0 microA) or pulse trains (5-msec pulse width, 1 to 64 Hz frequency, -0.2 to -5.4 V amplitude) either to individual capillaries or to whole populations of capillaries situated in these areas. Caution was exercised that stimuli did not cause muscle twitching. Red cell velocity in capillaries was measured from video recordings by the flying spot technique. Microelectrode stimulations caused flow stoppages in 4 out of 60 individual stimulated capillaries. Surface electrode stimulations had practically no effect on the mean red cell velocity in any of these populations but an appreciable effect on individual velocities in 7.5% of all capillaries in these populations. It is concluded that the phenomenon of electrically induced reductions of flow exists in the frog sartorius muscle, but it is scarce. It seems unlikely, therefore, that it represents a major mechanism of flow distribution in this tissue.

Intermediate size filaments and microfilaments in frog mesenteric capillaries.

Intermediate size filaments of mesenchymal origin in endothelial cells as well as tonofilaments could be identified by immunofluorescence. Both types of filaments form bundles which run in parallel to the main capillary axis. Filamentous actin could also be detected by this technique. The immunofluorescence of f-actin forms patch-like arrays irregularly distributed along the capillary and striated arrays which are oriented in some cases in parallel to the main capillary axis.

A new method for the simultaneous presentation of low and high magnifications of microscopic specimens: application to in vivo studies of mesenterial capillaries.

By combining an incident light microscope (C. Zeiss) with an inverted camera microscope (ICM 405, C. Zeiss) having a common optic axis, it was possible to present simultaneously two microscope images of one object. These images were recorded by two television cameras and displayed on a single monitor with the aid of a video mixer (Pieper, Schwerte). This method was applied to intravital microscopy of mesenterial capillaries (rabbit and frog). It allowed the choice of a vascular network as an overview with the incident light microscope from which a suitable capillary segment for an electrical stimulation experiment could be selected and magnified with the transmitted light microscope. The results of its application provided proof that electrically induced constrictions in capillaries are independent of proximally or distally occurring vascular reactions. Capillaries of mesenteries in rabbits and frogs were stimulated with direct currents ranging from +0.015 micro A to +20 micro A. The constrictions were found to be reversible and reproducible. In addition capillary constrictions could also be produced by topical application of histamine and serotonin droplets applied to the surface of the mesentery. These observations may help to resolve the question of whether capillary contractility in the sense of Stricker (1865, 1876) is a biological mechanism.

Distribution of oxygen partial pressure in the carotid body region and in the carotid body (rabbit).

In the specific tissue of the rabbit carotid body as well as in the connective tissue surrounding the organ, pO2 distribution was measured with membrane-covered needle electrodes (tip diameter 1-2 microns). The histograms resulting from measurements in the specific tissue were shifted to low pO2 values as compared to other tissues. The oxygen blowing test, i.e. exposure of the carotid body to humidified 100% oxygen was employed to decide upon the site of measurement: pO2 increased when the electrode measured in the surrounding tissue (type 1 response); pO2 remained stable or slightly decreased when the electrode sampled in the specific carotid body tissue (type 2 response). After the experiment, the electrode track was reconstructed from histological serial sections and the type of reaction was related to the type of tissue. Low pO2 values were found to prevail in the specific carotid body tissue, which leads to the conclusion that the amount of low pO2 values determines the degree of chemoreceptor activity.

[A simple method for preparing autologous and homologous FITC/plasma protein conjugates to be used in vital microscopy]. / Eine einfache Methode für die Präparation von autologen und homologen FITC/Plasma-Protein-Konjugaten für die Verwendung in der Vitalmikroskopie.

The present paper describes a method for preparing a spectroscopically uniform conjugate of FITC with the respective plasma of the animal. This plasma is used for vital microscopy investigations. In the case of rabbit plasma, the FITC preparation consists of two protein components with molecular weights of 67 000 and 75 000 Daltons. Apart from these components, no further components could be found after filtration of the FITC preparation via a Sephadex G-75 column. The various fractions of the eluate from the Sephadex G-75 column showed an identical spectrum. It is suggested that the conjugate be purified by filtration over Sephadex G-75 columns for preparation of the FITC conjugate. The stability of the preparation at 37 degrees C is satisfactory. The decrease of the fluorescent emission is about 1% per hour in relation to the emission maximum at 540 nm.