4D multiple-cathode ultrafast electron microscopy

Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging.In 4D ultrafast electron microscopy (UEM), ultrafast light pulses generate electron packets by photoemission at the cathode of an electron microscope, and these are used to probe a dynamic process initiated by heating or exciting the microscopic specimen with a second, synchronized ultrafast light pulse (1, 2). In conventional implementations, each pump pulse on the specimen is accompanied by one suitably delayed laser pulse on the cathode to generate one packet of electrons probing a single time point in the evolution of the specimen. A record of the full course of temporal evolution of the specimen is then constructed by repeating the experiment multiple times with variation of the delay time between the two light pulses, reading out a separate CCD image for each delay time. Thus, information about different time points in the dynamic response of the specimen is obtained from different excitation events. This implementation is ideally suited for a specimen that undergoes irreversible but sufficiently well-defined dynamics to allow a new specimen area to be used for each time point (Fig. 1A), or for a specimen that recovers fully to allow repeated identical excitations of the same area (Fig. 1B); see also Methodology. The applications of these two approaches are numerous, as highlighted in a recent review account of the work (3).Open in a separate windowFig. 1.Variant implementations of UEM. (A) Single-pulse UEM, which enables single-shot imaging of homogeneous specimens. (B) Stroboscopic UEM. (D) Multiple-cathode UEM. For comparison, we include, in C, the single-cathode, deflection method. See text for details.For the study of completely nonrepetitive dynamics, for example, a stochastic process in a heterogeneous sample that does not return to its initial configuration, a series of snapshots following a single excitation event can provide the only direct and detailed view of the evolution. Observing the effects of a single excitation pulse with video-mode imaging can currently reach millisecond-scale time resolution, far short of the time scale for many phenomena of interest in nanoscale materials science, chemistry, and physics. Nanosecond resolution has been reached (4) by combining one excitation pulse with a train of light pulses on a single cathode, with deflection of the imaging electrons after passing the specimen plane to direct each successive pulse to a new region of the detector (Fig. 1C). This nanosecond method has been successfully used with a high-speed electrostatic deflector array to obtain time sequences of irreversible and stochastic processes (5, 6).Here we demonstrate a technique that removes any limit on time resolution imposed by image deflection and in a single frame enables the capture of ultrafast phenomena. With this approach, it is possible to probe and distinctly record multiple time points in a dynamic process following a single initiation pulse. The probing electron packets are all generated by a single light pulse that impinges on multiple, spatially distinct, cathode surfaces (Fig. 1D). Time separations between packets in the electron-pulse train are adjusted by the cathode spatial and electrostatic configuration. In the present application, two electron packets, generated from two source locations at the same potential and separated in time by 19 picoseconds (ps), are recorded on each CCD frame after undergoing diffraction in a gold film following femtosecond heating. The packets originate from different cathode locations, pass through the same area of the specimen, and are recorded at distinct locations on the detector, thereby encoding two different time points in the evolution of the specimen. The results obtained provide the basis for exploration of expanded application of the multiple-cathode concept.     

Four-dimensional ultrafast electron microscopy

Electron microscopy is arguably the most powerful tool for spatial imaging of structures. As such, 2D and 3D microscopies provide static structures with subnanometer and increasingly with ångstrom-scale spatial resolution. Here we report the development of 4D ultrafast electron microscopy, whose capability imparts another dimension to imaging in general and to dynamics in particular. We demonstrate its versatility by recording images and diffraction patterns of crystalline and amorphous materials and images of biological cells. The electron packets, which were generated with femtosecond laser pulses, have a de Broglie wavelength of 0.0335 Å at 120 keV and have as low as one electron per pulse. With such few particles, doses of few electrons per square ångstrom, and ultrafast temporal duration, the long sought after but hitherto unrealized quest for ultrafast electron microscopy has been realized. Ultrafast electron microscopy should have an impact on all areas of microscopy, including biological imaging.     

Biomechanics of DNA structures visualized by 4D electron microscopy

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.     

Infrared PINEM developed by diffraction in 4D UEM

The development of four-dimensional ultrafast electron microscopy (4D UEM) has enabled not only observations of the ultrafast dynamics of photon–matter interactions at the atomic scale with ultrafast resolution in image, diffraction, and energy space, but photon–electron interactions in the field of nanoplasmonics and nanophotonics also have been captured by the related technique of photon-induced near-field electron microscopy (PINEM) in image and energy space. Here we report a further extension in the ongoing development of PINEM using a focused, nanometer-scale, electron beam in diffraction space for measurements of infrared-light-induced PINEM. The energy resolution in diffraction mode is unprecedented, reaching 0.63 eV under the 200-keV electron beam illumination, and separated peaks of the PINEM electron-energy spectrum induced by infrared light of wavelength 1,038 nm (photon energy 1.2 eV) have been well resolved for the first time, to our knowledge. In a comparison with excitation by green (519-nm) pulses, similar first-order PINEM peak amplitudes were obtained for optical fluence differing by a factor of more than 60 at the interface of copper metal and vacuum. Under high fluence, the nonlinear regime of IR PINEM was observed, and its spatial dependence was studied. In combination with PINEM temporal gating and low-fluence infrared excitation, the PINEM diffraction method paves the way for studies of structural dynamics in reciprocal space and energy space with high temporal resolution.Since its invention in the 1930s by Knoll and Ruska (1), the electron microscope has become a powerful tool in the fields of physics, chemistry, materials, and biology. A great variety of techniques related to the electron microscope has been developed in image, diffraction, and energy space (2, 3), with the spatial and energy resolutions of the transmission electron microscope now reaching 0.5 Å with Cs corrector (4) and sub-100 meV with electron monochromators (5, 6), respectively.To these capabilities of spatial and energy resolution has been added the high resolution in the fourth dimension (time) by the development of four-dimensional ultrafast electron microscopy (4D UEM) (7–9), currently enabling nanoscale dynamic studies with temporal resolution that is 10 orders of magnitude better than the millisecond range of video-camera-rate recording in conventional microscopes. In 4D UEM, ultrafast time resolution is reached by using two separate but synchronized ultrashort laser pulses, one to generate a probing electron pulse by photoemission at the microscope cathode and the other to excite the specimen into a nonequilibrium state. The state of the specimen within the window of time of the probe pulse can be observed by recording the probe electron packet scattered from the specimen in any of the different working modes of the microscope, such as image and diffraction (10), energy spectrum (11), convergent beam (12), or scanning transmission electron microscopy (TEM) (13). Scanning the time delay between arrival of the pump and probe pulses at the specimen, which is controlled by a precise optical delay line, allows the evolution of the specimen to be traced.One of the important techniques developed in, and unique to, UEM is photon-induced near-field electron microscopy (PINEM) (14). PINEM has extended the capability of UEM to observation of light–electron interactions near nanostructures or at an interface, which offers exciting prospects for the study of dynamics of photonics and plasmonics at the nanometer scale (15). The three-body interaction of photon, electron, and nanostructure relaxes momentum conservation and leads to efficient coupling between photons and electrons (16). In PINEM, an ultrashort optical pulse is used to excite evanescent electromagnetic fields near a nanostructure or at an interface. When the probe electron packet is in spatiotemporal overlap with these evanescent or scatter fields, some of its electrons can absorb/emit one or more scattered photons and then be detected by their contributions to displaced energy peaks in the electron energy spectrum. These displaced peaks appear as discrete sidebands to the zero-loss peak at separations given by the photon energy (hν) of the pump optical pulse. When using energy filtering to select for imaging only those electrons gaining energy, the resulting PINEM image reflects the strength and topology of the excited near field around the nanostructure or interface.The PINEM technique has been used to detect the evanescent near field surrounding a variety of structures with different materials properties and different geometries, such as carbon nanotubes (14), silver nanowires (14, 17), nanoparticles (16, 18), cells and protein vesicles (19), and several-atoms-thick graphene-layered steps (20). In addition, focused-beam PINEM has been used in scanning TEM mode to obtain induced near-field distributions for a copper grid bar (21), a nanometer gold tip (22), and a silver nanoparticle at the subparticle level (21). In a recent publication, three pulses, two optical and one electron, were introduced into the arsenal of techniques to gate the electron pulse and make its width only limited by the optical-pulse durations (23). Numerous general theoretical treatments (24–28) have successfully described the phenomenon, with detailed treatments quantitatively reproducing many unique features of these multifaceted experimental observations (17, 20–22).Despite the growing body of PINEM studies, almost all previously published PINEM results were obtained in the image mode of the electron microscope using optical pulses with wavelengths of 500–800 nm. An exception is a single unresolved PINEM spectrum for 1,038-nm excitation published from this laboratory (25). Because the PINEM response of a material is governed by its optical properties and dimensions relative to the wavelength of light, excitation wavelength is an important parameter largely remaining to be explored experimentally.Here we report the development of IR PINEM using excitation at the wavelength of 1,038 nm (photon energy 1.2 eV). The spatial- and fluence-dependent behavior of well-resolved IR PINEM induced at the edge of a copper grid bar is examined by combining nanometer-scale convergent-beam electron diffraction and diffraction-mode detection for electron-energy spectroscopy with an unprecedented energy resolution down to 0.63 eV at 200 keV. Different e-beam size effects were compared for PINEM generated by green and IR pump pulses. The spatial dependence of IR PINEM at the interface was studied at low-pulse fluence (linear regime) and high-pulse fluence (nonlinear regime). Diffraction of a gold crystal film was observed using the energy-resolved PINEM electrons produced by interaction with the scatter field of the adjacent copper grid edge. Notably, substantial PINEM peak amplitudes were achievable at dramatically lower fluence for IR pulses than for green pulses, opening up a possible path for studies of photosensitive materials. This general accessibility of strong PINEM signals is of particular importance for our primary interest of ultrafast dynamics, for which PINEM photon gating has the potential to vastly improve temporal resolution.All PINEM experiments reported here were performed on the California Institute of Technology UEM-2 apparatus. The operation voltage on UEM-2 is 200 keV. The laser system used emits a train of ∼220-fs pulses with wavelength of 1,038 nm, set to operate at a repetition rate of 1 MHz. The laser output was frequency-doubled two successive times to provide the 259-nm pulses used to generate the electron packet (probe beam) at the 200-keV microscope photocathode source. The residual 1,038-nm and 519-nm optical pulses were each available for use as the PINEM pump beam to excite the near-field plasmons at the interface. All of the experiments were carried out with polarization set to be perpendicular to the interface and in the single-electron regime (8) to eliminate space-charge effects. In diffraction mode, a camera length of 920 mm and a spectrometer entrance aperture of 1 mm were used to obtain a small collection angle for better energy resolution.     

Visualization of carrier dynamics in p(n)-type GaAs by scanning ultrafast electron microscopy

Four-dimensional scanning ultrafast electron microscopy is used to investigate doping- and carrier-concentration-dependent ultrafast carrier dynamics of the in situ cleaved single-crystalline GaAs(110) substrates. We observed marked changes in the measured time-resolved secondary electrons depending on the induced alterations in the electronic structure. The enhancement of secondary electrons at positive times, when the electron pulse follows the optical pulse, is primarily due to an energy gain involving the photoexcited charge carriers that are transiently populated in the conduction band and further promoted by the electron pulse, consistent with a band structure that is dependent on chemical doping and carrier concentration. When electrons undergo sufficient energy loss on their journey to the surface, dark contrast becomes dominant in the image. At negative times, however, when the electron pulse precedes the optical pulse (electron impact), the dynamical behavior of carriers manifests itself in a dark contrast which indicates the suppression of secondary electrons upon the arrival of the optical pulse. In this case, the loss of energy of material’s electrons is by collisions with the excited carriers. These results for carrier dynamics in GaAs(110) suggest strong carrier–carrier scatterings which are mirrored in the energy of material’s secondary electrons during their migration to the surface. The approach presented here provides a fundamental understanding of materials probed by four-dimensional scanning ultrafast electron microscopy, and offers possibilities for use of this imaging technique in the study of ultrafast charge carrier dynamics in heterogeneously patterned micro- and nanostructured material surfaces and interfaces.Recent advances in four-dimensional (4D) ultrafast electron microscopy (UEM) have made it possible to investigate nonequilibrium electronic and structural dynamics with atomic-scale spatial resolution and femtosecond temporal resolution (1). Unlike UEM, which operates in the transmission mode, scanning UEM techniques exploit the time evolution of secondary electrons (SEs) produced in the specimen, and provide additional marked advantages over the transmission mode. These include a relatively facile sample preparation requirement, an efficient heat dissipation, a lower radiation damage, and an accessibility to low-voltage environmental study (2, 3). Since its development this technique has been used to study carrier excitation dynamics in several prototypical semiconducting materials surfaces. In these studies, image contrast was monitored as a function of time, and it was found that Si exhibits a bright contrast in the image at positive times without appreciable dynamics at negative times, whereas CdSe displays bright contrast at positive times and dark contrast at negative times (2). However, the correlation between the measured time-dependent SE intensity and electronic structure of the material of interest remains elusive. Chemical doping is a widely used method to control the electronic properties of semiconducting materials by incorporating charge donating or accepting dopant atoms. It is a key element in developments involving modern semiconductor-based solid-state electronics.Here, we present a systematic study for the doping- and carrier-concentration-dependent carrier dynamics in the in situ cleaved GaAs(110) surface observed in the images obtained using scanning UEM. We show that the enhancement of the SE signal at time 0 is associated with the energy gained by the optical excitation, which increases SE production from the probing pulse, and this process mirrors the electronic doping characteristics of the semiconducting material. In contrast, the persistent dark contrast at both positive and negative times for carrier dynamics in GaAs(110) suggests an energy loss mechanism that involves strong suppression of SEs through carrier–carrier scatterings. Our simulations of the transient behavior further support this conclusion.A schematic representation of the experimental setup is given in Fig. 1. Electron pulses generated from a field-emission gun using femtosecond laser pulse irradiation are scanned across the specimen surface, which is illuminated with the optical pulse. The electrons emitted from the material surface are used to construct time-resolved images at various time delays between the optical pulse and the electron pulse. The detailed account of the experimental setup was described in previous publications from this laboratory (2–4), and thus here we briefly describe the imaging setup: the laser used in our experiments is an ytterbium-doped fiber laser system that generates ultrashort pulses at a central wavelength of 1,030 nm (measured pulse width of ∼400 fs). The second harmonic (photon energy of 2.4 eV) of the laser beam was directed to the sample at room temperature, whereas the quadrupled harmonic (photon energy of 4.8 eV) was used for the pulsed electron generation from the field-emission gun in SEM. For the series of experiments presented here, the pump laser fluence, repetition rate, and the data acquisition methodology were kept the same for comparison of samples with different doping characteristics. The pump laser fluence was deduced to be 69 μJ/cm², which is more than three orders of magnitude lower than that reported for the laser-induced damage threshold of a crystalline GaAs (∼0.1 J/cm² at a photon energy of 1.9 eV) (5). The emitted electrons from the material were measured using a positively biased Everhart-Thornley detector.Open in a separate windowFig. 1.Schematic representation of the scanning UEM at California Institute of Technology. Pulsed electrons are scanned over a specimen that is illuminated with an optical pulse, and SEs emitted from the material surface are detected to construct time-resolved images at various time delays between the optical and the electron pulse. In the case of a semiconducting material, at time 0, the optical pulse promotes electrons from the valence band to the conduction band, and immediately after that the electron pulse excites transiently populated conduction electrons above the vacuum level, resulting in an enhanced (bright contrast) SE emission. If SEs experience a material-dependent energy loss through the various channels of scattering processes involved while migrating toward the surface, then a decreased emission will result (dark contrast). Here, E_c, E_v, and E_vac are the energies of the bottom of the conduction band, the top of the valence band, and the vacuum level, respectively. Scale bars in the time-resolved images correspond to 50 μm.All scanning UEM images were acquired at a dwell time of 1 μs and were integrated 64 times to improve the signal-to-noise ratio. All experiments were conducted at a repetition rate of 4.2 MHz to ensure a full recovery of the material’s dynamical response before the arrival of a next pump pulse. Single crystals of GaAs (a direct band gap of 1.43 eV at room temperature) were all grown via Vertical Gradient Freeze method (purchased from MTI); the method is known to produce fewer defects during the growth, compared with those grown via the liquid encapsulated Czochralski method (6). The crystals were in situ cleaved in high vacuum (<1.5 × 10⁻⁶ Torr) to reduce the effects of contamination and formation of surface defects or adsorbates for the systematic study presented here. A clean (110) crystallographic orientation of GaAs does not possess any surface states within the band gap and thus a bulk-like band structure is expected at the surface without band-bending effects (7, 8). Cleavage along a direction perpendicular to the (001) orientation of GaAs exposes a fresh (110) plane. The cleaved surface was positioned at a working distance of 10 mm and perpendicular to the propagation direction of the pulsed primary electron beam with its energy of 30 keV.     

On the dynamical nature of the active center in a single-site photocatalyst visualized by 4D ultrafast electron microscopy

Understanding the dynamical nature of the catalytic active site embedded in complex systems at the atomic level is critical to developing efficient photocatalytic materials. Here, we report, using 4D ultrafast electron microscopy, the spatiotemporal behaviors of titanium and oxygen in a titanosilicate catalytic material. The observed changes in Bragg diffraction intensity with time at the specific lattice planes, and with a tilted geometry, provide the relaxation pathway: the Ti⁴⁺=O²⁻ double bond transformation to a Ti³⁺−O¹⁻ single bond via the individual atomic displacements of the titanium and the apical oxygen. The dilation of the double bond is up to 0.8 Å and occurs on the femtosecond time scale. These findings suggest the direct catalytic involvement of the Ti³⁺−O¹⁻ local structure, the significance of nonthermal processes at the reactive site, and the efficient photo-induced electron transfer that plays a pivotal role in many photocatalytic reactions.Single-site catalysts of both the thermally and photoactivated kind now occupy a prominent place in industrial- and laboratory-scale heterogeneous catalysis (1–8). Among the most versatile of these are the ones consisting of coordinatively unsaturated transition metal ions (Ti, Cr, Fe, Mn…) that occupy substitutional sites in well-defined, three-dimensionally extended, open-structure silicates of the zeolite type. The well-known and most widely used are the 4- or 5-coordinated Ti(IV) ions accommodated within the crystalline phase of silica, silicalite (9–14).Titanosilicates, especially, are used extensively both industrially and in the laboratory for a wide range of chemo-, regio-, and shape-selective oxidations of organic compounds (15–18). These single-site heterogeneous photocatalysts are quite distinct from those typified by TiO₂, SrTiO₃, and other titaniferous photocatalysts where the Ti(IV) ions are in 6-coordination; and where, in interpreting the processes involved in harnessing solar radiation, electronic band structure considerations hold sway in preference to the localized states (see, e.g., refs. 19, 20). It has been demonstrated (16–18, 21, 22) that single-site, coordinatively unsaturated Ti(IV)-centered photocatalysts are especially useful in the aerial oxidation of environmental pollutants in the photodegradation of NO (to N₂ and O₂), of H₂O (to H₂ and O₂), and in the photocatalytic reduction of CO₂ to yield methanol. There is an exigent need to explore the precise nature of the electronic, temporal, and spatial changes accompanying the initial act of photoabsorption that sets in train the ensuing elementary chemical processes that are of vital environmental significance in, for example, the utilization of anthropogenic CO₂ as a chemical feedstock (23).Here, we report the use of 4D ultrafast electron microscopy (UEM) (24–26) to trace the spatiotemporal behavior of the Ti(IV) and O²⁻ ions at the photocatalytic active center in the structurally well-characterized titanosilicate Na₄Ti₂Si₈O₂₂·4H₂O, known as JDF-L1 (27–29). JDF stands for Jilin–Davy–Faraday, as the crystalline solid described here was discovered and characterized in joint work involving Jilin University (P. R. China) and the Davy–Faraday Laboratory at the Royal Institution of Great Britain. L1 stands for the first layered catalyst formed during that collaboration; 5-coordinated solids containing Ti(IV) ions are rare among the hundred or so titaniferous minerals, the prime example being fresnoite, Ba₂Ti₂Si₂O₈. We choose this photocatalyst with 5-coordinated Ti because of its unique bonding structure. Our approach entails monitoring, at femtosecond resolution, the changes in intensities and anisotropies of Bragg (electron) diffraction reflections in such a manner as to retrieve the change in valency and the time scales involved in both the formation of Ti³⁺−O¹⁻ bond and the relaxation of the energy back to the local structure of the Ti = O bond in JDF-L1. Through these diffraction studies, and the associated Debye–Waller effect and structural factors anisotropies, it is found that a Ti³⁺−O¹⁻ bond is formed on the femtosecond time scale; whereas, the back relaxation from the site to the structure occurs on a much longer time scale, permitting ample time for reactivity involving Ti³⁺−O¹⁻, and indicating the potential significance of nonthermal processes in the photocatalytic activity at the reactive site.     

Nanomechanics and intermolecular forces of amyloid revealed by four-dimensional electron microscopy

The amyloid state of polypeptides is a stable, highly organized structural form consisting of laterally associated β-sheet protofilaments that may be adopted as an alternative to the functional, native state. Identifying the balance of forces stabilizing amyloid is fundamental to understanding the wide accessibility of this state to peptides and proteins with unrelated primary sequences, various chain lengths, and widely differing native structures. Here, we use four-dimensional electron microscopy to demonstrate that the forces acting to stabilize amyloid at the atomic level are highly anisotropic, that an optimized interbackbone hydrogen-bonding network within β-sheets confers 20 times more rigidity on the structure than sequence-specific sidechain interactions between sheets, and that electrostatic attraction of protofilaments is only slightly stronger than these weak amphiphilic interactions. The potential biological relevance of the deposition of such a highly anisotropic biomaterial in vivo is discussed.The intricate interplay of intermolecular forces stabilizing amyloid at the atomic level has yet to be fully elucidated (1). Amyloid fibrils are narrow (70–200 Å), elongated (1–3 μm), twisted (pitch  ～  1,000 ± 500 Å) aggregates containing a universal “cross-β” core structure (2) composed of arrays of β-sheets running parallel to the long axis of the fibrils (3). Their hierarchical structure is stabilized by three main protein–protein interfaces: (i) stacking of hydrogen-bonded β-strands within a single β-sheet (intrasheet), (ii) cross-β-sheet packing into a multisheet protofilament (intersheet), and (iii) lateral association of protofilaments (interprotofilament) (4). Each of these packing interfaces gives rise to characteristic diffraction pattern reflections corresponding to the intrasheet (4.8 Å), intersheet (8–12 Å, depending on sidechain volume), and interprotofilament (determined by chain length) spacings (5).By applying a laser-induced, temperature (T-) jump to amyloid, we can infinitesimally expand the material, thereby probing the intermolecular forces acting across each of the packing interfaces (6). Static, global heating, particularly of amyloid-like microcrystals (7), disrupts molecular structure, precluding such delicate perturbations. To capture the rapid expansion and recovery of an amyloid specimen, a precisely timed, pulsed (probe) electron beam, following the laser (pump) pulse, is used to generate a series of time-resolved diffraction patterns. By accurately measuring the movement (Δx) of the reflection (initially occurring at an equilibrium separation, x_e) upon initiation of the ultrafast temperature jump, we determine the relative expansion, or strain, ? = Δx/x_e. Atomistic simulations predict that the stretching elasticity of amyloid is linear for strains up to only ? ～ 0.1%, i.e., 10^?3 (8). The exquisite sensitivity and high spatiotemporal resolution of four-dimensional (4D) electron microscopy (9, 10) enables us to measure such minute deformations and directly probe, at the atomic level, the stiffness of the intermolecular forces stabilizing amyloid.     

Kikuchi ultrafast nanodiffraction in four-dimensional electron microscopy

Coherent atomic motions in materials can be revealed using time-resolved X-ray and electron Bragg diffraction. Because of the size of the beam used, typically on the micron scale, the detection of nanoscale propagating waves in extended structures hitherto has not been reported. For elastic waves of complex motions, Bragg intensities contain all polarizations and they are not straightforward to disentangle. Here, we introduce Kikuchi diffraction dynamics, using convergent-beam geometry in an ultrafast electron microscope, to selectively probe propagating transverse elastic waves with nanoscale resolution. It is shown that Kikuchi band shifts, which are sensitive only to the tilting of atomic planes, reveal the resonance oscillations, unit cell angular amplitudes, and the polarization directions. For silicon, the observed wave packet temporal envelope (resonance frequency of 33 GHz), the out-of-phase temporal behavior of Kikuchi's edges, and the magnitude of angular amplitude (0.3 mrad) and polarization elucidate the nature of the motion: one that preserves the mass density (i.e., no compression or expansion) but leads to sliding of planes in the antisymmetric shear eigenmode of the elastic waveguide. As such, the method of Kikuchi diffraction dynamics, which is unique to electron imaging, can be used to characterize the atomic motions of propagating waves and their interactions with interfaces, defects, and grain boundaries at the nanoscale.     

Attosecond electron pulses for 4D diffraction and microscopy

In this contribution, we consider the advancement of ultrafast electron diffraction and microscopy to cover the attosecond time domain. The concept is centered on the compression of femtosecond electron packets to trains of 15-attosecond pulses by the use of the ponderomotive force in synthesized gratings of optical fields. Such attosecond electron pulses are significantly shorter than those achievable with extreme UV light sources near 25 nm ( approximately 50 eV) and have the potential for applications in the visualization of ultrafast electron dynamics, especially of atomic structures, clusters of atoms, and some materials.     

Survey of large protein complexes in D. vulgaris reveals great structural diversity

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr ≈ 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate ≈10 times greater than that of previous “proteomic” screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.     

Direct role of structural dynamics in electron-lattice coupling of superconducting cuprates

The mechanism of electron pairing in high-temperature superconductors is still the subject of intense debate. Here, we provide direct evidence of the role of structural dynamics, with selective atomic motions (buckling of copper–oxygen planes), in the anisotropic electron-lattice coupling. The transient structures were determined using time-resolved electron diffraction, following carrier excitation with polarized femtosecond heating pulses, and examined for different dopings and temperatures. The deformation amplitude reaches 0.5% of the c axis value of 30 Å when the light polarization is in the direction of the copper–oxygen bond, but its decay slows down at 45°. These findings suggest a selective dynamical lattice involvement with the anisotropic electron–phonon coupling being on a time scale (1–3.5 ps depending on direction) of the same order of magnitude as that of the spin exchange of electron pairing in the high-temperature superconducting phase.     

Analysis of microvessels in pancreatic cancer: by light microscopy,confocal laser scan microscopy,and electron microscopy

Background/Purpose

The microvessel density (MVD) of most malignant tumors is considered to be strongly related to metastasis and prognosis. Weidner’s “hot spot method” for determining MVD is in general use, but it is possible that cells other than endothelial cells will also be stained. In our previous study, no correlations were observed between MVD determined by the “hot spot method” and prognosis/metastasis. But, using the “lumen method,” we found a correlation with the number of vessel structures only. In the present study, we analyzed the staining of microvessels in pancreatic cancer, using light microscopy, confocal laser scan microscopy (CLSM), and transmission electron microscopy (TEM).

Methods

Microvessel staining of pancreatic cancer with CD34, factor VIII, and CD45 antibodies was examined in consecutive slices by light microscopy. For CLSM, freshly resected specimens were immunostained with factor VIII and fluorescein isothiocynate. For TEM, specimens were fixed with 2.5% glutaraldehyde, treated with 1% osmium tetroxide, and embedded in epoxy resin.

Results

Staining of vessels with CD34 and factor VIII antibodies appeared similar under light microscopy. However, CD34-stained consecutive slices were judged not to reveal vessel structures, and some cells stained with CD45 antibody were similar in appearance to CD34-stained cells. Under CLSM, irregular arrangements of neovascularization, consisting of many branches, were observed, but many positively stained cells not identified as vessels were also seen. Microvessels were distinctly identified under TEM, but the types of individual cells could not be determined.

Conclusions

An integrated, reproducible method for the measurement of MVD is vital. For pancreatic cancer, the “lumen method” is recommended.

     

Scanning ultrafast electron microscopy

Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.     

Attosecond vacuum UV coherent control of molecular dynamics

High harmonic light sources make it possible to access attosecond timescales, thus opening up the prospect of manipulating electronic wave packets for steering molecular dynamics. However, two decades after the birth of attosecond physics, the concept of attosecond chemistry has not yet been realized; this is because excitation and manipulation of molecular orbitals requires precisely controlled attosecond waveforms in the deep UV, which have not yet been synthesized. Here, we present a unique approach using attosecond vacuum UV pulse-trains to coherently excite and control the outcome of a simple chemical reaction in a deuterium molecule in a non-Born–Oppenheimer regime. By controlling the interfering pathways of electron wave packets in the excited neutral and singly ionized molecule, we unambiguously show that we can switch the excited electronic state on attosecond timescales, coherently guide the nuclear wave packets to dictate the way a neutral molecule vibrates, and steer and manipulate the ionization and dissociation channels. Furthermore, through advanced theory, we succeed in rigorously modeling multiscale electron and nuclear quantum control in a molecule. The observed richness and complexity of the dynamics, even in this very simplest of molecules, is both remarkable and daunting, and presents intriguing new possibilities for bridging the gap between attosecond physics and attochemistry.The coherent manipulation of quantum systems on their natural timescales, as a means to control the evolution of a system, is an important goal for a broad range of science and technology, including chemical dynamics and quantum information science. In molecules, these timescales span from attosecond timescales characteristic of electronic dynamics, to femtosecond timescales characteristic of vibrations and dissociation, to picosecond timescales characteristic of rotations in molecules. With the advent of femtosecond lasers, observing the transition state in a chemical reaction (1), and controlling the reaction itself, became feasible. Precisely timed femtosecond pulse sequences can be used to selectively excite vibrations in a molecule, allow it to evolve, and finally excite or deexcite it into an electronic state not directly accessible from the ground state (2). Alternatively, interferences between different quantum pathways that end up in the same final state can be used to control the outcome of a chemical reaction (3–9).In recent years, coherent high harmonic sources with bandwidths sufficient to generate either attosecond pulse trains or a single isolated attosecond pulses have been developed that are also perfectly synchronized to the driving femtosecond laser (10–12). This new capability provides intriguing possibilities for coherently and simultaneously controlling both the electronic and nuclear dynamics in a molecule in regimes where the Born–Oppenheimer approximation is no longer valid, to select specific reaction pathways or products. Here, we realize this possibility in a coordinated experimental–theoretical study of dynamics in the simplest neutral molecule: deuterated hydrogen (D₂).The hydrogen molecule, as the simplest possible neutral molecule that can be fully described theoretically, has been the prototype molecule for understanding fundamental processes that lie at the heart of quantum mechanics (13–16). However, in such a small molecule, the coupled electron–nuclear dynamics are in the attosecond-to-few-femtosecond regime, whereas the electronically excited states lie in the vacuum UV (VUV) region of the spectrum. Because of the challenge of generating attosecond VUV waveforms using traditional laser frequency doubling or tripling in nonlinear crystals, it has not been possible to date to explore the dynamics of an electronically excited hydrogen molecule. Exploiting attosecond physics, however, only a handful of time-resolved experiments have been performed on H₂ (D₂), focusing mainly on controlling dissociation through electron localization in electronically excited ions (17–23). Recently it was realized that IR femtosecond laser pulses, in combination with a phased locked comb of attosecond VUV harmonics, can be used to control the excitation and ionization yields in He on attosecond timescales, by interfering electron wave packets (24, 25). These experiments extended the Brumer–Shapiro (3–9) two-pathway interference coherent control concept to the attosecond temporal and VUV frequency domain. More recently, it was shown that by manipulating the individual amplitude of VUV harmonics, it is possible to induce full electromagnetic transparency in He, by destructively interfering two electronic wave packets of the same amplitude and opposite phases (26). Other recent work used shaped intense femtosecond laser pulses to manipulate populations by controlling the oscillating charge distribution in a potassium dimer (27).In this paper, we demonstrate that we can coherently and simultaneously manipulate multistate electronic and multipotential-well nuclear wave packet dynamics to control the excitation, nuclear wave packet tunneling, and dissociation and ionization channels of the only electronically excited neutral molecule where full modeling of the coupled quantum dynamics is possible—H₂ (for practical reasons, we used deuterated hydrogen in this experiment). By combining attosecond pulse trains of VUV with two near-IR fields, together with strong-field control that exploits a combination of the two-pathway interference Brumer–Shapiro (3–9) and pump-dump Tannor–Rice (2) approaches, we demonstrate that we can selectively steer the ionization, vibration, and dissociation of D₂ through different channels. Interferences between electronic wave packets (evolving on attosecond timescales) are used to control the population of different electronic states of the excited neutral molecule, which can be switched on attosecond timescales. Then, by optimally selecting the excitation wavelengths and time delays, we can control the vibrational motion, total excitation, ionization yield, and desired ionization and dissociation pathways. State-of-the-art quantum calculations, which have only recently become feasible, allowed us to interpret this very rich set of quantum dynamics, including both the nuclear motion and the coherently excited electronic state interferences. Thus, we succeed in both observing and rigorously modeling multiscale coherent quantum control in the time domain. The observed richness and complexity of the dynamics, even in this very simplest of molecules, is both remarkable and daunting.We note that our approach for control, using a combination of phase-locked VUV and IR fields, where the VUV field consists of an attosecond pulse train with a 10-fs pulse envelope, is ideal for coherently exciting and manipulating electronic and vibrational states on attosecond time scales, while simultaneously retaining excellent spectral resolution necessary for state-selective attochemistry. Exciting electron dynamics in a molecule using a single, broad-bandwidth VUV attosecond pulse would simply excite many ionization/dissociation channels with little state selectivity, potentially masking the coherent electronic and nuclear quantum dynamics. For example, the bandwidth required to support an isolated 200-attosecond pulse around 15 eV is ∼5 eV. In contrast, the 10-fs VUV pulse train used here corresponds to a comb of VUV harmonics, each with a FWHM bandwidth of 183 meV, which can be tuned in the frequency domain (26) to coherently and selectively excite multiple electronic states.     

Observing (non)linear lattice dynamics in graphite by ultrafast Kikuchi diffraction

In materials, the nature of the strain–stress relationship, which is fundamental to their properties, is determined by both the linear and nonlinear elastic responses. Whereas the linear response can be measured by various techniques, the nonlinear behavior is nontrivial to probe and to reveal its nature. Here, we report the methodology of time-resolved Kikuchi diffraction for mapping the (non)linear elastic response of nanoscale graphite following an ultrafast, impulsive strain excitation. It is found that the longitudinal wave propagating along the c-axis exhibits echoes with a frequency of 9.1 GHz, which indicates the reflections of strain between the two surfaces of the material with a speed of ∼4 km/s. Because Kikuchi diffraction enables the probing of strain in the transverse direction, we also observed a higher-frequency mode at 75.5 GHz, which has a relatively long lifetime, on the order of milliseconds. The fluence dependence and the polarization properties of this nonlinear mode are entirely different from those of the linear, longitudinal mode, and here we suggest a localized breather motion in the a-b plane as the origin of the nonlinear shear dynamics. The approach presented in this contribution has the potential for a wide range of applications because most crystalline materials exhibit Kikuchi diffraction.Materials of graphite-type structure are prototype models for studies in solid-state physics (1–3), mainly because of their highly anisotropic bonding characteristics and their consequential unique properties (4, 5). Although the elastic properties of graphite and other carbon allotropes have been investigated for decades (6), a comprehensive and complete picture (7–9) still is lacking, especially when describing the nonlinear elastic properties, whose measurement requires homogeneous and large specimens (8).Using parallel-beam X-ray or electron illumination, the atomic structure of materials can be determined by measuring the position and intensity of Bragg spots. Time-resolved Bragg diffraction has been incorporated successfully to study longitudinal elastic motion, which involves compression/expansion of the unit cell along the direction perpendicular to the specimen’s surface. In graphite, which has an anisotropic thermal expansion, both longitudinal acoustic waves (10) and shear (transverse) waves may be generated as a result of the breaking of the translational symmetry at the level of thermoelastic forces (11).With convergent electron beam illumination, time-integrated Kikuchi diffraction provides a sensitive and precise measure of static lattice plane orientation (12). When time resolved, the evolution of Kikuchi diffraction patterns makes possible the investigation of both the longitudinal and transverse dynamics of the strain and associated lattice deformations (13, 14). This is because Kikuchi diffraction results from the scattering of electrons in the material and a large cone of wave vectors permits reflections that fulfill Bragg condition from different lattice planes; hence, it is possible to monitor the dynamics as the planes tilt and the lattice changes transversely. In contrast, these types of motions will not change the position (only the intensity) of Bragg spots in conventional diffraction experiments; for an illustration, see figure 2 in ref. 13.Open in a separate windowFig. 2.Kikuchi diffraction dynamics. (A) Oscillation dynamics of the ZOLZ plane (220): a single 75.5-GHz mode is detected. The black solid line is a monoexponential fit to mimic the incoherent thermal stress buildup. Note that the behavior in the negative time is a result of the long lifetime (milliseconds) of the 75.5-GHz mode (see text for details). (B) Oscillation dynamics of the HOLZ plane (142): a 9.1-GHz mode superimposed on the 75.5-GHz mode is detected. (C) Oscillation dynamics of the ZOLZ plane (220) and the HOLZ plane (31): echoes at 9.1-GHz frequency were observed for the HOLZ plane (31) (data are shifted by −0.05 mrad for clarity). For the ZOLZ plane (220), no transients were observed, as discussed in the text.Here, we use the Kikuchi diffraction approach in a convergent-beam ultrafast electron crystallography (CB-UEC) setup to investigate the acoustic wave dynamics of single crystals of graphite. By monitoring the temporal evolution of the Kikuchi pattern, it was possible to separate the longitudinal and transverse atomic motions. We observe longitudinal acoustic echoes propagating along the c-axis of the graphite unit cell, which represent the linear response and out-of-plane motion of the graphite lattice. The transverse lattice motion is observed as a higher-frequency mode at 75.5 GHz, and this mode exhibits a relatively long lifetime, on the order of milliseconds, and a polarization direction that rotates with the excitation fluence. The mode is an in-plane shear deformation of the graphite unit cell, and we suggest a localized breather motion in the a-b plane as its origin. In this work, the specimen was obtained from a natural graphite single crystal (naturally graphite) by mechanical cleavage (see Fig. 1B), and the thickness used is 220 nm.Open in a separate windowFig. 1.Kikuchi diffraction of graphite. (A) A representative pattern together with the indexed Kikuchi lines. Dashed and solid lines represent “deficiency” and “excess” lines (12), respectively. (B) Free-standing single crystalline graphite sample on a TEM grid. The white + marks the probed position. (C) Gray and colored dashed lines denote, respectively, lattice planes at equilibrium and when lattice deformation occurs. The blue lines represent the tilting of a ZOLZ plane (Left) and of a HOLZ plane (Right) as induced by an in-plane shear motion along the a-axis. The red lines represent the effect of a longitudinal motion along the c-axis; only for the HOLZ plane (Right) is there an effective tilting.The CB-UEC experiments were performed in a transmission geometry (15, 16), and the electron beam was focused down to a micrometer-length scale with a convergent angle of ∼3 mrad. Fig. 1A shows the measured CB diffraction pattern together with the indexed Kikuchi lines for graphite (17). The electron beam direction was set to 3.45° off the [0001] zone axis (Fig. 1A) to minimize the background from multiple electron scatterings.As a function of time, the shift of the Kikuchi line position with respect to the electron beam is proportional to the tilting of the corresponding lattice plane in the specimen; thus, lattice motion may be correlated directly with changes in the measured CB pattern. Lattice planes with different orientations respond differently to a certain lattice modulation (14). For instance, a lattice plane belonging to the zero-order Laue zone (ZOLZ), which is parallel to the c-axis, is sensitive to any tilt or shear motion developing in the a-b plane, whereas it is completely unaffected by motion along the c-axis (see schematics in Fig. 1C, Left). This is not the case for a lattice plane in high-order Laue zones (HOLZs) intersecting with the c-axis, which turns out to also be tilted by a lattice expansion/contraction along the c-axis (see schematics in Fig. 1C, Right).The experimental setup and the methodology for ultrafast dynamics measurements are detailed elsewhere (18–20). Briefly, structural dynamics are initiated by femtosecond laser pulses (120 fs, 800 nm, 45° incidence, p-polarized, 1.1 × 1.6-mm² spot size, 1 kHz) and probed by ultrashort electron pulses with kinetic energy of 30 keV, at different delay times between the arrival time of the excitation laser pulse and the electron pulse at the sample. The dynamics were recorded with time steps of 1 ps, or longer if needed. For each delay time, more than 50 patterns, each averaged over 10⁴ electron pulses, were recorded to achieve a high signal-to-noise level. It is worth noting that the use of the pump laser and the pulsed electron probe with a repetition rate of 1 kHz enables us to follow the dynamics under investigation up to 1 ms from time zero, which is defined as the moment at which the dynamics are initiated and both the optical and electron pulses overlap in space and time. This is possible to establish by monitoring the temporal response at the negative delay times (19). By using active cavity length control of the femtosecond laser oscillator, the repetition rate is synchronized to an external clock, yielding a frequency stability better than 1 Hz with a synchronization jitter below 1 ps.After determining the position of all Kikuchi lines (Supporting Information), we construct the temporal evolution of the tilting motion for each corresponding lattice plane by monitoring the change in position as a function of the delay time. Representative dynamics for the ZOLZ planes (2 20) and (22 0) and the HOLZ planes (1 42) and ( 31) are shown in Fig. 2, with time steps of 3 ps. Positive values of the tilt indicate that the lattice plane is inclining outward relative to the direct electron beam, whereas negative values indicate an inward inclination. Noted in Fig. 2A is the exponential fit (black solid curve), which describe the average incoherent thermal stress buildup (14). Experiments performed at the same sample position with a parallel electron beam illumination in a [0001]-zone axis result in a diffraction pattern that exhibits sharp Bragg spots, and the intensity of the spots is altered as the result of thermal-induced changes of the lattice, i.e., no oscillations are observed.Besides the thermal stress buildup, two major oscillations with distinct envelopes are observed in the Kikuchi pattern: a fast oscillation with a period of ∼13 ps and a slower one superimposed on the fast oscillation with a period of ∼110 ps. The amplitude of the fast oscillation is sensitive to the particular crystallographic direction (see Fig. 2 A and C for an example), whereas the slow one remains nearly the same for all associated lattice planes (Fig. 2 B and C).From fast Fourier transform (FFT) analysis, we determine the frequencies to be 75.5 GHz and 9.1 GHz, as displayed in Fig. 3A.^* To visualize the lattice motion from the measured Kikuchi pattern, we use the method of Fourier maps introduced by Yurtsever and Zewail (13). This method permits us to follow changes in both the amplitude and phase as a function of time. For each pixel of the diffraction pattern, we determine the amplitude and the phase in the Fourier spectra at the resonance frequencies of 9.1 GHz and 75.5 GHz. The images for the phase change are presented in Fig. 4 for both frequencies and for the three investigated excitation fluences. They clearly show the relative shift of the Kikuchi lines, from which details of the lattice motion can be deduced. From these Fourier maps, it is evident that the mode at 9.1 GHz involves only Kikuchi lines associated with the HOLZ planes, whereas the mode at 75.5 GHz affects the whole pattern, although with different amplitudes for the different planes. It also is clear from the maps that the polarization of the 75.5-GHz mode lies close to the direction [1 00] and exhibits a clear rotation with the excitation fluence. A more quantitative evaluation of these observations will be discussed below.Open in a separate windowFig. 3.FFT spectra and oscillation period. (A) FFT spectra of the dynamics after time zero for the ZOLZ plane (220) (Left) and for the HOLZ plane (31) (Right). The spectral resolution of the FFT is 0.65 GHz. (B) Close view of the oscillations at 75.5 GHz from the ZOLZ plane (220), measured with time steps of 1 ps (red) and 3 ps (blue). Both profiles exhibit the same sinusoidal behavior with identical periods and matching phase (see text).Open in a separate windowFig. 4.Fourier maps for the phases at the indicated resonance frequency. Shown are the phase images at 9.1 GHz (A) and 75.5 GHz (B) for the three investigated fluences, as obtained by Fourier transform of the time-resolved Kikuchi patterns. It is evident that the mode at 9.1 GHz involves only Kikuchi lines associated with HOLZ planes, whereas the mode at 75.5 GHz affects the whole pattern, with different amplitudes for different planes. The orange and red arrows in B are shown to follow the evolution of the Kikuchi lines (211) and (030), respectively. The yellowish region in B close to each Kikuchi line is a qualitative measure of the relative change. As the fluence increases, this region evolves preferentially toward the right part of the diffraction pattern and, hence, the direction of polarization, as shown by the white double arrows; a more quantitative evaluation of the amplitude is given in Fig. 5. The scale bar is in degrees.Careful control experiments were performed to verify the intrinsic origin of the 75.5-GHz mode. First, we compared the transients obtained using 3-ps steps with those recorded with smaller time steps of 1 ps, but at the same acquisition time per point. For both cases, we obtained the same 13.2-ps period with a precise phase matching (as shown in Fig. 3B). This rules out the effect of external periodic artifacts because the period, although the same in “real time,” must change in the “pump–probe” recording when the step duration changes; a more detailed discussion is presented in Supporting Information. Second, for the same diffraction pattern, the mode amplitude depends on the planes involved, i.e., the modulation is not the same across the entire pattern, as shown in Fig. 2. Third, the oscillations were absent when the excitation laser was blocked. Finally, to further verify the robustness of the mode across the specimen, we carried out the same measurements at another position on the graphite surface (with a thickness of 100 nm instead of 220 nm) and more than 1 mm away from the current one. We obtained the same oscillation dynamics with the identical frequency of 75.5 GHz.It follows that the weak-intensity oscillations observed in the negative delay-time region (Fig. 2) are a result of the long-lived 75.5-GHz mode, which survives until a new excitation from a following pump pulse (in a millisecond) resets the lattice in motion. When examining the phase of oscillations, we found no correlation between the phase observed before (small amplitude) and after (large amplitude) time zero, thus excluding the possibility of a one-frequency artifact oscillation. It is worth mentioning that direct monitoring of the output of our amplified laser shows no additional pulse between the pump pulses separated by 1 ms at a sensitivity level greater than 1%.In contrast to the long-lived mode, the 9.1-GHz mode and its higher-order harmonics (observed at 18.2, 27.3, and 36.5 GHz; Fig. 3A, Right) vanish at long delay times; the decay time is a few nanoseconds, typical of heat dissipation in solid systems. The observation that only the HOLZ planes exhibit the 9.1-GHz mode indicates that this mode is that of a wave with polarization and propagation direction along the c-axis, a direction that does not induce orientational modulation of the ZOLZ planes. This is supported further by the close resemblance of the temporal lineshape for this mode to the successive acoustic echoes created by an impulsively excited picosecond acoustic wave traveling between the two boundaries of a metal thin film along its normal (21–23).In this picture, a traveling sound wave is created, and its frequency fulfills the relation f = v/2l, where v is the speed of sound and l is the film thickness. With the measured thickness of l = 220 nm, which we determined from electron energy-loss spectroscopy, and knowing the frequency f = 9.1 GHz from FFT plots, we obtained the sound velocity of 4.0 km/s, which is in very good agreement with the value reported for the sound velocity of longitudinal acoustic waves propagating along the c-axis in graphite [v_LA[001] = 4.14 km/s (8)]. The behavior of this traveling longitudinal wave along the [0001] direction reflects the linear response of the lattice to the impulsive excitation, and a linear chain model (24) can describe the atomic motions involved.The lattice strain created by the excitation is within a region of the absorption length [33 nm at λ = 800 nm in graphite (25)] close to the front surface. From this point, this lattice deformation begins to propagate as a sound wave into the depth of the sample along the c-axis and undergoes multiple reflections by the front and back surfaces of the graphite plate. Because of the inhomogeneous excitation, this wave induces large surface displacement and considerable energy dissipation every time it reaches the boundaries.^† Because the laser absorption length initially makes the longitudinal spatial extension smaller than the sample thickness, the sound wave at the boundaries approaches a delta-like functional behavior, which results in several high-order harmonics in the FFT spectra.For both types of modes, we investigated the fluence dependence of the amplitude. This dependence can be visualized directly in the Fourier maps shown in Fig. 4. Whereas the amplitude of the 9.1-GHz mode exhibits a linear dependence on the excitation fluence, the mode at 75.5 GHz shows a much different behavior. For instance, the amplitude of the HOLZ plane (2 11) shows no discernable change as a function of the fluence in the range of 2.0–2.9 mJ/cm², whereas for the ZOLZ plane (03 0), the amplitude exhibits a definite nonlinear trend (at 2.0 mJ/cm², the mode is barely visible, whereas at 2.4 mJ/cm² and 2.9 mJ/cm², it has a well-defined amplitude).From these fluence-dependence experiments, it was possible to systematically monitor the oscillation amplitude along different directions of the Brillouin zone, represented in Fig. 5 for the excitation fluence of 2.0 mJ/cm² (Fig. 5 A and D), 2.4 mJ/cm² (Fig. 5 B and E), and 2.9 mJ/cm² (Fig. 5 C and F). The oscillation amplitude exhibits the same temporal evolution, within the time scale of 1 ns from the initial excitation, for all Kikuchi lines with a well-defined frequency of 75.5 GHz. Moreover, the maximum value remains approximately constant, within the measurement sensitivity, to 0.10 mrad for all fluences. By performing a temporal average of the amplitude profiles for each plane, we could determine the amplitude distribution along different crystallographic directions projected on the a-b plane of the graphite unit cell (Fig. 5 D–F).Open in a separate windowFig. 5.Temporal evolution and plane-specific projection of amplitude. (A–C) Temporal evolutions of the amplitudes at excitation fluences of 2.0 mJ/cm², 2.4 mJ/cm², and 2.9 mJ/cm², respectively, for the 75.5-GHz mode. Light green regions mark the time interval over which the oscillation amplitude has been averaged. (D–F) The average amplitudes derived from the temporal profiles shown in A–C are plotted along different crystallographic directions in the a-b plane of the graphite lattice. The intensity of the oscillation amplitude is represented by a shade modulation from red (intense) to blue (weak), and quantitatively described by the length of the black double-way arrows. The plane perpendicular to the deduced polarization is denoted by a green solid line. The orange lozenge in each panel outlines the graphite unit cell in the a-b plane.The direction of polarization can be obtained from the distribution of amplitudes. This is because lattice planes perpendicular to the polarization direction have strong oscillation amplitudes, whereas those lying close to the polarization direction have weak amplitudes. As evident from Fig. 5 A–C, both the ZOLZ and the HOLZ planes exhibit a synchronous temporal evolution with similar amplitudes, suggesting that the 75.5-GHz mode is a shear motion with a polarization direction preferentially lying in the a-b plane, as schematically depicted in Fig. 1C (blue colored).^‡ We note that an out-of-plane component would introduce a different temporal behavior for the ZOLZ and HOLZ planes, especially for those close to the polarization direction. We also note that as the excitation fluence increases, the 75.5-GHz mode changes its polarization direction rather than its total amplitude (Figs. 4 and ​and55).The above studies of ultrafast Kikuchi diffraction, dependence on the fluence, and the polarization properties provide the following picture for lattice dynamics. The linear mode at 9.1 GHz has its origin in a longitudinal wave, and the atomic motions can be understood simply by considering a linear chain of atoms, each of which represents a plane perpendicular to the c-axis. Because the absorption length of the optical pulse is smaller than the sample thickness, a delta function-like stress excitation is created, and in this regime, the presence of higher harmonics is expected, as observed experimentally and is evident in the FFT spectra.The nonlinear mode at 75.5 GHz is an in-plane shear deformation, as discussed above, created by lateral strain confinement (27) (see also Supporting Information). At a microscopic level, several authors (28–30) theoretically showed that elastically deformed carbon structures can support the existence of high-frequency and long-lived nonlinear localized modes in the form of breathers (31). These nonlinear modes are robust to local perturbations of the lattice and for a graphene sheet, are represented by the out-of-phase oscillation of neighboring atoms that are polarized within the a-b plane (32). Given the observed period of 13.2 ps and using the shear “speed” of ∼14.66 km/s (8), the length scale is ∼200 nm. The long lifetime of the breathers is a result of the fact that their frequency lies inside the gap of the phonon spectrum (29, 32); thus, their decay to other modes essentially is forbidden. The excitation of breathers has been reported experimentally in several systems, such as spin waves (33), optical lattices (34), and micromechanical cantilever arrays (35), and they also are expected for atomic systems because of the discreteness of the lattice structure, the presence of lattice defects, and the nonlinearity of the interaction between atoms.In conclusion, with CB ultrafast Kikuchi diffraction, it is shown that both the longitudinal and transverse (shear) elastic dynamics of the material can be observed and separated by following changes of selective diffraction reflections. The longitudinal motion along the c-axis is the result of a linear acoustic wave propagation, whereas the transverse motion in the a-b planes is associated with a nonlinear shear deformation. From knowledge of the fluence dependence, the polarization properties, and the long lifetime, we suggest a motion involving a localized breather on a length scale of a few hundred nanometers. The reported Fourier maps at the frequency of an individual mode display the atomic planes involved in the motion, and this feature is unique to Kikuchi diffraction dynamics. It is the basis for the potential use of the approach in a wide range of applications.     

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.     

Imaging conical intersection dynamics during azobenzene photoisomerization by ultrafast X-ray diffraction

X-ray diffraction is routinely used for structure determination of stationary molecular samples. Modern X-ray photon sources, e.g., from free-electron lasers, enable us to add temporal resolution to these scattering events, thereby providing a movie of atomic motions. We simulate and decipher the various contributions to the X-ray diffraction pattern for the femtosecond isomerization of azobenzene, a textbook photochemical process. A wealth of information is encoded besides real-time monitoring of the molecular charge density for the cis to trans isomerization. In particular, vibronic coherences emerge at the conical intersection, contributing to the total diffraction signal by mixed elastic and inelastic photon scattering. They cause distinct phase modulations in momentum space, which directly reflect the real-space phase modulation of the electronic transition density during the nonadiabatic passage. To overcome the masking by the intense elastic scattering contributions from the electronic populations in the total diffraction signal, we discuss how this information can be retrieved, e.g., by employing very hard X-rays to record large scattering momentum transfers.

Diffraction signals reveal the momentum transfer experienced by an incident photon through interference with one or more scatterers. Traditionally, X-ray diffraction is used to determine the structure of crystalline matter. A momentum-space image, provided by the scattered photons, is recorded, allowing for the reconstruction of the real-space crystal structure. Diffraction patterns are dominated by the Bragg peaks, arising from the interaction of multiple scatterers in a long-range crystalline order. The relevant material quantity is the electronic charge density, from which the X-ray photons elastically scatter.The advent of free-electron X-ray light sources (1, 2) has added two game-changing ingredients to diffraction experiments. The first is a peak brilliancy that can exceed third-generation synchrotron sources by nine orders of magnitude (3). The high number of photons in the beam allows for a significant reduction of the sample size, while still achieving the necessary ratio of scattered photons per object to record diffraction patterns. Structure determination of nanocrystals (4, 5), aligned gas-phase molecular samples (6), and macromolecular structures (7, 8) were reported. Attempts toward imaging single molecules at free-electron lasers were made (9), although some challenges remain (8). The second ingredient is temporal resolution, enabled by subfemtosecond pulse durations (1 fs = 10−15 s), also available from tabletop setups through, e.g., high harmonic generation (10) or laser-driven plasma sources (11). Time-resolved diffraction movies can monitor the electronic charge density evolution during a chemical process (12–19). A complementary technique is ultrafast electron diffraction, where the X-rays are replaced by bright electron pulses that scatter from the total (nuclear + electronic) charge density (20–24).Diffraction from matter in nonstationary states, such as molecules undergoing a chemical transformation, involves physical processes that go beyond elastic scattering from instantaneous snapshots of the electronic ground state density. This is especially true for excited molecules, being in a time-evolving superposition of many-body states. Inelastic scattering from different electronic states (25–27), as well as electronic and vibrational coherences (27–30), contributes to the signal.Here we study theoretically the diffractive imaging of vibronic coherences that emerge at conical intersections (CoIns). These are regions of degeneracy in electronic potential energy surfaces (PESs), where the movement of electrons and nuclei become strongly coupled, enabling ultrafast radiationless relaxation channels back to the ground state (31, 32). Processes like the primary event of vision (33) or the outcome of photochemical reactions (23), among many others, are determined by the occurrence and properties of CoIns. A textbook example of CoIn dynamics is the optical switching between cis and trans azobenzene (Fig. 1A): azobenzene can be switched selectively on a femtosecond timescale between both isomers (34–36) with high quantum yield, it exhibits interesting photophysics like a violation of Kasha’s rule (37, 38), and it has found broad application as the photoactive unit in switching the activity of pharmaceutical compounds (39) or neurons (optogenetics) (40). There is a large body of experimental and theoretical literature on azobenzene, and the previous references are by no means exhaustive. For a deeper overview of scientific progress about azobenzene photophysics, we refer the reader to ref. 38 and section 8 of its supplement.Open in a separate windowFig. 1.Molecular structures and electron densities of azobenzene. (A) Isomerization scheme with the (Left) trans and (Right) cis geometry. Both processes can be selectively achieved with high quantum yield. In this work, we simulate the cis→trans process. (B) Real-space electronic densities at a structure close to the CoIn at 90° of CNNC torsion, with the ground state (96 electrons; Left), the transition state (1 electron; Middle), and the excited state (96 electrons; Right) density.Three-vibrational-mode PESs for the photoisomerization of azobenzene were reported in ref. 41. The nuclear space consists of the carbon–nitrogen–nitrogen–carbon (CNNC) torsion angle and the two CNN bending angles between the azo unit and the benzene rings. CNNC torsion is the reactive coordinate that connects the cis and trans minima at 0° and 180°, respectively, while the two CNN angles are responsible for the symmetry breaking that leads to the minimum energy CoIn seam (41). We perform nuclear wave packet simulations of the cis→trans isomerization, yielding the total nuclear + electronic molecular wave function. Using the quantum-electrodynamical formalism for time-resolved diffraction signals developed in refs. 27 and 42, we follow the isomerization via its diffraction snapshots. The focus of this paper is on the coherence contributions to the diffraction signal and their distinctly different shapes and magnitudes compared to the other contributions. This results in a real-space movie of the molecular transition charge density at the CoIn.     

Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to "colorize" detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging.     

Filming the formation and fluctuation of skyrmion domains by cryo-Lorentz transmission electron microscopy

Magnetic skyrmions are promising candidates as information carriers in logic or storage devices thanks to their robustness, guaranteed by the topological protection, and their nanometric size. Currently, little is known about the influence of parameters such as disorder, defects, or external stimuli on the long-range spatial distribution and temporal evolution of the skyrmion lattice. Here, using a large (7.3 × 7.3?μm²) single-crystal nanoslice (150 nm thick) of Cu₂OSeO₃, we image up to 70,000 skyrmions by means of cryo-Lorentz transmission electron microscopy as a function of the applied magnetic field. The emergence of the skyrmion lattice from the helimagnetic phase is monitored, revealing the existence of a glassy skyrmion phase at the phase transition field, where patches of an octagonally distorted skyrmion lattice are also discovered. In the skyrmion phase, dislocations are shown to cause the emergence and switching between domains with different lattice orientations, and the temporal fluctuation of these domains is filmed. These results demonstrate the importance of direct-space and real-time imaging of skyrmion domains for addressing both their long-range topology and stability.In a noncentrosymmetric chiral lattice, the competition between the symmetric ferromagnetic exchange, the antisymmetric Dzyaloshinskii–Moriya interaction, and an applied magnetic field can stabilize a highly ordered spin texture, presenting as a hexagonal lattice of spin vortices called skyrmions (1–4).Magnetic skyrmions have been experimentally detected in materials having the B20 crystal structure such as MnSi (5), Fe_1?xCo_xSi (6, 7), FeGe (8), and Cu₂OSeO₃ (9) and, recently, also on systems like GaV₄S₈ (10) and beta-Mn-type alloys (11). Small-angle neutron scattering studies of bulk solids evidenced the formation of a hexagonal skyrmion lattice confined in a very narrow region of temperature and magnetic field (T-B) in the phase diagram (5, 6). In thin films and thinly cut slices of the same compounds, instead, skyrmions can be stabilized over a wider T-B range as revealed by experiments using cryo-Lorentz transmission electron microscopy (LTEM) (12, 13). Furthermore, it was proposed and recently observed that skyrmions can also exist as isolated objects before the formation of the ordered skyrmion lattice (14, 15). A recent resonant X-ray diffraction experiment also suggested the formation of two skyrmion sublattices giving rise to regular superstructures (16).In a 2D landscape, long-range ordering can be significantly altered by the presence of defects and disorder. Indeed, the competition between order and disorder within the context of lattice formation continues to be an issue of fundamental importance. Condensed matter systems are well known to provide important test beds for exploring theories of structural order in solids and glasses. An archetypal and conceptually relevant example is the superconducting vortex lattice, where real-space imaging studies allow direct access to the positional correlations and local coordination numbers (17–19). Up until now, however, analogous studies of skyrmion lattices have not been reported even though (as for superconducting vortices) it is well known that defects and dislocations present in a sample can pin the motion of skyrmions induced by external perturbations such as an electric field (20) or a magnetic field (16). This competition between disorder and elasticity will clearly give rise to a complex energy landscape promoting diverse metastable states (21) and superstructures (22, 23). Furthermore, previous imaging studies of skyrmion lattices could probe only the short-range order due to limitations in the size of the imaged area and its homogeneity.In this paper, by systematic observations using cryo-LTEM, we reveal the magnetic field-dependent evolution of the skyrmion-related spin textures in a Cu₂OSeO₃ thin plate and study their long-range ordering properties imaging up to ∼1,000 lattice constants. The different phases of the spin textures are analyzed with state-of-the-art methods to unravel their spatial properties. At low magnetic fields, the coexistence of two helical domains is observed, in contrast to previous studies (9); the angle between the two helices’ axis is retrieved via a reciprocal space analysis. At the magnetic field close to the helical–skyrmion phase transition, evidence for a glassy skyrmion phase is found via cross-correlation analysis, a method that has recently been applied to the analysis of both X-rays and electron diffraction patterns to retrieve information on the local order and symmetry of colloidal systems (24–26). In this phase, we reveal also patches of octagonally distorted skyrmion lattice crystallites. In the skyrmion phase, by locating the position of each skyrmion and generating an angle map of the hexagonal unit cell they formed, we obtain a direct-space distortion map of the skyrmion lattice. This distortion map evidences the presence of orientation-disordered skyrmion lattice domains present within the single-crystalline sample. Each domain boundary coincides with a dislocation formed by a seven–five or a five–eight–five Frenkel-type defect. The number of such dislocations decreases with increasing magnetic field, and large single-domain regions are formed. The formation of these mesoscopic domains was also filmed with camera-rate (millisecond) time resolution. The presence of differently oriented skyrmion lattice domains was observed in spatially separated regions, or in the same area of the sample but at a different moment in time. Based on our observation, we propose an alternative scenario for the appearance of split magnetic Bragg peaks reported in ref. 16. Instead of the formation of regular superstructures of coexisting misoriented skyrmion lattices in real space, we suggest that the splitting is caused by a spatial or temporal integration of an orientation-fluctuating skyrmion lattice. This result highlights the importance of a direct-space, real-time probe for assessing the dynamical topological properties of a large number of skyrmions.