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We have recently introduced a microsecond time-resolved version of cryo-electron microscopy (cryo-EM) to enable the observation of the fast conformational motions of proteins. Our technique involves locally melting a cryo sample with a laser beam to allow the proteins to undergo dynamics in liquid phase. When the laser is switched off, the sample cools within just a few microseconds and revitrifies, trapping particles in their transient configurations, in which they can subsequently be imaged. We have previously described two alternative implementations of the technique, using either an optical microscope or performing revitrification experimentsin situ. Here, we show that it is possible to obtain near-atomic resolution reconstructions fromin siturevitrified cryo samples. Moreover, the resulting map is indistinguishable from that obtained from a conventional sample within our spatial resolution. Interestingly, we observe that revitrification leads to a more homogeneous angular distribution of the particles, suggesting that revitrification may potentially be used to overcome issues of preferred particle orientation.SynopsisNear-atomic resolution reconstructions can be obtained fromin situmelted and revitrified cryo samples. Revitrification results in a more homogeneous angular distribution.

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that involves melting a cryo sample with a laser beam to allow protein dynamics to briefly occur in liquid, before trapping the particles in their transient configurations by rapidly revitrifying the sample. With a time-resolution of just a few microseconds, this approach is notably fast enough to study domain motions that are typically associated with the activity of proteins, but which have previously remained inaccessible. Here, we add crucial details to the characterization of our method. We show that single-particle reconstructions of apoferritin and cowpea chlorotic mottle virus (CCMV) from revitrified samples are indistinguishable from those in conventional samples, demonstrating that melting and revitrification leaves the particles intact and that they do not undergo structural changes within the spatial resolution afforded by our instrument. We also characterize how rapid revitrification affects the properties of the ice, showing that revitrified samples exhibit comparable amounts of beam-induced motion. Our results pave the way for microsecond time-resolved studies of the conformational dynamics of proteins and open up new avenues to study the vitrification process and address beam-induced specimen movement.SynopsisMicrosecond melting and revitrification of cryo samples preserves the structure of embedded particles. The beam-induced motion of revitrified samples is comparable to that of conventional cryo samples.

Reshaping plasmonic nanoparticles with laser pulses has been extensively researched as a tool for tuning their properties. However, in the absence of direct observations of the processes involved, ...

Proteins typically undergo conformational dynamics on the microsecond to millisecond timescale as they perform their function, which is much faster than the time-resolution of cryo-electron microscopy and has thus prevented real-time observations. Here, we propose a novel approach for microsecond time-resolved cryo-electron microscopy that involves rapidly melting a cryo specimen in situ with a laser beam. The sample remains liquid for the duration of the laser pulse, offering a tunable time window in which the dynamics of embedded particles can be induced in their native liquid environment. After the laser pulse, the sample vitrifies in just a few microseconds, trapping particles in their transient configurations, so that they can subsequently be characterized with conventional cryo-electron microscopy. We demonstrate that our melting and revitrification approach is viable and affords microsecond time resolution. As a proof of principle, we study the disassembly of particles after they incur structural damage and trap them in partially unraveled configurations.

The manipulation of liquids at nanoscale dimensions is a central goal of the emergent nanofluidics field. Such endeavors extend to nanodroplets, which are ubiquitous objects involved in many technological applications. Here, we employ time-resolved electron microscopy to elucidate the formation of so-called jumping nanodroplets on a graphene surface. We flash-melt a thin gold nanostructure with a laser pulse and directly observe how the resulting nanodroplet contracts into a sphere and jumps off its substrate, a process that occurs in just a few nanoseconds. Our study provides the first experimental characterization of these morphological dynamics through real-time observation and reveals new aspects of the phenomenon. We observe that friction alters the trajectories of individual droplets. Surprisingly, this leads some droplets to adopt dumbbell-shaped geometries after they jump, suggesting that they spin with considerable angular momentum. Our experiments open up new avenues for studying and controlling the fast morphological dynamics of nanodroplets through their interaction with structured surfaces.

Cryo-electron microscopy is a form of transmission electron microscopy that has been used to determine the 3D structure of biological specimens in the hydrated state and with high resolution. We report the development of 4D cryo-electron microscopy by integrating the fourth dimension, time, into this powerful technique. From time-resolved diffraction of amyloid fibrils in a thin layer of vitrified water at cryogenic temperatures, we were able to detect picometer movements of protein molecules on a nanosecond time scale. Potential future applications of 4D cryo-electron microscopy are numerous, and some are discussed here. © 2013 American Chemical Society.

Cationic zirconocene complexes are active species in Ziegler-Natta catalysis for olefin polymerization. Their structure and metal-ligand bond strength strongly influence their activity. In the present work, the infrared multiphoton dissociation (IRMPD) spectrum of mass selected Zr(C(5)H(5))(2)(OH)(CH(3)CN)(+) cations was obtained in the 300-1500 cm(-1) fingerprint range by coupling a Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an electrospray ionization (ESI) source and the infrared free electron laser (IR-FEL) at the Centre Laser Infrarouge d'Orsay (CLIO). The experimental efforts are complemented by quantum chemical calculations at the MP2 and B3LYP levels using the 6-311G* basis set. Vibrational assignments of transitions observed in the IRMPD spectra to modes of the Zr-O-H, C(5)H(5), and CH(3)CN moieties are based on comparison to calculated linear absorption spectra. Both the experimental data and the calculations provide unprecedented information about structure, metal-ligand bonding, charge distribution, and binding energy of the complex.

Protonated furan (C4H5O+, furanH+) and protonated pyrrole (C4H6N+, pyrroleH+) are generated by chemical ionization of the respective parent molecules in the cell of an FT-ICR mass spectrometer using CH5+/C2H5+ as protonating agents. The protonation site is investigated by resonant infrared multiphoton dissociation (IRMPD) spectroscopy in the 900–1700 cm−1 fingerprint range employing the free electron laser (FEL) at the Centre Laser Infrarouge Orsay (CLIO). Comparison with quantum chemical calculations at the B3LYP/6-311G(2df, 2pd) level of theory demonstrates unambiguously that only the Cα protonated isomers are observed, which correspond to the global minima on the potential energy surfaces of both protonated heterocyclic molecules. Spectroscopic features corresponding to protonation at the Cβ atom or at the heteroatom are not detected. The IRMPD spectra correspond to the first spectroscopic identification of both protonated heterocyclic molecules in the gas phase. During the course of the experiments, the IRMPD spectrum of the furan radical cation (C4H4O+, furan+) has been detected as well. Comparison of the IR spectra of the neutral molecules with the IRMPD spectra of the radical cation and the protonated species reveals the effects of both ionization and protonation on the structural properties of these fundamental heterocyclic molecules.

Microhydrated fluorobenzene cation clusters, C 6 H 5 F + –(H 2 O) n with n = 1 and 2, were characterized by IR photodissociation spectra in the O–H stretch range and UB3LYP/6-31G* calculations. The intermolecular C 6 H 5 F + –H 2 O potential features several minima with charge–dipole orientation and comparable binding energies ( D 0 ∼ 9 ± 1 kcal/mol). The C 6 H 5 F + –H 2 O spectrum is consistent with a structure in which the O atom of H 2 O approaches the C 6 H 5 F + cation from above the aromatic plane. The C 6 H 5 F + –(H 2 O) 2 spectrum reveals the presence of two isomers, in which either a (H 2 O) 2 dimer or two single H 2 O ligands are attached to C 6 H 5 F + . The detected C 6 H 5 F + –(H 2 O) 1,2 complexes were unreactive with respect to nucleophilic substitution.

Our investigation of the phenylalanine/serine (Phe/Ser) protonated dimer suggests that the intermolecular interaction between the two amino acids is more complex than could have been anticipated from previous studies of similar systems. Isomer-specific infrared (IR) spectra, rec-orded at an internal temperature of ~10 K, demonstrate the presence of at least five isomers with non-zwitterionic structures. Moreover, isotopic substitution experiments provide evidence for different protonation sites among the-se isomers.

Nanofluidics involves the study of fluid transport in nanometer-scale structures. We report the direct observation of fluid dynamics in a single zinc oxide nanotube with the high spatial and temporal resolution of four-dimensional (4D) electron microscopy. The nanotube is filled with metallic lead, which we melt in situ with a temperature jump induced by a heating laser pulse. We then use a short electron pulse to create an image of the ensuing dynamics of the hot liquid. Single-shot images elucidate the mechanism of irreversible processes, whereas stroboscopic diffraction patterns provide the heating and cooling rates of single nanotubes. The temporal changes of the images enable studies of the viscous friction involved in the flow of liquid within the nanotube, as well as studies of mechanical processes such as those that result in the formation of extrusions.

We present a radio frequency (RF)/high voltage pulse generator designed to provide suitable waveforms for the operation of a planar multipole ion trap/time-of-flight mass spectrometer. Our generator supplies a RF signal to two pairs of trapping electrodes, allowing ions to be stored in between them. Subsequently, the RF is rapidly switched off and high voltage extraction pulses are applied to the trap electrodes in order to obtain a time-of-flight spectrum of the stored ions. The quenching of the RF and the extraction pulses are synchronized to the RF phase, ensuring well-defined ejection conditions. [http://dx.doi.org/10.1063/1.4802630]

We present a technique for in situ visualization of the biomechanics of DNA structural networks using 4D electron microscopy. Vibrational oscillations of the DNA structure are excited mechanically through a short burst of substrate vibrations triggered by a laser pulse. Subsequently, the motion is probed with electron pulses to observe the impulse response of the specimen in space and time. From the frequency and amplitude of the observed oscillations, we determine the normal modes and eigenfrequencies of the structures involved. Moreover, by selective “nano-cutting” at a given point in the network, it was possible to obtain Young’s modulus, and hence the stiffness, of the DNA filament at that position. This experimental approach enables nanoscale mechanics studies of macromolecules and should find applications in other domains of biological networks such as origamis.

We present a novel, hybrid ion trap/time-of-flight mass spectrometer that is based on a planar multipole design. Compared with Paul trap/time-of-flight instruments, this design possesses the principal advantages of higher injection efficiency and more homogeneous extraction fields. We demonstrate the viability of the concept and describe the characterization of a first prototype. Ions can be injected into the trap with little mass discrimination and stored for several minutes. A resolution of over 1300 is achieved in reflectron mode, and the influence of the RF amplitude and pressure on the resolution is analyzed. We suggest several applications in which this new instrument could offer advantages over existing technology.

Keywords: arenes ; aromatic substitution ; carbocations ; IR spectroscopy ; polycycles Reference EPFL-ARTICLE-225598doi:10.1002/anie.200701838 Record created on 2017-02-13, modified on 2017-05-12

We present a spectroscopic investigation of the excited-state dynamics of the phenylalanine (Phe)/serine (Ser) protonated dimer in the gas phase. Using an ultraviolet (UV) laser pulse, we promote individual isomers to the S1 state and probe their fate with an infrared (IR) pulse. We find that the S1 state has a lifetime of ∼70 ns and undergoes intersystem crossing (ISC) to the T1 state. Time-resolved IR spectra allow us to follow the structural evolution of the dimer. In the S1 state, the different isomers retain the hydrogen-bonding pattern of the ground state. Intersystem crossing triggers a sudden increase of the vibrational energy, so that the dimers can overcome isomerization barriers and explore large parts of the potential energy surface (PES). Their broad IR spectra largely resemble one another and indicate that the dimers adopt a molten structure.

Atomic-resolution electron microscopy is a crucial tool to elucidate the structure of matter. Recently, fast electron cameras have added the time domain to high-resolution imaging, allowing static images to be acquired as movies from which sample drift can later be removed computationally and enabling real-time observations of atomic-scale dynamics on the millisecond time scale. Even higher time resolution can be achieved with short electron pulses, yet their potential for atomic-resolution imaging remains unexplored. Here, we generate high-brightness microsecond electron pulses from a Schottky emitter whose current we briefly drive to near its limit. We demonstrate that drift-corrected imaging with such pulses can achieve atomic resolution in the presence of much larger amounts of drift than with a continuous electron beam. Moreover, such pulses enable atomic-resolution observations on the microsecond time scale, which we employ to elucidate the crystallization pathways of individual metal nanoparticles as well as the high-temperature transformation of perovskite nanocrystals.

Die wesentliche Zielsetzung des Computerprogramms ist die Berechnung des kostenoptimalen regionalen Elektrizitatsversorgungssystems in einem Planungszeitraum von 20 Jahren. Die optimale Losung wird beschrieben durch die installierten und generierten Leistungen der Stromerzeugungsanlagen in den verschiedenen Zeitintervallen. Weitere Ziele sind: Der Programmablauf ist so zu gestalten, daβ eine einfache und eindeutige Bedienerfuhrung durch den Anwender gewahrleistet ist. Das Programm ist auf einem handelsublichen Mikrocomputer zu implementieren. Dadurch werden die Kosten der Hardware in vertretbaren Grenzen gehalten und der Transfer des Programmes in ein Entwicklungsland erleichtert. Die Anpassungsfahigkeit des Programms auf die unterschiedlichen Erfordernisse der Entwicklungslander ist zu gewahrleisten.

Die im Kap. 3 entwickelten Kostenfunktionen werden im folgenden guantifiziert. Die Kostenermittlung basiert in der Regel auf den Daten ausgefuhrter Anlagen bzw. Projekte.

Die Aufgabe der im folgenden Abschnitt beschriebenen Fallstudien ist es, das entwickelte Modell zu testen, d.h. die Ubereinstimmung des Modells mit der Realitat zu uberprufen. Fur konkrete Untersuchungsobjekte werden Strategien zur Ausbauplanung der landlichen Stromversorgung errechnet. Die Ziele des folgenden Abschnittes sind die Darstellung und Interpretation der Ergebnisse, die durch die Anwendung des entwickelten Computermodells auf ausgewahlte Regionen in Entwicklungslandern errechnet wurden, die Schluβfolgerungen aus den Ergebnissen darzustellen und zu entscheiden, ob fur die Strategien zum kostenoptimalen Ausbau der regionalen Elektrizitatsversorgung Aussagen getroffen werden konnen, die unabhangig vom Fallbeispiel fur jedes Entwicklungsland gelten.

Das Modell gliedert sich in die Bereiche Stromerzeugung, Strombedarf, Energieverteilung, Netzregelung und Optimierung. Die ubergeordnete Modellstruktur ist in Abb. 2 dargestellt.

Im Rahmen des folgenden Kapitels werden ausgewahlte Modelle zur Planung der Elektrizitatsversorgung in Entwicklungslandern dargestellt. Die getroffene Auswahl berucksichtigt wichtige Modelle und verdeutlicht deren unterschiedliche Zielsetzungen und Anwendungsgebiete. Zunachst werden Struktur und Ablauf der Modelle erlautert, gefolgt von einer Darstellung der in diesen Modellen verwandten Rechenverfahren zur Optimierung.

Watching lead flow at the nanoscale Microfluidic devices have recently become useful in commercial chemical synthesis. But what about fluid dynamics at the nanometer scale? Lorenz and Zewail used an electron microscope with nanosecond time resolution to capture images of molten lead flowing through a nanotube. They flash-melted the metal with a laser pulse to begin their flow measurements at a precise time point. The experiments offered insights into viscous friction as well as heat-transfer dynamics in a channel one-thousandth as wide as a strand of hair. Science , this issue p. 1496

In ultrafast transmission electron microscopy, time zero can be accurately determined by making use of the photon-induced near-field electron microscopy (PINEM) effect, which causes electrons interacting with the near fields of a nanoparticle to coherently gain or lose energy in multiples of the photon energy when the laser pump and electron probe pulse overlap in time. If the instrument is not equipped with an energy filter, which is required to observe the PINEM effect, the response of a sample is frequently monitored instead. However, the gradual or delayed onset of this response can render an accurate measurement as challenging. Here, we demonstrate a simple and accurate method for determining time zero without an energy filter that is based on the observation that the outline of a nanoparticle blurs when the electron and laser pulse overlap in time. We show that this phenomenon arises from the PINEM effect, which causes some electrons to gain a large energy spread, thus blurring the image due to the chromatic aberration of the imaging system. This effect can also be used to characterize the instrument response and determine the laser polarization in situ. Furthermore, it may find applications for mapping out the near fields of a nanoparticle without the help of an energy filter.

A generally accepted understanding of the anomalous properties of water will only emerge if it becomes possible to systematically characterize water in the deeply supercooled regime, from where the anomalies appear to emanate. This has largely remained elusive because water crystallizes rapidly between 160 K and 232 K. Here, we present an experimental approach to rapidly prepare deeply supercooled water at a well-defined temperature and probe it with electron diffraction before crystallization occurs. We show that as water is cooled from room temperature to cryogenic temperature, its structure evolves smoothly, approaching that of amorphous ice just below 200 K. Our experiments narrow down the range of possible explanations for the origin of the water anomalies and open up new avenues for studying supercooled water.

We report here a new technique for spectroscopic studies of protonated, gas-phase biomolecules and demonstrate its utility by measuring highly resolved electronic and infrared spectra of peptides of up to 17 amino acids. After UV excitation of an aromatic chromophore of a protonated peptide, a CO(2) laser further excites the molecules, increasing their vibrational energy and hence their dissociation rate, allowing detection of the UV excitation by monitoring the resulting photofragments. We show that addition of the CO(2) laser excitation increases the fragmentation yield on the time scale of our experiments by as much as 2 orders of magnitude, significantly enhancing the sensitivity of UV photofragment spectroscopy. We also demonstrate that this approach can be applied in an IR-UV double-resonance scheme, allowing measurement of conformer-specific infrared spectra of protonated peptides.

The characterization of the interactions of Zn2+ ions with imidazole ligands is vital for understanding the function of a plethora of zinc enzymes at the molecular level. The infrared multiple photon dissociation (IRMPD) spectrum of mass selected Zn2+(imidazole)(4) cations, [ZnIm(4)](2+), was obtained in the 670-1840 cm(-1) fingerprint range by coupling the infrared free electron laser (IR-FEL) at the Centre Laser Infrarouge d'Orsay (CLIO) with a quadrupole ion trap mass spectrometer equipped with an electrospray ionization (ESI) source. The experimental efforts are complemented by quantum chemical calculations for [ZnIm(n)](2+) with n = 1-4 at the B3LYP level using basis sets ranging from cc-pVDZ to aug-cc-pVTZ. By comparison with calculated linear absorption spectra, the transitions observed in the IRMPD spectrum are assigned to vibrational modes of the imidazole ligands. In combination, the experimental data and the calculations provide detailed information about structure, metal-ligand bonding, charge distribution, and binding energy of the [ZnIm(4)](2+) complex in the gas phase. The superior abundance of the n = 4 complex of [ZnIm(n)](2+) in the mass spectra of the ESI source is indicative of the preferred coordination number CN = 4 for Zn2+ interacting with imidazole ligands in both the gas and the liquid phase. Comparison of the IR spectra of [ZnIm(n)](2+) with that of bare Im reveals the impact of the strong Zn2+-Im interaction on the electronic, geometric, and vibrational structure of the aromatic ligands upon sequential filling of the first coordination shell. Comparison with the structural and vibrational properties of the imidazole cation demonstrates that the metal-to-ligand charge transfer in [ZnIm(n)](2+) is dominated by sigma donation, whereas contributions from pi donation are minor. (C) 2011 Elsevier B.V. All rights reserved.

Plasmonic nanoparticles in aqueous solution have long been known to fragment under irradiation with intense ultrafast laser pulses, creating progeny particles with diameters of a few nanometers. However, the mechanism of this process is still intensely debated, despite numerous experimental and theoretical studies. Here, we use in situ electron microscopy to directly observe the femtosecond laser-induced fragmentation of gold nanoparticles in water, revealing that the process occurs through ejection of individual progeny particles. Our observations suggest that the fragmentation mechanism involves Coulomb fission, which occurs as the femtosecond laser pulses ionize and melt the gold nanoparticle, causing it to eject a highly charged progeny droplet. Subsequent Coulomb fission events, accompanied by solution-mediated etching and growth processes, create complex fragmentation patterns that rapidly fluctuate under prolonged irradiation. Our study highlights the complexity of the interaction of plasmonic nanoparticles with ultrafast laser pulses and underlines the need for in situ observations to unravel the mechanisms of related phenomena., Direct observation of Coulomb fission of plasmonic nanoparticles in water.

We present here the design of a new tandem mass spectrometer that combines an electrospray ion source with a cryogenically cooled ion trap for spectroscopic studies of cold, gas-phase ions. The ability to generate large ions in the gas phase without fragmentation, cool them to approximately 10 K in an ion trap, and perform photofragment spectroscopy opens up new possibilities for spectroscopic characterization of large biomolecular ions. The incorporation of an ion funnel, together with a number of small enhancements, significantly improves the sensitivity, signal stability, and ease of use compared with the previous instrument built in our laboratory.

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that affords microsecond time resolution. It involves melting a cryo sample with a laser beam to allow dynamics of the embedded particles to occur. Once the laser beam is switched off, the sample revitrifies within just a few microseconds, trapping the particles in their transient configurations, which can subsequently be imaged to obtain a snap shot of the dynamics at this point in time. While we have previously performed such experiments with a modified transmission electron microscope, we here demonstrate a simpler implementation that uses an optical microscope. We believe that this will make our technique more easily accessible and hope that it will encourage other groups to apply microsecond time-resolved cryo-EM to study the fast dynamics of a variety of proteins.

Thanks to their high brightness, field emitters are the electron sources of choice in most high-end electron microscopes. Under typical operating conditions, the available emission current from these emitters is largely limited by practical considerations, and extracting significantly larger currents is usually not possible without reducing the lifetime of the emitter or even damaging it. Such limitations may, however, not apply if the emitter is only briefly subjected to extreme operating conditions so that damage can be outrun. Here, we demonstrate that it is possible to temporarily operate a Schottky emitter far outside its stable operating regime and significantly increase its emission current. We do so by locally heating the tip of the emitter with a microsecond laser pulse, which boosts the emission current by a factor of 3.7 to nearly 450 μA. We believe that the generation of intense microsecond electron pulses from a field emitter will particularly benefit the atomic-resolution imaging of fast processes that occur on the microsecond timescale.