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Yeast morphology and counting are highly important in fermentation as they are often associated with productivity and can be influenced by process conditions. At present, time-consuming and offline methods are utilized for routine analysis of yeast morphology and cell counting using a haemocytometer. In this study, we demonstrate the application of an in situ microscope to obtain a fast stream of pseudohyphae images from agitated sample suspensions of a Saccharomyces cerevisiae strain, whose morphology in cell clusters is frequently found in the bioethanol fermentation industry. The large statistics of microscopic images allow for online determination of the principal morphological characteristics of the pseudohyphae, including the number of constituent cells, cell-size, number of branches, and length of branches. The distributions of these feature values are calculated online, constituting morphometric monitoring of the pseudohyphae population. By providing representative data, the proposed system can improve the effectiveness of morphological characterization, which in turn can help to improve the understanding and control of bioprocesses in which pseudohyphal-like morphologies are found.

To ensure high quality output of biotechnological processes, relevant process parameters need to be monitored. As bioprocesses are increasingly executed in single use bioreactors, there is an increasing demand for new sensors applicable to these processes. In this work, we investigate different approaches for continuous non-invasive cell growth monitoring, especially for single use bioreactor applications. Therefore, the permittivity of the cell culture is used as a measure for the biomass. In a first step, a measuring procedure based on the transmission measurement of an electromagnetic wave is investigated. It appears that the penetration depth of this sensor is not sufficient for a noninvasive measurement through the polymer wall of a single use bioreactor. Therefore, alternative setups based on magnetic induction are investigated. The initial setup is very simple. It consists of a planar coil connected to an impedance analyzer. The coil is attached to the outside of the polymer foil of the single use bioreactor and an impedance spectrum is measured. To evaluate the sensor, E. coli cultivations are performed in a modified cultivation setup, which enables measurements through the polymer foil of a Sartorius BIOSTAT® CultiBag RM, and additionally allows sampling of culture medium for optical density reference measurements. The resonance peak of the coil in the impedance spectrum, is observed as measure for the optical density. Regardless of the simple sensor construction, we found a good correlation between optical density and the damping ratio of the resonance peak. However, the sensor signal shows saturation towards high optical densities. Therefore, an LTCC coil producing a higher magnetic flux density in the culture medium is investigated subsequently. This sensor shows a linear response up to high optical densities, but the sensitivity is reduced compared to the former used coil and therefore scattering of the data is increased. However, to increase the sensitivity, a linear variable differential transformer is realized. Using this setup, the influence of the primary magnetic flux is eliminated from the measuring voltage. This approach delivers the most promising results, as the sensor response is linear up to high optical densities and data scattering is low.

We present a novel automated system for morphology analysis of red blood cells (RBC) under flow. RBC concentrates collected by blood banks for transfusions are stored for periods of up to several weeks, during which time a number of changes occur, collectively termed the storage lesion. Typically the extent of hemolysis is the defining criterion to determine the acceptability of the RBCs for transfusions. Morphological changes are related with biochemical alteration during the storage of RBCs. The typical blood smear procedure for determining such changes is a labor-intensive and potentially biased manual process. The advantage of the flow morphometry system presented here is that it provides fully automated morphological classification of RBCs with large sample numbers in a short time. Our system uses a commercially available flow cell and flow conditions that prevent adhesion of RBCs, thus eliminating the need for blocking agents such as albumin that affect the distribution of cell shapes. Our morphometry results are validated by comparison with standard biochemical assays (hemolysis, ATP) for blood from 17 donors stored under blood bank conditions for 13 weeks. We show that the percentage of spherocytes present can be used to estimate the status of RBC concentrates. © 2017 International Society for Advancement of Cytometry.

Background: The automatic segmentation of pseudohyphal cell-aggregates from brightfield microscopy images for counting forming cells is a challenging task due to the heterogeneous optical appearances of the cells as they may lie on different focal planes. The current cell counting method is based on a time-consuming manual counting of stained cells on a hemocytometer and in most cases, it represents estimates of low statistical significance due to the effort needed to prepare and analyze many samples. In this work, we evaluated the effectiveness of a marker-controlled watershed algorithm for automatic segmentation of pseudohyphae from brightfield microscopic images. The cell heterogeneity problem was addressed by processing intracellular contents of focused and defocused cells to extract initial foreground markers for the watershed method. By properly segmenting cells of different classes within a pseudohypha allows increasing the number of cells analyzed contributing thus to more reliable estimates. To facilitate the evaluation of the proposal by acquiring images containing a diversity of cells´ appearances, we utilized in situ microscopy, an imaging system used to capture images directly from suspensions.Results: The performance of the method was evaluated on 120 portraits of a yeast exhibiting a diversity of pseudohyphal morphologies. Automatic results were compared with manual references obtained by visual inspection of the images. Despite the simultaneous occurrence of a representative mixture of focused, over-, and under-focused cells, the method produced robust results with an average segmentation sensitivity, specificity, and accuracy of 76%, 89%, and 76%, respectively. On average, each microscopic image was processed within 3 s.Conclusions: Our approach was capable to segment pseudohyphae formed by cells exhibiting a large diversity of appearances. The application of a marker-controlled watershed algorithm as a simple, yet effective technique for segmenting pseudohyphae demonstrated satisfactory overall performance to support automated analysis of pseudohyphal cell-aggregates from brightfield images.

In industrial yeast fermentation processes, single-cell yeast suspensions are usually preferable to cells in aggregates, as single cells exhibit a larger contact area with the nutrient medium, which in many cases helps optimize the process. In addition to affecting fermentation time and efficiency, cell aggregates (e.g., pseudohyphal yeast morphology) may also impair centrifugation systems, one of the most expensive and complex steps of the production process that involves the recovery of yeast cells for subsequent fermentation cycles. To date, no standard technique allows for a systematic diagnosis of yeast morphology in real time during sugarcane biofuel fermentation. Accordingly, we investigate an in situ microscope (ISM) for online monitoring of the density and morphology of an industrial Saccharomyces cerevisiae strain widely used in Brazilian distilleries (PE-2). During batch and repeated batch sugarcane molasses fermentation, the instrument revealed single cells, budding yeast cells, and pseudohyphae, all in a variety of sizes and shapes. The ISM image analysis indicated that the volume of single yeast cells increased by roughly 40% over the lag phase before budding and remained approximately constant thereafter. Pseudohyphae with three and more cells appeared mostly during the stationary phase. Cooling problems were simulated by raising the temperature from 33 to 45 °C. During this thermal stress, single cells as well as budding cells and pseudohyphae forming cells became smaller and exhibited intracellular inhomogeneities. From these results, we conclude that an ISM is a useful tool for monitoring yeast morphology during sugarcane fermentation. Atypical morphologies can be detected early and be used as an automatic warning system.

This paper addresses some key issues related to the automation of fermentation process analysis in the context of industrial-scale ethanol production from sugarcane substrates. As the current methods for the determination of cell density and viability are time consuming and laborious, high resolution in situ microscopy (0.5µm) is proposed as a promising alternative. Laboratory-scale experiments presented here show that this imaging technique allows automatic, on-line, and real-time monitoring of yeast cells suspended in sugarcane molasses used in the ethanol industry. In particular, the feasibility of cell concentration measurements of Saccharomyces cerevisiae SA-1 in industrial sugarcane molasses is demonstrated. Automated concentration measurements exhibit a linear correlation with manual reference values using a Neubauer chamber from 3×106 cells/mL up to a saturation level at approximately 2×108 cells/mL. Furthermore, it was demonstrated that the microscopic resolution of this technique, combined with its large statistics, allows a morphological assessment of the size, shape and some internal structures of the yeast cells. On average, the accuracy of the algorithm´s yeast cells classification was 0.80. The results obtained suggest that the ISM is a suitable tool to perform in-line sugarcane fermentation monitoring.

The migratory behaviour and spatial area use of sympatric Arctic charr Salvelinus alpinus and brown trout Salmo trutta were investigated during their marine feeding migration. The likelihood of finding individuals of both species in the inner or outer fjord areas was dependent on water temperature in the inner area (especially for S. alpinus), the temperature difference between the inner and outer areas (especially for S. trutta) and fish fork length (both species). The strongest predictor was the water temperature in the inner area, and particularly S. alpinus left this area and moved to the outer areas with increasing temperatures in the inner area. At 8° C in the inner area, the likelihood of finding S. alpinus in the outer areas was >50%. This predictor had a smaller effect on S. trutta, and the likelihood of finding S. trutta in the outer areas only started to increase at around 14° C. The relationships between temperature and area use did not correspond to the species' optimal growth temperatures, but to their previously documented temperature preferences. Individuals of both species used mainly the littoral fjord areas, and to a lesser extent the pelagic areas. In conclusion, temperature differences between the inner and outer marine areas probably resulted in the segregated area use between the species, because water temperatures or factors influenced by temperature affected their migratory behaviour and habitat use differently. The results indicate that increased marine temperatures with global warming may lead to increased spatial overlap between S. trutta and S. alpinus, which again may lead to increased interspecific competition during their marine phase, and with S. alpinus probably being the more negatively affected.

Background: In the activated sludge process, problems of filamentous bulking and foaming can occur due to overgrowth of certain filamentous bacteria. Nowadays, these microorganisms are typically monitored by means of light microscopy, commonly combined with staining techniques. As drawbacks, these methods are susceptible to human errors, subjectivity and limited by the use of discontinuous microscopy. The in situ microscope appears as a suitable tool for continuous monitoring of filamentous bacteria, providing real-time examination, automated analysis and eliminating sampling, preparation and transport of samples. In this context, a proper image processing algorithm is proposed for automated recognition and measurement of filamentous objects. Methods: This work introduces a method for real-time evaluation of images without any staining, phase-contrast or dilution techniques, differently from studies present in the literature. Moreover, we introduce an algorithm which estimates the total extended filament length based on geodesic distance calculation. For a period of twelve months, samples from an industrial activated sludge plant were weekly collected and imaged without any prior conditioning, replicating real environment conditions. Results: Trends of filament growth rate-the most important parameter for decision making-are correctly identified. For reference images whose filaments were marked by specialists, the algorithm correctly recognized 72 % of the filaments pixels, with a false positive rate of at most 14 %. An average execution time of 0.7 s per image was achieved. Conclusions: Experiments have shown that the designed algorithm provided a suitable quantification of filaments when compared with human perception and standard methods. The algorithm's average execution time proved its suitability for being optimally mapped into a computational architecture to provide real-time monitoring. CA extern

The present study demonstrates the application of in situ microscopy for monitoring the growth of filamentous bacteria which can induce disturbances in an industrial activated sludge process. An in situ microscope (ISM) is immersed directly into samples of activated sludge with Microthrix parvicella as dominating species. Without needing further preparatory steps, the automatic evaluation of the ISM-images generates two signals: the number of individual filaments per image (ISM–filament counting) and the total extended filament length (TEFL) per image (ISM–online TEFL). In this first version of the image-processing algorithm, closely spaced crossing filament-segments or filaments within bulk material are not detected. The signals show highly linear correlation both with the standard filament index and the TEFL. Correlations were further substantiated by comparison with real-time polymerase chain reaction (real-time PCR) measurements of M. parvicella and of the diluted sludge volume index. In this case study, in situ microscopy proved to be a suitable tool for straightforward online-monitoring of filamentous bacteria in activated sludge systems. With future adaptation of the system to different filament morphologies, including cross-linking filaments, bundles, and attached growth, the system will be applicable to other wastewater treatment plants.

International audience; This work aims at checking the possibility of estimating mammalian cell viability from images provided by an in situ microscope (ISM). It was found that images of cells in bioreactors obtained by a high-resolution ISM contain a certain part of cells which exhibits strong morphological similarity with images of dying cells or dead cells obtained by epifluorescence. Cell images of this fraction have less homogeneous texture and less smooth borders as compared to regular cells. Modifications of intracellular organelles and irregularities of the plasma membrane can explain such visual features. Therefore, by only using the texture effect, a criterion is proposed in order to distinguish living cells from the other ones. It is based on the variability of the inside part of the image of the cell. A quantitative estimate of viability (ev) is then calculated from a set of images obtained for each sample. The viabilities obtained from the conventional flow cytometry method are inside the 5% confidence interval of these estimations.

Cell concentration and cell vitality are key parameters to be monitored during cell cultivation processes. Common techniques used for these purposes are often based on sterile sampling and subsequent off line measurements. Extraction and preparation of samples is labour-intensive and risk-entailing. These disadvantages are avoided if the cell culture is directly inspected by using an in-situ technique, e.g. an in-situ microscope (ISM). An ISM delivers a wealth of image data which can be evaluated so as to provide automatic monitoring of the cell density and of morphological parameters as the cell-size. In-situ microscopy can either employ periodic opening and closing of a probe chamber inside the reactor or, alternatively, flash illumination and optical depth of field in order to define a virtual probe zone. Here, we describe optics and software of an advanced version of such an ISM with unprecedented resolution and frame rate. Fast collection of online-galleries of individual cell-portraits even at low cell concentrations enables online morphological analysis without sample extraction. Cell density data obtained by the ISM and traditional counting are shown in comparison, revealing the advantage of the ISM with respect to statistic deviations.

With the objective to develop both characterization methods and test systems for blood and tissue compatibility, some polymers, e.g., polyethylene (PE), polypropylene (PP), and ethylene/propylene/diene-ter-polymer (EPDM) of different shapes, e.g., beads, films, tubes, fibers, tubings, and microtome slices, were grafted with a variety of 15 monomers in order to introduce hydrophobic, hydrophilic, and ionizable groups. The grafted surface was characterized morphologically by the surface area (Brunauer-Emmett-Teller: BET value), by scanning electron microscopy (SEM), and by profilographic measurements. Surface grafting was controlled by frustrated multiple internal reflexion (FMIR)–ir measurements, by determination of the critical surface tension, and by energy-dispersive x-ray analysis (EDXA) combined with SEM. The EDXA–SEM method was found to be a helpful tool to characterize the homogeneity and penetration profile of surface grafting. The tissue compatibility was tested by implanting test samples under the skin of rats. Blood compatibility was determined via in vitro test systems based on the determination of single clotting factors.

In the literature the incidence of cardiac involvement in blunt chest trauma varies considerably. This reflects the diagnostic problems encountered in polytraumatised patients. We report the case of an 18 year old man who suffered bilateral pulmonary contusion and traumatic myocardial infarction following a motorbike accident. The myocardial infarct was diagnosed by means of ECG, cardiac enzymes and echocardiography. When the diagnosis was made the time for successful interventional treatment had lapsed. A coronary angiography was performed after stabilisation which revealed a proximal dilatation of the left anterior descending artery. Left ventricular function was severely impaired (ejection fraction 26%). Due to the pulmonary contusion respiratory support was required for 14 days. The course was further complicated by left ventricular failure with low output.

The oxidation of 13 liquid olefins with either high-voltage or radio frequency (RF) glow discharges has been studied. The reactions were carried out by making the oxygen plasma reach the low vapor pressure substrate. Product formation has proved to be selective—epoxides, aldehydes, ketones, and low quantities of carboxylic acids being the most important species. Fragmentation products were only observed in the traps of the RF system for the most volatile olefin of this study. This indicates that, except for this case, the most relevant interaction of this study has been the heterogeneous reaction of the plasma with the liquid. Total conversion, i.e., mass transformed against initial mass of substrate, has been studied as a function of temperature of the liquid and oxygen flow rate in the reactor, this ranging from 15 to 53.6 mmol/hr in the high-voltage system and 13 to 270 mmol/hr in the RF device. The optimum conversions were 27 to 99%. A correlation between these results and the behavior of the O( 3 P) population in the discharge allows us to conclude that this is the most relevant species to the oxidation process. A discussion relating the structures of the olefins with the composition of the mixtures produced by the oxidation is also presented.

The migratory behaviour and spatial area use of sympatric Arctic charr Salvelinus alpinus and brown trout Salmo trutta were investigated during their marine feeding migration. The likelihood of finding individuals of both species in the inner or outer fjord areas was dependent on water temperature in the inner area (especially for S. alpinus), the temperature difference between the inner and outer areas (especially for S. trutta) and fish fork length (both species). The strongest predictor was the water temperature in the inner area, and particularly S. alpinus left this area and moved to the outer areas with increasing temperatures in the inner area. At 8° C in the inner area, the likelihood of finding S. alpinus in the outer areas was50%. This predictor had a smaller effect on S. trutta, and the likelihood of finding S. trutta in the outer areas only started to increase at around 14° C. The relationships between temperature and area use did not correspond to the species' optimal growth temperatures, but to their previously documented temperature preferences. Individuals of both species used mainly the littoral fjord areas, and to a lesser extent the pelagic areas. In conclusion, temperature differences between the inner and outer marine areas probably resulted in the segregated area use between the species, because water temperatures or factors influenced by temperature affected their migratory behaviour and habitat use differently. The results indicate that increased marine temperatures with global warming may lead to increased spatial overlap between S. trutta and S. alpinus, which again may lead to increased interspecific competition during their marine phase, and with S. alpinus probably being the more negatively affected.

This work reviews the state-of-the-art in image-based in situ methods with regard to their potential use for fermentation of Saccharomyces cerevisiae in sugarcane wine. The integration of real time information from fermentation tanks in the control strategies has high potential to promote better fermentative performance. While several image-based techniques for the measurement of cell concentration have been established, a reliable and consistent viability measurement still remains a challenging task. Reagent-free methods that estimate viability from information contained in micrograph images are reviewed. Nevertheless, the inherent complexity of the sugarcane syrup medium imposes extra challenges regarding its representation in microscopic images and their evaluation by real time image analysis.

A new technique is presented which allows the use of a front-end sensor head for in situ and on-line characterization of cell concentration and cell size during fermentation. An epifluorescence microscope is mounted in a port of a bioreactor viewing directly into the agitated broth. Still images from cells are generated using pulsed illumination. They are directly visualized on a monitor and used for automatic image analysis. The cell concentration and morphological information are determined by counting and evaluating the cell images with respect to their depth from focus characteristic. An in situ microscope was successfully tested during yeast fermentations and yielded results which correlated well with results from a hemocytometer. (c) 1995 John Wiley & Sons, Inc.

International audience; Current state of the art to determine the viability of animal cell suspension cultures is based on sampling and subsequent counting using specific staining assays. We demonstrate for the first time a noninvasive in situ imaging cytometry capable of determining the statistics of a morphologic transition during cell death in suspension cultures. To this end, we measure morphometric inhomogeneity--defined as information entropy--in cell in situ micrographs. We found that the cells are partitioned into two discrete entropy states broadened by phenotypical variability. During the normal course of a culture or by inducing cell death, we observe the transition of cells between these states. As shown by comparison with ex situ diagnostics, the entropy transition happens before or while the cytoplasmatic membrane is loosing its ability to exclude charged dyes. Therefore, measurement of morphometric inhomogeneity constitutes a noninvasive assessment of viability in real time. Biotechnol.

BackgroundTwo of the key parameters to be monitored during cellcultivation processes are cell concentration and viability.Until today, this is very often done off-line by sterilesampling and subsequent counting using a hemocyt-ometer or an electronic cell counter. Cell biology lacks ameasurable quantity by which single cells in suspensioncan be non-invasively diagnosed as dead or alive. How-ever, it would be of significance for process monitoringand in the light of initiatives like PAT if cell density aswell as viability could be determined directly and on-line.Optical measurement of cell density byin situ micro-scopy eliminates the need for sampling and allows forcontinuous monitoring of this key parameter; see e.g.[1,2]; Guez et al. [1] describe an in situ microscope(ISM) which does not use any moving mechanical partswithin or outside the fermentation vessel. It transmits inreal time images taken directly in the stirred suspensionwithin the bioreactor. Image data is processed and eval-uated to provide monitoring of cell-density and mor-phological parameters, e.g. cell size, by means ofassessing the obtained in situ cell-micrographs.Previously, we have extendedin situ microscopytowards viability assessment of suspended cells [3,5].Now, we present new findings on this topic and showthat in cultures of suspended cells, cell-death corre-sponds to measurable changes in morphometric para-meters as e.g. variance, contrast or entropy of thegreyvalues of in situ cell-micrographs. As an example,here we show viability determination via greyvaluedispersion.Material and methodsWe use a custom built high resolution ISM (HS Man-nheim) with water immersion objective, 40x magnifica-tion, numerical aperture 0.75 equipped with optical fiberillumination. Data acquisition is at 0.3– 15 frames persecond, frames have 1293x1040 pixels; primary dataanalysis results in cell micrographs (Figure 1a). We haveapplied the system to bench top and larger bioreactors(see e.g. [4]) and worked mainly with Jurkat, CHO andHybridoma cells.For the experiment presented in Figure 1a/b, cells arecultivated in a Biostat C30 (Sartorius BBI Systems) withthe ISM inserted in one of the existing probe ports. Pro-prietary hybridoma cells (InVivo BioTech Services) arecultured in serum free ISF-1 (InVivo BioTech Services;Biochrom AG) and monitored over the full length ofthe fermentation (not shown).For the experiment presented in Figure 1c, cells arecultivated in a custom built autoclavable steel bench topreactor(HSMannheim)with25mmporttoaccommo-date the ISM and a working volume of 0.7 L. To testreal time and viability determination capabilities of thesystem over a wide range of viabilities in a short time,cells were challenged with 3% Ethanol at 42 hours. Cellcounts (not shown) and viability were determined by insitu microscopy and, as reference, by means of a ViCellcell viability analyser (Beckman Coulter) and Flow Cyto-metry (Partec) using Annexin V / FITC and PI staining.Jurkat cells (DSMZ ACC 282) were cultured in 90%RPMI 1640 + 10% FBS.

Mass spectroscopic analysis of neutrals and ions from a deposition plasma shows that the decomposition of the organometallic precursor compounds [La(thd)3, Cu(acac)2, and Al isopropoxide] in the plasma .starts with the abstraction of complete ligands. The mass spectra of plasma ions sensitively indicate the incomplete oxidation of the organic fragments with increasing organometallic partial pressure. The concentration of carbon-rich ions in the oxygen-based deposition plasma correlates with the carbon content of the deposited oxide films. Specific emissions of the precursor compounds (e.g., Cu atomic lines and LaO bands) can be used to monitor the precursor partial pressure; however, there is some interference with sputter emission from the deposited films. During La2O3 deposition, optical emission of oxidation products (e.g., OH, CO, CO2 bands) was used to regulate the precursor partial pressure in the discharge with a closed-loop control circuit.

A computer-controlled system for measurements of Langmuir probe characteristics is described. Fast data collection (one point in 200 mu s, measurement of the total characteristic in less than 1 s) enables measurements to be made in a time which is short compared with changes of the probe surface condition. Various operation modes facilitate an effective check of the probe surface contamination. The test measurements using conducting (Cu) and dielectric (Al2O3) deposits illustrate the possibility of application of this probe assembly to PECVD of organometallic compounds.

Thin films of magnesium oxide have been deposited on glass, quartz, stainless steel, and nickel by plasma enhanced chemical vapour deposition using 2,2,6,6-tetramethyl-heptanedionato-3,5-magnesium(II) (Mg(thd)2) as precursor. The films show (100)-orientation when deposited at temperatures ≧673 K. The influence of experimental parameters on deposition rate and film properties has been studied.

Improved methods for Al2O3 metallization by Cu are described. Good adhesion between Cu and Al2O3 substrate depends on the formation of chemical bonds between the substrate and the metallic layer. The temperature needed for the formation of a CuAl2O4 spinel interface is reduced from 1050°C to 900°C by the addition of various oxides. The adhesion between the CuAl2O4 interface and deposited Cu is stronger then the tensile strength of pure Cu. Plasma techniques for the formation of a Cu containing interface are also described. Bombardment of a Cu film with Xe+ ions in a rf-glow discharge implants Cu atoms into the substrate to a depth of 5 nm, as determined by SIMS depth profiling. Methods for reduction of the CuAl2O4 surface for subsequent metallization are also presented.

The P300 waveform has been associated with cognitive activity during information processing, and the hippocampus has been discussed as a possible generator of this waveform. This being the case some would argue that it should be possible to record the P300 with a shorter latency and greater amplitude from an electrode placed as near the hippocampus as possible. Under local anaesthesia we inserted a specially constructed needle electrode through the left nostril in the recessus pharyngeus close to the hippocampus. The results did not support the hippocampal hypothesis. Nasopharyngeally recorded P300's were found to have significantly decreased amplitudes and extended latencies when compared to vertex recordings.

In the past years, in situ microscopy has been demonstrated as a technique for monitoring the concentration and morphology of moving microparticles in agitated suspensions. However, up until now, this technique can only achieve a high resolution if a certain manual or automated effort is established for continuous precise focusing. Therefore, the application of in situ microscopes (ISMs) as sensors is inhibited in the cases where unattended operation is required. Here, we demonstrate a high-resolution ISM which, unlike others, is built as an entirely rigid construction, requiring no adjustments at all. This ISM is based on a specially designed water immersion objective with numerical aperture = 0.75 and a working distance of 15 μm. The objective can be built exclusively from off-the-shelf parts and the front surface directly interfaces with the moving suspension. We show various applications of the system and demonstrate the imaging performance with submicron resolution within moving suspensions of microorganisms.

International audience; An in situ microscope (ISM) device is utilised in this study to monitor hybridoma cells concentration in a stirred bioreactor. It generates images by using pulsed illumination of the liquid broth synchronised with the camera frame generation to avoid blur from the cell's motion. An appropriate image processing isolates the sharp objects from the blurred ones that are far from the focal plane. As image processing involves several parameters, this paper focuses on the robustness of the results of the cells counting. This stage determines the applicability of the measuring device and has seldom been tackled in the presentations of ISM devices. Calibration is secondly performed for assessing the cell-concentration from the cell automated numeration provided by the ISM. Flow cytometry and hemacytometer chamber were used as reference analytical methods. These measures and the output of the image processing allow estimating a single calibration parameter: the reference volume per image equal to 1.08 × 10 −6 mL. In these conditions, the correlation coefficient between both reference and ISM data sets becomes equal to 0.99. A saturation of this system during an ultrasonic wave perfusion phase that deeply changes the culture conditions is observed and discussed. Principal component analysis (PCA) is used to undergo the robustness study and the ISM calibration step.

In the literature the incidence of cardiac involvement in blunt chest trauma varies considerably. This reflects the diagnostic problems encountered in polytraumatised patients. We report the case of an 18 year old man who suffered bilateral pulmonary contusion and traumatic myocardial infarction following a motorbike accident. The myocardial infarct was diagnosed by means of ECG, cardiac enzymes and echocardiography. When the diagnosis was made the time for successful interventional treatment had lapsed. A coronary angiography was performed after stabilisation which revealed a proximal dilatation of the left anterior descending artery. Left ventricular function was severely impaired (ejection fraction 26%). Due to the pulmonary contusion respiratory support was required for 14 days. The course was further complicated by left ventricular failure with low output.

Thin films of yttria‐stabilized zirconia were deposited by plasma‐enhanced chemical vapor deposition on quartz Si(100), Si(111), Ni, and the steels V2A and hastelloy at substrate temperatures (Ts): 673–873 K. The metal β‐diketonates Y(thd)3 and Zr(thd)4 were used as precursors. The fully stabilized fluorite‐type cubic structure was obtained over a wide range of yttria contents from 3.5 to 80 mol % (Ts=773 K). The quality of the films depended on the match of the thermal expansion coefficients of substrate and deposit.

In this study we have measured exposure levels to quartz in different parts of the slate industry in Alta, Northern Norway. Full shift personal samples were collected from the breathing zones of outdoor and indoor workers in the slate quarries and a slate factory. The quartz content of respirable dust was between 7 and 41%. The slate factory had the lower quartz levels although 41% of total and 73% of respirable samples were above the Norwegian TLV for quartz. The average concentration of total quartz in the slate factory was 0.27 mg/m3 and the average concentration of respirable quartz was 0.12 mg/m3. Outdoor in the quarries the average levels of quartz were 0.58 and 0.13 mg/m3 for total and respirable quartz, respectively. From the beginning of the last decade most of the quarry-workers have built quarry halls to protect themselves against a cold winter climate. Inside in these quarry halls the average levels were 1.74 mg/m3 total quartz and 0.46 mg/m3 respirable quartz. Assessment of historical exposure showed that 32 of totally 45 quarry workers with available exposure history had a lifetime inhaled quartz dose of more than 10 g. There is reason to fear that silicosis will be an increasing problem among quarry workers if efforts to reduce quartz exposure are not put into effect.

A new depth-from-focus technique is introduced that requires only a single image to determine the distance of simple shaped objects from the focal plane. The technique has been applied to evaluate the concentration of cells in a bioreactor during a fermentation process. Since the low-intensity fluorescent light gathered by a light-amplifying camera results in images of low signal-to-noise ratio, an adaptive smoothing filter is used. A sharpness criterion derived from bandpass decomposition of the image in a Laplacian pyramid is used to define a virtual measuring volume. In this volume, process parameters such as cell concentration, cell size and intensity of cell fluorescence are evaluated. The technique is also suitable for other types of simple objects.

The horizontal relationship of the jaws has been defined as the angles or distances between the reference planes of the craniofacial complex and points A and B, which are representative of the anterior limits of the denture bases. The aims of this study were (1) to examine statistically and geometrically the different cephalometric measurements which are used to indicate the A-P jaw relationship, and (2) to provide a more reliable parameter by means of comparative cephalometric analyses with various clinical examples. The APDI and Wits appraisal are parameters for evaluating the anteroposterior relationship of the dentition rather than the jaws. FH to AB plane angle (FABA) may provide not only a reliable cephalometric measurement of the anteroposterior relationship of the jaws but also a clue to the facial profile.

A process has been developed for coating PTFE with copper. It consists of a plasma pretreatment with reactive gases such as O2 or CF4/O2 followed by a plasma deposition of a thin metallic layer of Pd, Pt, Au, or Cu and subsequent electroless and electrochemical copper plating. The coatings show excellent adhesion properties, limited only by the cohesive strength of the substrate. The PTFE surfaces have been studied with scanning electron microscopy (SEM), and x-ray photoelectron spectroscopy (XPS) after different steps of the pretreatment. SEM reveals directly the roughening of the PTFE surface by the plasma etching step. XPS results for Pd-activated surfaces point to a chemical interaction between Pd and the fluorine atoms of the PTFE. This new process of metallization is a simple technique, avoids hazardous chemicals, requires no decontamination and can equally well be applied to other polymers.

This study was undertaken to ascertain the changes of fetuses in craniofacial dimension and to evaluate the growth and development during fetal period. Lateral cephalograms by soft X-ray were taken from 64 fetuses and were measured in linear and angular aspects. The following conclusions were obtained. 1. The linear increase rates of anterior cranial base length exceeded those of the posterior cranial base length. 2. Growth increments on horizontal dimension were greater than vertical dimension in the maxilla. 3. The cranial base angle was almost constant after fetal period 13 weeks with the average angle of 130 degrees. 4. The angle between anterior cranial base and palatal plane was decreased gently during fetal period.

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.

Doppler-free two-photon spectroscopy of several states of6, 7Li has been achieved by placing an atomic beam inside the dye laser cavity. The hyperfine structure splitting of the 4S state, the fine structure splitting of the 3D level as well as the isotope shifts of the 2S–4S and 2S-3D transition have been measured, by scanning the single-mode laser frequency and detecting the fluorescence light. In addition, signal detection by three-photon ionization via the resonant 3D state was accomplished.

An automatic Michelson-type fringe counting interferometer (Lambda-meter) allowing for interpolation to 1/50 of a wavelength is described. The movable part of the interferometer consists of a carriage which slides on two polished steel bars and transports two corner cube retroreflectors. A missing-fringe digital control logic identifies and instantaneously corrects for laser interference fringe dropouts, caused by short-time laser light instabilities. By exact superposition of the beams of a He 22 Ne laser stabilized to a line of I 2 and the laser to be measured in the interferometer, absolute wavelength measurements with a precision of 1 to 2 parts in 10 8 were performed. Real-time control of the measurement, data acquisition and calculation of the wavelength are provided by a microcomputer.

Das Durchschlagverhalten dunner Kunststoff-Folien wurde bei Frequenzen zwischen 1 Hz und 50 Hz und bei Gleichspannung experimentell untersucht. Aus den Ergebnissen kann gefolgert werden, das bei Einbettung von Elektroden und Probe in feste Isolierstoffe keine Teilentladungen vor dem Durchschlag der Probe auftreten. Bei einer Spannungssteigerung von mehr als 1 kV/s ist die Durchschlagspannung bei Gleichspannung gleich dem Scheitelwert der Durchschlagspannung bei 50 Hz. Aus diesem kann dann bei bekannter Probendicke die „wahre” Durchschlagfestigkeit errechnet werden.