Stimulated electron energy loss and gain in an electron microscope without a pulsed electron gun.

We report on a novel way of performing stimulated electron energy-loss and energy-gain spectroscopy (sEELS/sEEGS) experiments that does not require a pulsed gun. In this scheme, a regular scanning transmission electron microscope (STEM) equipped with a conventional continuous electron gun is fitted with a modified EELS detector and a light injector in the object chamber. The modification of the EELS detector allows one to expose the EELS camera during tunable time intervals that can be synchronized with nanosecond laser pulses hitting the sample, therefore allowing us to collect only those electrons that have interacted with the sample under light irradiation. Using  ∼ 5 ns laser pulses of  ∼ 2 eV photon energy on various plasmonic silver samples, we obtain evidence of sEELS/sEEGS through the emergence of up to two loss and gain peaks in the spectra at  ±â€¯2 and  ±â€¯4 eV. Because this approach does not involve any modification of the gun, our method retains the original performances of the microscope in terms of energy resolution and spectral imaging with and without light injection. Compared to pulsed-gun techniques, our method is mainly limited to a perturbative regime (typically no more that one gain event per incident electron), which allows us to observe resonant effects, in particular when the plasmon energy of a silver nanostructure matches the laser photon energy. In this situation, EELS and EEGS signals are enhanced in proportion to n+1 and n, respectively, where n is the average plasmon population due to the external illumination. The n term is associated with stimulated loss and gain processes, and the term of 1 corresponds to conventional (spontaneous) loss. The EELS part of the spectrum is therefore an incoherent superposition of spontaneous and stimulated EEL events. This is confirmed by a proper quantum-mechanical description of the electron/light/plasmon system incorporating light-plasmon and plasmon-electron interactions, as well as inelastic plasmon decay.

Spatial modulation of above-the-gap cathodoluminescence in InP nanowires.

We report the observation of light emission on wurtzite InP nanowires excited by fast electrons. The experiments were performed in a scanning transmission electron microscope using an in-house-built cathodoluminescence detector. Besides the exciton emission, at 850 nm, emission above the band gap from 400 to 800 nm was observed. In particular, this broad emission presented systematic periodic modulations indicating variations in the local excitation probability. The physical origin of the detected emission is not clear. Measurements of the spatial variation of the above-the-gap emission points to the formation of leaky cavity modes of a plasmonic nature along the nanowire length, indicating the wave nature of the excitation. We propose a phenomenological model, which fits closely the observed spatial variations.

Capturing the signature of single atoms with the tiny probe of a STEM.

With their first scanning transmission electron microscope (STEM), Albert Crewe and his collaborators have succeeded 40 years ago in bringing to reality a dream for all electron microscopists, to see individual atoms. In the derivation of Crewe's pioneering work, the present review describes various historical and present steps, involving continuous instrumental and methodological developments as well as the preparation of suitable specimens. They have lead to the identification of individual atoms by electron energy-loss spectroscopy (EELS) and to the demonstration of atom-by-atom spectroscopy. Beyond these spectacular successes which open wide fields of use, most recent technical achievements, such as the introduction of monochromators on the incident electron beam or of optical spectrometers for recording spectra (in the visible as well as in the X-ray domain), will undoubtedly lead to refine the accessible signature of single atoms and molecules.

Enhanced Eshelby twist on thin Wurtzite InP nanowires and measurement of local crystal rotation.

We have performed a detailed study of the lattice distortions of InP wurtzite nanowires containing an axial screw dislocation. Eshelby predicted that this kind of system should show a crystal rotation due to the dislocation induced torque. We have measured the twisting rate and the dislocation Burgers vector on individual wires, revealing that nanowires with a 10-nm radius have a twist up to 100% larger than estimated from elasticity theory. The strain induced by the deformation has a Mexican-hat-like geometry, which may create a tube-like potential well for carriers.

Multi-dimensional and multi-signal approaches in scanning transmission electron microscopes.

Developments in instrumentation are essential to open new fields of science. This clearly applies to electron microscopy, where recent progress in all hardware components and in digitally assisted data acquisition and processing has radically extended the domains of application. The demonstrated breakthroughs in electron optics, such as the successful design and practical realization and the use of correctors, filters and monochromators, and the permanent progress in detector efficiency have pushed forward the performance limits, in terms of spatial resolution in imaging, as well as for energy resolution in electron energy-loss spectroscopy (EELS) and for sensitivity to the identification of single atoms. As a consequence, the objects of the nanoworld, of natural or artificial origin, can now be explored at the ultimate atomic level. The improved energy resolution in EELS, which now encompasses the near-IR/visible/UV spectral domain, also broadens the range of available information, thus providing a powerful tool for the development of nanometre-level photonics. Furthermore, spherical aberration correctors offer an enlarged gap in the objective lens to accommodate nanolaboratory-type devices, while maintaining angström-level resolution for general characterization of the nano-object under study.

Extending the analysis of EELS spectrum-imaging data, from elemental to bond mapping in complex nanostructures.

Multiple least squares fitting has been employed for long time in elemental electron energy-loss spectroscopy (EELS) analysis, in particular in biology, but with the hypothesis of a rather stable shape for the used core-loss signals. In the present case, we explore its use for identifying the variations in the edges' fine structures in complex boron nitride samples and in particular for mapping the bonding types of boron in such samples. Details about this improved procedure applied to data acquired in the spectrum-imaging mode are reported here.

Imaging Si nanoparticles embedded in SiO(2) layers by (S)TEM-EELS.

Fabrication of systems in which Si nanoparticles are embedded in a thin silica layer is today mature for non-volatile memory and opto-electronics applications. The control of the different parameters (position, size and density) of the nanoparticles population is a key point to optimize the properties of such systems. A review of dedicated transmission electron microscopy (TEM) methods, which can be used to measure these parameters, is presented with an emphasis on those relying on electron energy-loss spectroscopy (EELS). Defocused bright-field imaging can be used in order to determine topographic information of a whole assembly of nanoparticles, but it is not efficient for looking at individual nanoparticles. High-resolution electron imaging or dark-field imaging can be of help in the case of crystalline particles but they always provide underestimated values of the nanocrystals population. EELS imaging in the low-energy-loss domain around the Si plasmon peak, which gives rise to strong signals, is the only way to visualize all Si nanoparticles within a silica film and to perform reliable size and density measurements. Two complementary types of experiments are investigated and discussed more extensively: direct imaging with a transmission electron microscope equipped with an imaging filter (EFTEM) and indirect imaging from spectrum-imaging data acquired with a scanning transmission electron microscope equipped with a spectrometer (STEM-PEELS). The direct image (EFTEM) and indirect set of spectra (STEM-PEELS) are processed in order to deliver images where the contribution of the silica matrix is minimized. The contrast of the resulting images can be enhanced with adapted numerical filters for further morphometric analysis. The two methods give equivalent results, with an easier access for EFTEM and the possibility of a more detailed study of the EELS signatures in the case of STEM-PEELS. Irradiation damage in such systems is also discussed.

Improving energy resolution of EELS spectra: an alternative to the monochromator solution.

In this paper, we propose a numerical method which can routinely improve the energy resolution down to 0.2-0.3eV of electron energy-loss spectra acquired in a transmission electron microscope. The method involves measurement of the point-spread function (PSF) corresponding to the spectrometer aberration and to the incident energy spread, and then an inversion of this PSF so as to restore the spectrum. The chosen algorithm is based on an iterative calculation of the maximum likelihood solution known to be very robust against small errors in the PSF used. Restorations have been performed on diamond and graphite C-K edges acquired with an initial energy resolution of around 1eV. After reconstruction, the sharp core exciton lines become clearly visible for both compounds and the final energy resolution is estimated to be about 200-300meV. In the case of graphite, restorations involving both energy resolution and angular resolution have been successfully conducted. Finally, restorations of Fe L(2,3) and O-K edges measured for various iron oxides will be shown.

Costimulation of N-methyl-D-aspartate and muscarinic neuronal receptors modulates gap junctional communication in striatal astrocytes.

Cocultures of neurons and astrocytes from the rat striatum were used to determine whether the stimulation of neuronal receptors could affect the level of intercellular communication mediated by gap junctions in astrocytes. The costimulation of N-methyl-D-asparte (NMDA) and muscarinic receptors led to a prominent reduction of astrocyte gap junctional communication (GJC) in coculture. This treatment was not effective in astrocyte cultures, these cells being devoid of NMDA receptors. Both types of receptors contribute synergistically to this inhibitory response, as the reduction in astrocyte GJC was not observed after the blockade of either NMDA or muscarinic receptors. The involvement of a neuronal release of arachidonic acid (AA) in this inhibition was investigated because the costimulation of neuronal NMDA and muscarinic receptors markedly enhanced the release of AA in neuronal cultures and in cocultures. In addition, both the reduction of astrocyte GJC and the release of AA evoked by NMDA and muscarinic receptor costimulation were prevented by mepacrine, a phospholipase A(2) inhibitor, and this astrocyte GJC inhibition was mimicked by the exogenous application of AA. Metabolites of AA formed through the cyclooxygenase pathway seem to be responsible for the effects induced by either the costimulation of NMDA and muscarinic neuronal receptors or the application of exogenous AA because, in both cases, astrocyte GJC inhibition was prevented by indomethacin. Altogether, these data provide evidence for a neuronal control of astrocytic communication and open perspectives for the understanding of the modalities through which cholinergic interneurons and glutamatergic inputs affect local circuits in the striatum.

Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades.

Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Antiproliferative properties of sphingosine-1-phosphate in human hepatic myofibroblasts.

Sphingosine-1-phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg-1 and Edg-3. S1P stimulated thymidine incorporation and induced activation of extracellular signal-regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P-evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3-kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin-sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood-brain barrier.

Element-selective single atom imaging.

Electron energy-loss spectroscopy (EELS) is widely used to identify elemental compositions of materials studied by microscopy. We demonstrate that the sensitivity and spatial resolution of EELS can be extended to the single-atom limit. A chemical map for gadolinium (Gd) clearly reveals the distribution of Gd atoms inside a single chain of metallofullerene molecules (Gd@C82) generated within a single-wall carbon nanotube. This characterization technique thus provides the "eyes" to see and identify individual atoms in nanostructures. It is likely to find broad application in nanoscale science and technology research.

Pleiotropic effects of lysophosphatidic acid on striatal astrocytes.

Lysophosphatidic acid (LPA) is a potent lipid mediator that is likely involved in diverse functions in the brain. Several recent studies have suggested that astrocytes are important target cells for LPA. In the present study, we have identified the signal transduction pathways activated following LPA stimulation in mouse striatal astrocytes in primary culture. In cells prelabeled with myo-[3H]inositol, LPA stimulated the formation of [3H]inositol phosphates (EC50 = 0.7 microM). This effect was reproduced neither by other lysophospholipids nor by phosphatidic acid. Astrocyte pretreatment with pertussis toxin partially abolished this LPA response indicating the involvement of a Gi/Go protein. In [3H]adenine-prelabeled cells, LPA strongly inhibited the formation of [3H]cyclic AMP induced by forskolin (EC(50) = 0.3 microM) and by isoproterenol and PACAP-38. These inhibitory effects were strongly reduced by pertussis toxin treatment. Although with a lesser potency (EC50 = 5 microM), LPA also stimulated the release of [3H]arachidonic acid from [3H]arachidonic acid-prelabeled astrocytes. This latter effect was totally inhibited by mepacrine, did not involve a pertussis toxin-sensitive G protein, and was highly dependent on external calcium. LPA also stimulated the activity of both extracellular signal-regulated kinases (Erk) Erk1 and Erk2 by a mechanism involving a Gi/Go protein. Surprisingly, in contrast to that observed in fibroblasts, LPA was totally ineffective in stimulating DNA synthesis. These results provide additional evidence in favor of an important physiological role of LPA in the astrocytic functions.

Functional coupling of the NK1 tachykinin receptor to phospholipase D in chinese hamster ovary cells and astrocytoma cells.

In [3H]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK1 tachykinin receptor, the selective NK1 agonist [Pro9]substance P ([Pro9]SP) time and concentration dependently stimulated the formation of [3H]phosphatidylethanol in the presence of ethanol. This [Pro9]SP-induced activation of phospholipase D (PLD) was blocked by NK1 receptor antagonists and poorly or not mimicked by NK2 and NK3 agonists, respectively. In confirmation of previous observations, [Pro9]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro9]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a Gi/Go protein is not involved in these different signaling pathways. The activation of PLD by [Pro9]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro9]SP-evoked response was neither affected by Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK1 receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro9]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK1 receptors.

Interleukin-1 enhances the ATP-evoked release of arachidonic acid from mouse astrocytes.

During neuropathological states associated with inflammation, the levels of cytokines such as interleukin-1beta (IL-1beta) are increased. Several studies have suggested that the neuronal damage observed in pathogenesis implicating IL-1beta are caused by an alteration in the neurochemical interactions between neurons and astrocytes. We report here that treating striatal astrocytes in primary culture with IL-1beta for 22-24 hr enhances the ATP-evoked release of arachidonic acid (AA) with no effect on the ATP-induced accumulation of inositol phosphates. The molecular mechanism responsible for this effect involves the expression of P2Y2 receptors (a subtype of purinoceptor activated by ATP) and cytosolic phospholipase A2 (cPLA2, an enzyme that mediates AA release). Indeed, P2Y2 antisense oligonucleotides reduce the ATP-evoked release of AA only from IL-1beta-treated astrocytes. Further, both the amount of cPLA2 (as assessed by Western blotting) and the release of AA resulting from direct activation of cPLA2 increased fourfold in cells treated with IL-1beta. We also report evidence indicating that the coupling of newly expressed P2Y2 receptors to cPLA2 is dependent on PKC activity. These results suggest that during inflammatory conditions, IL-1beta reveals a functional P2Y2 signaling pathway in astrocytes that results in a dramatic increase in the levels of free AA. This pathway may thus contribute to the neuronal loss associated with cerebral ischemia or traumatic brain injury.

Endothelin stimulates phospholipase D in striatal astrocytes.

In primary cultures of mouse striatal astrocytes prelabeled with [3H]myristic acid, endothelin (ET)-1 induced a time-dependent formation of [3H]phosphatidic acid and [3H]diacylglycerol. In the presence of ethanol, a production of [3H]phosphatidylethanol was observed, indicating the activation of a phospholipase D (PLD). ET-1 and ET-3 were equipotent in stimulating PLD activity (EC50 = 2-5 nM). Pretreatment of the cells with pertussis toxin partially abolished the effect of ET-1, indicating the involvement of a Gi/G(o) protein. Inhibition of protein kinase C by Ro 31-8220 or down-regulation of the kinase by a long-time treatment with phorbol 12-myristate 13-acetate (PMA) totally abolished the ET-1-induced stimulation of PLD. In contrast, a cyclic AMP-dependent process is not involved in the activation of PLD, because the ET-1-evoked formation of [3H]phosphatidylethanol was not affected when cells were coincubated with either isoproterenol, 8-bromo-cyclic AMP, or forskolin. Acute treatment with PMA also stimulated PLD through a protein kinase C-dependent process. However, the ET-1 and PMA responses were additive. Furthermore, the ET-1-evoked response, contrary to that of PMA, totally dependent on the presence of extracellular calcium. These results suggest that at least two distinct mechanisms are involved in the control of PLD activity in striatal astrocytes. Finally, ET-1, ET-3, and PMA also stimulated PLD in astrocytes from the mesencephalon, the cerebral cortex, and the hippocampus.

Practical aspects of electron energy loss spectroscopy (EELS) in biology.

New commercially available electron microscopes accessories permit an easy Electron Energy Loss Spectroscopy (EELS) analysis with good sensitivity and accuracy. But EELS still remains a complex method whose potentiality is seldomly completely exploited. Beside nanoanalysis, several possibilities of producing new images are described. The scattering power image for beam-sensitive specimens, the separation of elastic and inelastic components and the mean free path ratio are among these possibilities. Mass thickness measurement for example can benefit a lot from these techniques. In the nanoanalysis field, a particular emphasis is given to the spectrum-image acquisition method. This method is very convenient to obtain chemical maps of different elements from the same area of the specimen. The background effects and the errors in its estimation are often underevaluated. This can provoke spurious effects in chemical maps. The spectrum-image offers several ways to avoid this inconveniency.

Synergistic effects of acetylcholine and glutamate on the release of arachidonic acid from cultured striatal neurons.

The activation of muscarinic and NMDA receptors by carbachol and NMDA, respectively, stimulated the release of [3H]arachidonic acid ([3H]AA) from cultured striatal neurons. Striking synergistic effects were observed when both agonists were coapplied. This synergistic response was suppressed by atropine or (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine hydrogen maleate and inhibited by magnesium. It was markedly reduced in the absence of external calcium and suppressed by mepacrine. NMDA strongly elevated the intracellular calcium concentration ([Ca2+]i), but carbachol was ineffective. Ionomycin, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, or potassium depolarization, which increased [Ca2+]i but was ineffective on [3H]AA release, also potentiated the carbachol response. Sphingosine and Ro 31-8220 suppressed the responses evoked by carbachol, NMDA, or both agonists. However, no synergistic responses could be observed when phorbol 12-myristate 13-acetate was associated with either carbachol or NMDA. Together, these results suggest that both the massive influx of calcium induced by NMDA and the coupling of muscarinic receptors with a putative phospholipase A2 are required for the strong synergistic effects of carbachol and NMDA on [3H]AA release. Synergistic effects were also observed with acetylcholine and glutamate in the presence of magnesium, further revealing the physiological relevance of this process.

Glial receptors and their intervention in astrocyto-astrocytic and astrocyto-neuronal interactions.

As shown on cultured astrocytes from the mouse, in the presence of adenosine deaminase, 2-chloroadenosine by acting on A1-adenosine receptors potentiated the activation of phospholipase C induced by the alpha 1-adrenergic agonist, methoxamine. This potentiation required the presence of external calcium and was blocked by pertussis toxin. Moreover, this potentiation resulted from a cascade of events: activation (by calcium and protein kinase C) of a phospholipase A2 coupled to A1-adenosine receptors, release of arachidonic acid, which inhibited the reuptake of glutamate into astrocytes and finally additional activation of phospholipase C by externally accumulated glutamate through metabotropic receptors. The effects of 2-chloroadenosine and methoxamine were respectively mimicked by somatostatin and substance P while endothelins reproduced the combined effects of 2-chloroadenosine and methoxamine. Conditioned media from treated astrocytes enriched in glutamate stimulated phospholipase C in cultured striatal neurones. In addition, glutamate alone was also found to stimulate phospholipase A2 in astrocytes through receptors exhibiting a pharmacological profile distinct from metabotropic receptors coupled to phospholipase C and the glutamate response was potentiated by ATP. Moreover, the neuronal arachidonic acid production evoked by glutamate was potentiated by acetylcholine. Finally, the combined application of 2-chloroadenosine and methoxamine on striatal astrocytes reduced the permeability of gap junctions between astrocytes and this response was mimicked by arachidonic acid. Together, these results emphasized the contribution of astrocytes in the regulation of glutamatergic transmission.

Muscarinic cholinergic agonists stimulate arachidonic acid release from mouse striatal neurons in primary culture.

In cultured striatal neurons from embryonic mice, carbachol was found to stimulate the release of arachidonic acid (AA) EC50 = 87 microM) and formation of inositol phosphates (IPs) (EC50 = 54 microM). Both responses were reproduced by muscarinic but not nicotinic agonists, and both exhibited the same pharmacological profile toward four muscarinic antagonists. Furthermore, both responses were insensitive to pertussis toxin, providing additional evidence for the involvement of the same muscarinic receptor(s), most probably of the m1 subtype. Both carbachol-evoked responses were also highly sensitive to the presence of external calcium. The calcium ionophore ionomycin, ineffective alone on AA release, strongly potentiated the carbachol response. In contrast, ionomycin alone stimulated the formation of IPs but did not significantly modify the carbachol response. Protein kinase C activation positively regulated the carbachol-evoked release of AA because this response was markedly potentiated by phorbol 12-myristate 13-acetate (PMA) and was abolished by sphingosine and Ro 31-8220. In contrast, PMA markedly inhibited the carbachol-evoked formation of IPs. The carbachol-evoked release of AA was not mimicked by the combined applications of ionomycin and PMA, which suggests that phospholipase C stimulation alone is not sufficient to trigger AA release. Taken together, these results suggest that the coupling of m1 receptors to a putative phospholipase A2 that is positively regulated by protein kinase C and by calcium is necessary for the carbachol-evoked release of AA.(ABSTRACT TRUNCATED AT 250 WORDS)