Rapidly excised and cryofixed rat tissue

All preparation efforts of biological samples in electron microscopy are focused to preserve structures as close as possible to the native state. To achieve this goal with tissues, it is of advantage to have a very short time between excision and fixation. The most common approach is chemical fixation: cross-linking of the tissue samples with aldehydes followed by postfixation with osmium tetroxide. Here, the fastest approach for tissue samples is perfusion. However, the diffusion of the fixation solution from blood vessels into the depth of the tissue is still slow and does not allow an overall instant fixation of a single cell. As a result, osmotic effects become evident (swelling or shrinkage of cell organelles). Another possibility is to take a tissue sample from the experimental animal. Excision of tissue can last quite some time, which results in even more pronounced autolytic induced osmotic effects. Furthermore, the animal does not survive the procedure in most cases. Alternatively, microbiopsies are an elegant technique to rapidly excise small quantities of tissue. Some tissues, such as liver and muscle, may be obtained using a non-lethal approach. To avoid the artifacts introduced by chemical fixation, high-pressure freezing of microbiopsies (brain, liver, kidney, and muscle) is a powerful alternative to chemical fixation. Here, we describe the microbiopsy method, and high-pressure freezing/freeze-substitution (HPF/FS) as a follow-up procedure. Cryosectioning of high-pressure frozen samples is optimally preserving the ultrastructure; however, it is not considered to be a routine approach yet.

Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

The effect of arts speech therapy on cerebral oxygenation and low frequency hemodynamic oscillations measured using functional near-infrared spectroscopy

Introduction: As a previous study revealed, arts speech therapy (AST) affects cardiorespiratory interaction [1]. The aim of the present study was to investigate whether AST also has effects on brain oxygenation and hemodynamics measured non-invasively using near-infrared spectroscopy (NIRS). Material and methods: NIRS measurements were performed on 17 subjects (8 men and 9 women, mean age: 35.6 ± 12.7 y) during AST. Each measurement lasted 35 min, comprising 8 min pre-baseline, 10 min recitation and 20 min post-baseline. For each subject, measurements were performed for three different AST recitation tasks (recitation of alliterative, hexameter and prose verse). Relative concentration changes of oxyhemoglobin (Δ[O2Hb]) and deoxyhemoglobin (Δ[HHb]) as well as the tissue oxygenation index (TOI) were measured using a Hamamatsu NIRO300 NIRS device and a sensor placed on the subjects forehead. Movement artifacts were removed using a novel method [2]. Statistical analysis (Wilcoxon test) was applied to the data to investigate (i) if the recitation causes changes in the median values and/or in the Mayer wave power spectral density (MW-PSD, range: 0.07–0.13 Hz) of Δ[O2Hb], Δ[HHb] or TOI, and (ii) if these changes vary between the 3 recitation forms. Results: For all three recitation styles a significant (p < 0.05) decrease in Δ[O2Hb] and TOI was found, indicating a decrease in blood flow. These decreases did not vary significantly between the three styles. MW-PSD increased significantly for Δ[O2Hb] when reciting the hexameter and prose verse, and for Δ[HHb] and TOI when reciting alliterations and hexameter, representing an increase in Mayer waves. The MW-PSD increase for Δ[O2Hb] was significantly larger for the hexameter verse compared to alliterative and prose verse Conclusion: The study showed that AST affects brain hemodynamics (oxygenation, blood flow and Mayer waves). Recitation caused a significant decrease in cerebral blood flow for all recitation styles as well as an increase in Mayer waves, particularly for the hexameter, which may indicate a sympathetic activation. References 1. D. Cysarz, D. von Bonin, H. Lackner, P. Heusser, M. Moser, H. Bettermann. Am J Physiol Heart Circ Physiol, 287 (2) (2004), pp. H579–H587 2. F. Scholkmann, S. Spichtig, T. Muehlemann, M. Wolf. Physiol Meas, 31 (5) (2010), pp. 649–662

Software Cartography: Thematic Software Visualization with Consistent Layout

Software visualizations can provide a concise overview of a complex software system. Unfortunately, as software has no physical shape, there is no `natural' mapping of software to a two-dimensional space. As a consequence most visualizations tend to use a layout in which position and distance have no meaning, and consequently layout typically diverges from one visualization to another. We propose an approach to consistent layout for software visualization, called Software Cartography, in which the position of a software artifact reflects its vocabulary, and distance corresponds to similarity of vocabulary. We use Latent Semantic Indexing (LSI) to map software artifacts to a vector space, and then use Multidimensional Scaling (MDS) to map this vector space down to two dimensions. The resulting consistent layout allows us to develop a variety of thematic software maps that express very different aspects of software while making it easy to compare them. The approach is especially suitable for comparing views of evolving software, as the vocabulary of software artifacts tends to be stable over time. We present a prototype implementation of Software Cartography, and illustrate its use with practical examples from numerous open-source case studies.

A differential concentration-dependent effect of IVIg on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms

Background Polymorphonuclear neutrophils (PMN) play a key role in host defences against invading microorganisms but can also potentiate detrimental inflammatory reactions in case of excessive or misdirected responses. Intravenous immunoglobulins (IVIg) are used to treat patients with immune deficiencies and, at higher doses, in autoimmune, allergic and systemic inflammatory disorders. Methodology/Principal Findings We used flow cytometry to examine the effects of IVIg on PMN functions and survival, using whole-blood conditions in order to avoid artifacts due to isolation procedures. IVIg at low concentrations induced PMN activation, as reflected by decreased L-selectin and increased CD11b expression at the PMN surface, oxidative burst enhancement, and prolonged cell survival. In contrast, IVIg at higher concentrations inhibited LPS-induced CD11b degranulation and oxidative burst priming, and counteracted LPS-induced PMN lifespan prolongation. Conclusions/Significance IVIg appears to have differential, concentration-dependent effects on PMN, possibly supporting the use of IVIg as either an anti-microbial or an anti-inflammatory agent.

Restricted or severely hindered diffusion of intramyocellular lipids in human skeletal muscle shown by in vivo proton MR spectroscopy

Although magnetic resonance spectroscopy can be used as a unique tool to study molecular diffusion, it is rarely used to measure the diffusion properties of intramyocellular and extramyocellular lipids. Lipids have very low apparent diffusion coefficients (ADCs), which make these measurements difficult and necessitate strong diffusion gradients and long diffusion times. Consequence is that these measurements have inherently low signal-to-noise ratio and are prone to artifacts. The addition of physiological triggering and individual storage and processing of the spectra is seen to be a possible approach to maximize signal intensity and achieve high reproducibility of the experiments. Thus, the optimized measurement protocol was used to investigate the diffusion properties of lipids in human skeletal muscle in vivo. At a diffusion time of about 110 ms, intramyocellular lipids show a significantly lower ADC (2.0 × 10(-6) mm(2)/s, 95% confidence interval 1.10 × 10(-6) to 2.94 × 10(-6) mm(2)/s) than extramyocellular lipids (1.58 × 10(-5) mm(2)/s, 95% confidence interval 1.41 × 10(-5) to 1.75 × 10(-5) mm(2)/s). Because the chemical properties of both lipid pools can be assumed to be similar, the difference can only be attributed to restricted or severely hindered diffusion in the intramyocellular droplets.

Assessment of coronary artery disease by post-mortem cardiac MR

Minimally invasive or virtual autopsies are being advocated as alternative to traditional autopsy, but have limited abilities to detect coronary artery disease. It was the objective of this study to assess if the occurrence of chemical shift artifacts (CSA) along the coronary arteries on non-contrast, post-mortem cardiac MR may be used to investigate coronary artery disease.

High-resolution imaging of 2D outer membrane protein F (OmpF) crystals by atomic force microscopy

In this chapter the methodological bases are provided to achieve subnanometer resolution on two-dimensional (2D) membrane protein crystals by atomic force microscopy (AFM). This is outlined in detail with the example of AFM studies of the outer membrane protein F (OmpF) from the bacterium Escherichia coli (E. coli). We describe in detail the high-resolution imaging of 2D OmpF crystals in aqueous solution and under near-physiological conditions. The topographs of OmpF, and stylus effects and artifacts encountered when imaging by AFM are discussed.

ARIS-Campaign: Intercomparison of three ground based 22 GHz radiometers for middle atmospheric water vapor at the Zugspitze in winter 2009

This paper presents the Alpine Radiometer Intercomparison at the Schneefernerhaus (ARIS), which took place in winter 2009 at the high altitude station at the Zugspitze, Germany (47.42° N, 10.98° E, 2650 m). This campaign was the first direct intercomparison between three new ground based 22 GHz water vapor radiometers for middle atmospheric profiling with the following instruments participating: MIRA 5 (Karlsruhe Institute of Technology), cWASPAM3 (Max Planck Institute for Solar System Research, Katlenburg-Lindau) and MIAWARA-C (Institute of Applied Physics, University of Bern). Even though the three radiometers all measure middle atmospheric water vapor using the same rotational transition line and similar fundamental set-ups, there are major differences between the front ends, the back ends, the calibration concepts and the profile retrieval. The spectrum comparison shows that all three radiometers measure spectra without severe baseline artifacts and that the measurements are in good general agreement. The measurement noise shows good agreement to the values theoretically expected from the radiometer noise formula. At the same time the comparison of the noise levels shows that there is room for instrumental and calibration improvement, emphasizing the importance of low elevation angles for the observation, a low receiver noise temperature and an efficient calibration scheme. The comparisons of the retrieved profiles show that the agreement between the profiles of MIAWARA-C and cWASPAM3 with the ones of MLS is better than 0.3 ppmv (6%) at all altitudes. MIRA 5 has a dry bias of approximately 0.5 ppm (8%) below 0.1 hPa with respect to all other instruments. The profiles of cWASPAM3 and MIAWARA-C could not be directly compared because the vertical region of overlap was too small. The comparison of the time series at different altitude levels show a similar evolution of the H2O volume mixing ratio (VMR) for the ground based instruments as well as the space borne sensor MLS.

Training synaesthesia

Abstract. Synaesthetic inducers such as graphemes are typically cultural artifacts. Thus, a learning component seems evident in synaesthesia (Simner et al, 2009 Brain 132 57 – 64). Normally, synaesthetes report to have their experiences since they can remember. Nevertheless, a recent training study suggests that synaesthesia can be mimicked in non-synaesthetes. To date, the role of learning during the development of synaesthesia is still debated. It is not clear whether synaesthesia can be learned or trained at all. To address this question, we compared a non-adaptive and an adaptive training for their effectiveness. We assessed their impact on two types of priming tasks, before and after the training. We found stronger priming in the adaptive training group suggesting that adaptive training is more efficient to mimic synaesthesia.

The esophageal ECG as a novel technique for ambulant heart rhythm monitoring

Introduction: Diagnosing arrhythmias by conventional Holter-ECG can be cumbersome because of artifacts, skin irritation and poor P-waves. In contrast, esophageal electrocardiography (eECG) is promising due to the anatomic relationship of the esophagus to the atria and its favorable bioelectric properties. Methods used: In an ambulant setting, we recorded eECGs from 10 volunteers with a novel, highly-miniaturized eECG recorder that is worn discretely behind the ear (1.5×1.8×5cm, 22grams). The device continuously records two eECG leads during 3 days with 500Hz sampling frequency and 24-bit resolution. Results: Mean ± SD recording time was 21.7±19.6 hours (max. 60 hours). Test persons were not limited in daily activities (e.g. eating, speaking) and only complained mild discomfort during probe insertion, which subsided later on. During 99.8% of time, the recorder acquired signals appropriate for further analysis. In unfiltered data, QRS complexes and P-waves were identifiable during >98% of time. P waves had higher amplitudes as compared to surface ECG (0.71 ± 0.42mV vs. 0.16 ± 0.03mV, p = 0.004). No complications occurred. Conclusion: Ambulatory eECG recording is safe, well tolerated and promising due to excellent P-wave detection, overcoming some limitations of conventional Holter ECG.

Fine structure of hippocampal mossy fiber synapses following rapid high-pressure freezing

Synapses of hippocampal neurons play important roles in learning and memory processes and are involved in aberrant hippocampal function in temporal lobe epilepsy. Major neuronal types in the hippocampus as well as their input and output synapses are well known, but it has remained an open question to what extent conventional electron microscopy (EM) has provided us with the real appearance of synaptic fine structure under in vivo conditions. There is reason to assume that conventional aldehyde fixation and dehydration lead to protein denaturation and tissue shrinkage, likely associated with the occurrence of artifacts. However, realistic fine-structural data of synapses are required for our understanding of the transmission process and for its simulation. Here, we used high-pressure freezing and cryosubstitution of hippocampal tissue that was not subjected to aldehyde fixation and dehydration in ethanol to monitor the fine structure of an identified synapse in the hippocampal CA3 region, that is, the synapse between granule cell axons, the mossy fibers, and the proximal dendrites of CA3 pyramidal neurons. Our results showed that high-pressure freezing nicely preserved ultrastructural detail of this particular synapse and allowed us to study rapid structural changes associated with synaptic plasticity.

Bullous pemphigoid: simple measures for a complex disease

Simple descriptive population data are potentially helpful in understanding how bullous pemphigoid (BP) originates and evolves over time. Before embarking with etiological correlations, artifacts and biases should be ruled out. Ideally, epidemiological data should be complemented by immunological and genetic analyses aimed at providing better insight into the causation and prognosis of BP.

Time course based artifact identification for independent components of resting-state FMRI

In functional magnetic resonance imaging (fMRI) coherent oscillations of the blood oxygen level-dependent (BOLD) signal can be detected. These arise when brain regions respond to external stimuli or are activated by tasks. The same networks have been characterized during wakeful rest when functional connectivity of the human brain is organized in generic resting-state networks (RSN). Alterations of RSN emerge as neurobiological markers of pathological conditions such as altered mental state. In single-subject fMRI data the coherent components can be identified by blind source separation of the pre-processed BOLD data using spatial independent component analysis (ICA) and related approaches. The resulting maps may represent physiological RSNs or may be due to various artifacts. In this methodological study, we propose a conceptually simple and fully automatic time course based filtering procedure to detect obvious artifacts in the ICA output for resting-state fMRI. The filter is trained on six and tested on 29 healthy subjects, yielding mean filter accuracy, sensitivity and specificity of 0.80, 0.82, and 0.75 in out-of-sample tests. To estimate the impact of clearly artifactual single-subject components on group resting-state studies we analyze unfiltered and filtered output with a second level ICA procedure. Although the automated filter does not reach performance values of visual analysis by human raters, we propose that resting-state compatible analysis of ICA time courses could be very useful to complement the existing map or task/event oriented artifact classification algorithms.

In Vitro and In Vivo Imaging Characteristics Assessment of Polymeric Coils Compared with Standard Platinum Coils for the Treatment of Intracranial Aneurysms

BACKGROUND AND PURPOSE:Conventional platinum coils cause imaging artifacts that reduce imaging quality and therefore impair imaging interpretation on intraprocedural or noninvasive follow-up imaging. The purpose of this study was to evaluate imaging characteristics and artifact production of polymeric coils compared with standard platinum coils in vitro and in vivo.MATERIALS AND METHODS:Polymeric coils and standard platinum coils were evaluated in vitro with the use of 2 identical silicon aneurysm models coiled with a packing attenuation of 20% each. DSA, flat panel CT, CT, and MR imaging were performed. In vivo evaluation of imaging characteristics of polymeric coils was performed in experimentally created rabbit carotid bifurcation aneurysms. DSA, CT/CTA, and MR imaging were performed after endovascular treatment of the aneurysms. Images were evaluated regarding visibility of individual coils, coil mass, artifact production, and visibility of residual flow within the aneurysm.RESULTS:Overall, in vitro and in vivo imaging showed relevantly reduced artifact production of polymeric coils in all imaging modalities compared with standard platinum coils. Image quality of CT and MR imaging was improved with the use of polymeric coils, which permitted enhanced depiction of individual coil loops and residual aneurysm lumen as well as the peri-aneurysmal area. Remarkably, CT images demonstrated considerably improved image quality with only minor artifacts compared with standard coils. On DSA, polymeric coils showed transparency and allowed visualization of superimposed vessel structures.CONCLUSIONS:This initial experimental study showed improved imaging quality with the use of polymeric coils compared with standard platinum coils in all imaging modalities. This might be advantageous for improved intraprocedural imaging for the detection of complications and posttreatment noninvasive follow-up imaging.