Analysis of acute brain slices by electron microscopy: A correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

Electron microscopy of RecA-DNA complexes: Two different states, their functional significance and relation to the solved crystal structure

Seven different electron microscopy techniques habe been employed to study the RecA protein of E. coli. This review provides a summary of the conclusions that have been drawn from these studies, and attempts to relate these observations to models for the role of RecA protein in homologous recombination.

Synapses on motoneuron dendrites in the brachial section of the frog spinal cord: a computer-aided electron microscopic study of cobalt-filled cells.

Cobalt-labelled motoneuron dendrites of the frog spinal cord at the level of the second spinal nerve were photographed in the electron microscope from long series of ultrathin sections. Three-dimensional computer reconstructions of 120 dendrite segments were analysed. The samples were taken from two locations: proximal to cell body and distal, as defined in a transverse plane of the spinal cord. The dendrites showed highly irregular outlines with many 1-2 microns-long 'thorns' (on average 8.5 thorns per 100 microns 2 of dendritic area). Taken together, the reconstructed dendrite segments from the proximal sites had a total length of about 250 microns; those from the distal locations, 180 microns. On all segments together there were 699 synapses. Nine percent of the synapses were on thorns, and many more close to their base on the dendritic shaft. The synapses were classified in four groups. One third of the synapses were asymmetric with spherical vesicles; one half were symmetric with spherical vesicles; and one tenth were symmetric with flattened vesicles. A fourth, small class of asymmetric synapses had dense-core vesicles. The area of the active zones was large for the asymmetric synapses (median value 0.20 microns 2), and small for the symmetric ones (median value 0.10 microns 2), and the difference was significant. On average, the areas of the active zones of the synapses on thin dendrites were larger than those of synapses on large calibre dendrites. About every 4 microns 2 of dendritic area received one contact. There was a significant difference between the areas of the active zones of the synapses at the two locations. Moreover, the number per unit dendritic length was correlated with dendrite calibre. On average, the active zones covered more than 4% of the dendritic area; this value for thin dendrites was about twice as large as that of large calibre dendrites. We suggest that the larger active zones and the larger synaptic coverage of the thin dendrites compensate for the longer electrotonic distance of these synapses from the soma.

Post-detection analysis for grating-based ultra-small angle X-ray scattering.

Until recently, the hard X-ray, phase-sensitive imaging technique called grating interferometry was thought to provide information only in real space. However, by utilizing an alternative approach to data analysis we demonstrated that the angular resolved ultra-small angle X-ray scattering distribution can be retrieved from experimental data. Thus, reciprocal space information is accessible by grating interferometry in addition to real space. Naturally, the quality of the retrieved data strongly depends on the performance of the employed analysis procedure, which involves deconvolution of periodic and noisy data in this context. The aim of this article is to compare several deconvolution algorithms to retrieve the ultra-small angle X-ray scattering distribution in grating interferometry. We quantitatively compare the performance of three deconvolution procedures (i.e., Wiener, iterative Wiener and Lucy-Richardson) in case of realistically modeled, noisy and periodic input data. The simulations showed that the algorithm of Lucy-Richardson is the more reliable and more efficient as a function of the characteristics of the signals in the given context. The availability of a reliable data analysis procedure is essential for future developments in grating interferometry.

Indirect hydrogen monitoring by chemi-ionisation following an associative ionisation pathway after metastable helium atoms production by electron impact ionisation: Protonation and deuteration of carrier gas

Gas chromatography (GC) is an analytical tool very useful to investigate the composition of gaseous mixtures. However, hydrogen (H2) detection after a GC separation is only possible with a Thermal Conductivity Detector (TCD), a Helium Ionisation Detector (HID) or expensive Atomic Emission Detector (AED). Recently, indirect H2 detection by GC coupled to mass spectrometry (MS) was demonstrated but the mechanism of carrier gas protonation remained unclear. With electron impact as ionisation source of MS and helium (He) as GC carrier gas, H2 is not ionised according the expected Penning ionisation neither according to the Associative ionisation. Rearrangement ionisation (RI) was found to be the main channel for H2 and D2 ionisation under GC-MS conditions used in most of laboratories using GC-MS, leading to the formation of [He−H]+ and [He−D]+ ions.

PENELOPE-2008: A Code System for Monte Carlo Simulation of Electron and Photon Transport

The computer code system PENELOPE (version 2008) performs Monte Carlo simulation of coupledelectron-photon transport in arbitrary materials for a wide energy range, from a few hundred eV toabout 1 GeV. Photon transport is simulated by means of the standard, detailed simulation scheme.Electron and positron histories are generated on the basis of a mixed procedure, which combinesdetailed simulation of hard events with condensed simulation of soft interactions. A geometry packagecalled PENGEOM permits the generation of random electron-photon showers in material systemsconsisting of homogeneous bodies limited by quadric surfaces, i.e., planes, spheres, cylinders, etc. Thisreport is intended not only to serve as a manual of the PENELOPE code system, but also to provide theuser with the necessary information to understand the details of the Monte Carlo algorithm.

Focused ion beam scanning electron microscopy in biology.

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB-SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three-dimensional data, FIB-SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block-face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo-) transmission electron microscopy. Here, we will present an overview of the development of FIB-SEM and discuss a few points about sample preparation and imaging.

X-ray diffraction, thermal analysis, and Raman scattering study of K2BeF4 and comparation to other member of the (beta)-K2SO4 family with ferroelectric -paraelectric transition

Thermal analysis, powder diffraction, and Raman scattering as a function of the temperature were carried out on K2BeF4. Moreover, the crystal structure was determined at 293 K from powder diffraction. The compound shows a transition from Pna21 to Pnam space group at 921 K with a transition enthalpy of 5 kJ/mol. The transition is assumed to be first order because the compound shows metastability. Structurally and spectroscopically the transition is similar to those observed in (NH4)2SO4, which suggests that the low-temperature phase is ferroelectric. In order to confirm it, the spontaneous polarization has been computed using an ionic model.

Electron capture and emission by the Ti acceptor level in GaP

Previously reported results on deep level optical spectroscopy, optical absorption, deep level transient spectroscopy, photoluminescence excitation, and time resolved photoluminescence are reviewed and discussed in order to know which are the mechanisms involved in electron capture and emission of the Ti acceptor level in GaP. First, the analysis indicates that the 3T1(F) crystal¿field excited state is not in resonance with the conduction band states. Second, it is shown that both the 3T2 and 3T1(F) excited states do not play any significant role in the process of electron emission and capture.

The effect of substrate on high-temperature annealing of GaN epilayers: Si versus sapphire

We have studied the effects of rapid thermal annealing at 1300¿°C on GaN epilayers grown on AlN buffered Si(111) and on sapphire substrates. After annealing, the epilayers grown on Si display visible alterations with craterlike morphology scattered over the surface. The annealed GaN/Si layers were characterized by a range of experimental techniques: scanning electron microscopy, optical confocal imaging, energy dispersive x-ray microanalysis, Raman scattering, and cathodoluminescence. A substantial Si migration to the GaN epilayer was observed in the crater regions, where decomposition of GaN and formation of Si3N4 crystallites as well as metallic Ga droplets and Si nanocrystals have occurred. The average diameter of the Si nanocrystals was estimated from Raman scattering to be around 3¿nm. Such annealing effects, which are not observed in GaN grown on sapphire, are a significant issue for applications of GaN grown on Si(111) substrates when subsequent high-temperature processing is required.

Raman microprobe characterization of electrodeposited S-rich CuIn(S,Se)2 for photovoltaic applications: Microstructural analysis

This article reports a detailed Raman scattering and microstructural characterization of S-rich CuIn(S,Se)2 absorbers produced by electrodeposition of nanocrystalline CuInSe2 precursors and subsequent reactive annealing under sulfurizing conditions. Surface and in-depth resolved Raman microprobe measurements have been correlated with the analysis of the layers by optical and scanning electron microscopy, x-ray diffraction, and in-depth Auger electron spectroscopy. This has allowed corroboration of the high crystalline quality of the sulfurized layers. The sulfurizing conditions used also lead to the formation of a relatively thick MoS2 intermediate layer between the absorber and the Mo back contact. The analysis of the absorbers has also allowed identification of the presence of In-rich secondary phases, which are likely related to the coexistence in the electrodeposited precursors of ordered vacancy compound domains with the main chalcopyrite phase, in spite of the Cu-rich conditions used in the growth. This points out the higher complexity of the electrodeposition and sulfurization processes in relation to those based in vacuum deposition techniques.

Analysis of S-rich CuIn(S,Se)2 layers for photovoltaic applications: Influence of the sulfurization temperature on the crystalline properties of electrodeposited and sulfurized CuInSe2 precursors

This paper reports the microstructural analysis of S-rich CuIn(S,Se)2 layers produced by electrodeposition of CuInSe2 precursors and annealing under sulfurizing conditions as a function of the temperature of sulfurization. The characterization of the layers by Raman scattering, scanning electron microscopy, Auger electron spectroscopy, and XRD techniques has allowed observation of the strong dependence of the crystalline quality of these layers on the sulfurization temperature: Higher sulfurization temperatures lead to films with improved crystallinity, larger average grain size, and lower density of structural defects. However, it also favors the formation of a thicker MoS2 interphase layer between the CuInS2 absorber layer and the Mo back contact. Decreasing the temperature of sulfurization leads to a significant decrease in the thickness of this intermediate layer and is also accompanied by significant changes in the composition of the interface region between the absorber and the MoS2 layer, which becomes Cu rich. The characterization of devices fabricated with these absorbers corroborates the significant impact of all these features on device parameters as the open circuit voltage and fill factor that determine the efficiency of the solar cells.

Analysis by optical absorption and transmission electron microscopy of the strain inhomogeneities in InGaAs/InP strained layers

Optical absorption spectra and transmission electron microscopy (TEM) observations on InGaAs/InP layers under compressive strain are reported. From the band¿gap energy dispersion, the magnitude of the strain inhomogeneities. Is quantified and its microscopic origin is analyzed in view of the layer microstructure. TEM observations reveal a dislocation network at the layer interface the density of which correlates with ¿¿. It is concluded that local variations of dislocation density are responsible for the inhomogeneous strain field together with another mechanism that dominates when the dislocation density is very low.

Microstructure and secondary phases in coevaporated CuInS2 films: Dependence on growth temperature and chemical composition

The microstructure of CuInS2-(CIS2) polycrystalline films deposited onto Mo-coated glass has been analyzed by Raman scattering, Auger electron spectroscopy (AES), transmission electron microscopy, and x-ray diffraction techniques. Samples were obtained by a coevaporation procedure that allows different Cu-to-In composition ratios (from Cu-rich to Cu-poor films). Films were grown at different temperatures between 370 and 520-°C. The combination of micro-Raman and AES techniques onto Ar+-sputtered samples has allowed us to identify the main secondary phases from Cu-poor films such as CuIn5S8 (at the central region of the layer) and MoS2 (at the CIS2/Mo interface). For Cu-rich films, secondary phases are CuS at the surface of as-grown layers and MoS2 at the CIS2/Mo interface. The lower intensity of the MoS2 modes from the Raman spectra measured at these samples suggests excess Cu to inhibit MoS2 interface formation. Decreasing the temperature of deposition to 420-°C leads to an inhibition in observing these secondary phases. This inhibition is also accompanied by a significant broadening and blueshift of the main A1 Raman mode from CIS2, as well as by an increase in the contribution of an additional mode at about 305 cm-1. The experimental data suggest that these effects are related to a decrease in structural quality of the CIS2 films obtained under low-temperature deposition conditions, which are likely connected to the inhibition in the measured spectra of secondary-phase vibrational modes.

Micro-Raman study of stress distribution in local isolation structures and correlation with transmission electron microscopy

Stress in local isolation structures is studied by micro‐Raman spectroscopy. The results are correlated with predictions of an analytical model for the stress distribution and with cross‐sectional transmission electron microscopy observations. The measurements are performed on structures on which the Si3N4 oxidation mask is still present. The influence of the pitch of the periodic local isolation pattern, consisting of parallel lines, the thickness of the mask, and the length of the bird"s beak on the stress distribution are studied. It is found that compressive stress is present in the Si substrate under the center of the oxidation mask lines, with a magnitude dependent on the width of the lines. Large tensile stress is concentrated under the bird"s beak and is found to increase with decreasing length of the bird"s beak and with increasing thickness of the Si3N4 film.