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.     

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.     

4D visualization of embryonic, structural crystallization by single-pulse microscopy

In many physical and biological systems the transition from an amorphous to ordered native structure involves complex energy landscapes, and understanding such transformations requires not only their thermodynamics but also the structural dynamics during the process. Here, we extend our 4D visualization method with electron imaging to include the study of irreversible processes with a single pulse in the same ultrafast electron microscope (UEM) as used before in the single-electron mode for the study of reversible processes. With this augmentation, we report on the transformation of amorphous to crystalline structure with silicon as an example. A single heating pulse was used to initiate crystallization from the amorphous phase while a single packet of electrons imaged selectively in space the transformation as the structure continuously changes with time. From the evolution of crystallinity in real time and the changes in morphology, for nanosecond and femtosecond pulse heating, we describe two types of processes, one that occurs at early time and involves a nondiffusive motion and another that takes place on a longer time scale. Similar mechanisms of two distinct time scales may perhaps be important in biomolecular folding.     

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.     

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.     

Four-dimensional ultrafast electron microscopy of phase transitions

Reported here is direct imaging (and diffraction) by using 4D ultrafast electron microscopy (UEM) with combined spatial and temporal resolutions. In the first phase of UEM, it was possible to obtain snapshot images by using timed, single-electron packets; each packet is free of space-charge effects. Here, we demonstrate the ability to obtain sequences of snapshots ("movies") with atomic-scale spatial resolution and ultrashort temporal resolution. Specifically, it is shown that ultrafast metal-insulator phase transitions can be studied with these achieved spatial and temporal resolutions. The diffraction (atomic scale) and images (nanometer scale) we obtained manifest the structural phase transition with its characteristic hysteresis, and the time scale involved (100 fs) is now studied by directly monitoring coordinates of the atoms themselves.     

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.     

Photon gating in four-dimensional ultrafast electron microscopy

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon–electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a “single” light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a “second” optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM.In ultrafast electron microscopy (UEM) (1–3), electrons generated by photoemission at the cathode of a transmission electron microscope are accelerated down the microscope column to probe the dynamic evolution of a specimen initiated by an ultrafast light pulse. The use of femtosecond lasers to generate the electron probe and excite the specimen has made it possible to achieve temporal resolution on the femtosecond time scale, as determined by the cross-correlation of the optical and electron pulses. One important method in the UEM repertoire is photon-induced near-field electron microscopy (PINEM) (4, 5), in which the dynamic response detected by the electron probe is the pump-induced charge density redistribution in nanoscale specimens (6).Photon–electron coupling is the basic building block of PINEM, which takes place in the presence of nanostructures when the energy-momentum conservation condition is satisfied (4, 5). This coupling leads to inelastic gain/loss of photon quanta by electrons in the electron packet, which can be resolved in the electron energy spectrum (5, 7, 8). This spectrum consists of discrete peaks, spectrally separated by multiples of the photon energy (n?ω), on the higher and lower energy sides of the zero loss peak (ZLP) (4) (Fig. 1). The development of PINEM enables the visualization of the spatiotemporal dielectric response of nanostructures (9), visualization of plasmonic fields (4, 5) and their spatial interferences (10), imaging of low atomic number nanoscale materials (11), characterization of ultrashort electron packets (12, 13), and imaging of different biological structures (14).Open in a separate windowFig. 1.Concept of photon gating in 4D electron microscopy. (A) The microscope column with one electron (dark blue) and two optical (red) pulses focused onto the specimen. The wavefunctions of the three pulses are schematically shown at the top. One optical pulse is coincident with the electron pulse at the specimen to generate a PINEM signal. The resulting light blue PINEM pulse is sliced out from other electrons for detection as an energy spectrum, an image, or a diffraction signal (see the text). The second optical pulse initiates the dynamics to be probed. (B) Electron energy spectrum generated at the specimen plane when optical and electron pulses arrive simultaneously. The gain energy range is shaded light blue. (C) Illustration for the temporal pulse sequence, two optical and one electron pulse for ultrafast time-resolved PINEM measurements.As shown by Park et al. (5), the PINEM intensity (I_PINEM) is given by the square modulus of the field integral F˜0 (i.e., IPINEM∝|F˜0|2), in the weak interaction limit. The near field of a nanoparticle leads to the scattering of the electron packet, which can be treated rigorously using the Schrödinger equation/Mie scattering theory. It follows that PINEM images the object and displays its field characteristics depending on its shape, the polarization and wavelength of optical excitation, and the width of pulses used. For a spherical nanoparticle, the field integral at point (x, y) in the specimen plane is simplified to give (6)F˜0≈−iE˜0⁡cos⁡ϕχs 23 a3(Δk)2K[Δkb],[1]where E˜0 is the electric field amplitude of the incident light, ? the light polarization angle, a the particle radius, b=x2+y2 the impact parameter, K the modified Bessel function of the second type, Δk the momentum change of the electron, and χ_s = 3(ε ? 1)/(ε + 2), where χ_s is the material susceptibility and ε the dielectric function.In previous studies of the parameters in Eq. 1, only E˜0 was time dependent. The PINEM intensity, at a given point in space, was a function only of the time delay between the optical and electron pulses, providing, for the pulse lengths currently used, a cross-correlation profile when this delay was scanned across the time of temporal coincidence, or t = 0 (4, 5, 9, 13). Hitherto, PINEM has not been used to study the ultrafast dynamics of matter. Here, we follow the strategy of using the PINEM gain electrons generated by a first optical pulse, whose delay relative to the electron pulse is maintained at t = 0, to probe dynamics initiated by introduction of a second optical pulse on the specimen, as proposed theoretically in ref. 15. By this approach, we were able to optically gate the electron pulse (i.e., create an electron pulse that only lasts for the duration of the optical pulse) and achieve significant enhancement of the temporal resolution (see the second paragraph below).The concept of the experiment is illustrated by Fig. 1A, in which the electron pulse in blue and one optical pulse (P₁) in red are shown arriving at the specimen plane simultaneously. Interaction between photon and electron in the presence of the specimen “slices out” the light blue pulse of gain electrons, which are separated from all other electrons by energy dispersion or filtering to be detected according to microscope settings in spectroscopy, imaging, or diffraction mode, as illustrated schematically at the bottom of the column. Note, it is possible to obtain PINEM diffraction, but this is not the subject of this paper. A second, or pump, optical pulse (P₂) is shown below the specimen, having already triggered the dynamics of interest. A series of time axes is plotted in Fig. 1C showing examples of characteristic sequences of pulse arrival times at the specimen plane during the experiment, with the pump arrival defining the zero of time.A striking feature of this technique that was alluded to above is the potential for high temporal resolution, unlimited by the electron pulse duration, because the optical pulse acts as a temporal gate for a longer electron pulse. In the weak interaction limit, the duration of the pulse of PINEM electrons emulates that of the optical pulse that created it (15), as clearly shown in Fig. 1A. When these photon-gated electrons are used to probe dynamics triggered by a second ultrafast optical pulse, the time resolution is determined by the cross-correlation of the two optical pulses. This paves the way for the realization of attosecond electron microscopy, as done in all-optical spectroscopy (16) but with the spatial resolution being that of atomic motions. As suggested in Fig. 1A, we envisage the use of the photon-gated electron pulses, in imaging or in diffraction mode, for the study of a variety of optically initiated material processes, either of the nanostructure or of its surrounding media.The PINEM signal can be directly monitored to detect changes in any of the specimen optical or physical properties expressed in Eq. 1. Here, we demonstrate the use of the time-resolved PINEM technique where it is shown that the photoinduced dielectric response of VO₂—which is strongly related to the lattice symmetry (17)—manifests itself in a change in PINEM intensity. We relate the changes in optical properties of the polycrystalline VO₂ nanoparticles to the phase transition dynamics from initial (monoclinic) insulator phase to (tetragonal) metal phase, the subject of numerous previous studies.Vanadium dioxide has been discussed as an active metamaterial (18) and one of the best candidates for solid-state ultrafast optical switches in photonics applications (19, 20) due to its unique structural photoinduced phase transition behavior (21). This phase transition has been examined by investigating the change in the heat capacity through thermal excitation (22, 23), whereas its ultrafast dynamics has been studied by optical spectroscopy (24, 25), THz spectroscopy (26, 27), X-ray diffraction (28, 29), ultrafast electron crystallography (30), and electron microscopy (31).     

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.     

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.     

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.     

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.     

From the Cover: Goniometer-based femtosecond crystallography with X-ray free electron lasers

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β₂-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.Using extremely bright, short-timescale X-ray pulses produced by X-ray free-electron lasers (XFELs), femtosecond crystallography (FX) is an emerging method that expands the structural information accessible from very small or very radiation-sensitive macromolecular crystals. Central to this method is the “diffraction before destruction” (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1–3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs–long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second. Exposing small crystals to these intense ultrashort pulses circumvents the dose limitations of conventional X-ray diffraction experiments (4) and may produce useful data to resolutions beyond what is achievable at synchrotrons (5). This innovation provides a pathway to obtain atomic information from proteins that only form micrometer- to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable “diffraction before reduction” data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7–11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotrons.A single X-ray pulse from the LCLS damages the illuminated sample volume and also some of the sample in the immediate vicinity, requiring the combination of measurements from discrete volumes or units of crystalline material to obtain a complete dataset (14). The first FX experiments were carried out in vacuum at the LCLS using a gas dynamic virtual nozzle (GDVN) liquid injector (15), which delivered crystals of submicron to a few microns in size, suspended in carrier solution to a series of X-ray pulses produced at up to a 120-Hz repetition rate. These pioneering experiments demonstrated the utility of serial femtosecond crystallography (SFX) and the use of crystals of less than 5 μm in size, often termed “nanocrystals” (NCs), for macromolecular structure determination to high resolution (16, 17). As NCs may be a ubiquitous but generally overlooked outcome of commercial crystallization screens that fail to produce larger crystals (18), FX may open up many systems to crystallographic analysis. However, to develop FX into a generally applicable method, a number of challenges in the areas of sample preparation, data collection, and data processing must be overcome.Obtaining a sufficient supply of crystals in an appropriate carrier solution is a first hurdle to conducting a SFX experiment. In addition to the GDVN (2, 3, 14, 16, 17, 19, 20), other injectors such as a nanoflow electrospinning injector (21) and a lipidic cubic phase (LCP) injector (22), have been developed that have a reduced flow rate and lower sample consumption. However, because injectors deliver a continuous stream of solution containing a random distribution of crystals, and the X-ray pulses are extremely short, often only a small percentage of pulses hit a crystal and produce a useful diffraction pattern. Carrying out these experiments at room temperature avoids the difficulties associated with cryoprotection, and datasets obtained at ambient temperatures can provide insight on the functional motions of protein molecules (23). However, there are different and often more complex optimization steps associated with specific injector technologies. Solutions containing a mixture of crystal sizes may require filtering to avoid clogging in the injector nozzle, and delicate crystals may be damaged from the pressures and shear forces of the delivery process itself (24). For experiments conducted in vacuo, stream formation may be disrupted by solution bubbling, drying, or freezing as it exits the injector and enters the vacuum chamber. Drop-on-demand methods that deliver single drops containing crystals to individual X-ray pulses have the potential to significantly reduce sample consumption are in development, such as acoustic and micropiezo activated technologies, but implementation has been complicated by a variety of factors, including difficulties imposed by viscous solutions and unpredictable trajectories of drops that contain crystals of varied shapes and sizes.Here, we describe an alternative strategy for FX experiments that leverages the well-established benefits of the highly automated goniometer-based setups used at state-of-the-art microfocus synchrotron beam lines, and expands these technologies to take full advantage of the unique capabilities of XFEL sources. Key to this approach is the coupling of highly automated instrumentation with specialized sample containers and customized software for efficient data collection with minimal sample consumption. High-density sample containers, such as microfluidic chips or microcrystal traps (25) for room temperature studies or grids for experiments at cryogenic temperatures, hold samples in known locations. These sample holders enable very rapid and precise positioning of crystals into the X-ray interaction region for consistent production of diffraction patterns. To optimize data completeness and resolution, data may be collected using a range of crystal sizes with a variety of X-ray beam sizes, and different regions of larger crystals may be exposed in different orientations. When small crystals align with the sample holder in a preferred orientation, exposing each crystal at varied angles to the holder surface may take advantage of this effect to enhance completeness. When the X-ray pulse mean diameter is greater than about 8 μm or is highly attenuated, the protein crystal may remain intact after exposure to the X-ray pulse and still diffract, but usually to a lower resolution as a result of radiation damage. In these cases, it is possible to rotate the crystal and collect additional diffraction patterns to use as an aid in indexing and scaling the partially recorded reflections of the initial still diffraction pattern.     

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.     

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.     

Biological imaging with 4D ultrafast electron microscopy

Advances in the imaging of biological structures with transmission electron microscopy continue to reveal information at the nanometer length scale and below. The images obtained are static, i.e., time-averaged over seconds, and the weak contrast is usually enhanced through sophisticated specimen preparation techniques and/or improvements in electron optics and methodologies. Here we report the application of the technique of photon-induced near-field electron microscopy (PINEM) to imaging of biological specimens with femtosecond (fs) temporal resolution. In PINEM, the biological structure is exposed to single-electron packets and simultaneously irradiated with fs laser pulses that are coincident with the electron pulses in space and time. By electron energy-filtering those electrons that gained photon energies, the contrast is enhanced only at the surface of the structures involved. This method is demonstrated here in imaging of protein vesicles and whole cells of Escherichia coli, both are not absorbing the photon energy, and both are of low-Z contrast. It is also shown that the spatial location of contrast enhancement can be controlled via laser polarization, time resolution, and tomographic tilting. The high-magnification PINEM imaging provides the nanometer scale and the fs temporal resolution. The potential of applications is discussed and includes the study of antibodies and immunolabeling within the cell.     

Breaking resolution limits in ultrafast electron diffraction and microscopy

Ultrafast electron microscopy and diffraction are powerful techniques for the study of the time-resolved structures of molecules, materials, and biological systems. Central to these approaches is the use of ultrafast coherent electron packets. The electron pulses typically have an energy of 30 keV for diffraction and 100-200 keV for microscopy, corresponding to speeds of 33-70% of the speed of light. Although the spatial resolution can reach the atomic scale, the temporal resolution is limited by the pulse width and by the difference in group velocities of electrons and the light used to initiate the dynamical change. In this contribution, we introduce the concept of tilted optical pulses into diffraction and imaging techniques and demonstrate the methodology experimentally. These advances allow us to reach limits of time resolution down to regimes of a few femtoseconds and, possibly, attoseconds. With tilted pulses, every part of the sample is excited at precisely the same time as when the electrons arrive at the specimen. Here, this approach is demonstrated for the most unfavorable case of ultrafast crystallography. We also present a method for measuring the duration of electron packets by autocorrelating electron pulses in free space and without streaking, and we discuss the potential of tilting the electron pulses themselves for applications in domains involving nuclear and electron motions.     

Use of transmission electron microscopy to identify nanocrystals of challenging protein targets

The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as “hits.” We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.The emergence of X-ray free electron laser (X-FEL)–based serial femtosecond crystallography holds the promise of solving the 3D structure of proteins that can only crystallize as “nanocrystals” (NCs) or are highly sensitive to radiation damage (1–5). NCs appropriate for X-FEL experiments are considered to be 200 nm to 2 μm in size (6). This size is constrained primarily by the requirements of the NC delivery system to the X-FEL beam. In addition to allowing for structure resolution of NCs by X-FEL experiments, they provide the advantage of requiring no crystal cryoprotection because these experiments are performed at room temperature (3, 7). Given the opportunities that X-FELs offer to the field of crystallography, efficient methodologies to detect NCs from single crystallography drops and to optimize these identified conditions yielding NCs will be essential for future developments in structural biology. Current methods to detect the presence of NCs include dynamic light scattering (DLS), bright-field microscopy, birefringence microscopy, and intrinsic tryptophan UV fluorescence imaging, as well as technologies that rely upon second harmonic generation, such as second order nonlinear imaging of chiral crystals (SONICC) (8, 9) and X-ray powder diffraction. However, limitations of these imaging techniques include (i) ineffective detection of crystals smaller than 5 μm (8, 10), (ii) false-positive conditions as a result of interference from precipitate backgrounds (8, 10), and (iii) false-negative conditions resulting from the lack of tryptophan residues in the case of UV fluorescence and from the lack of chiral centers in the case of SONICC (11). Although DLS can accurately measure the size distribution of nanometer-sized protein aggregates, it is unable to distinguish unambiguously between amorphous and crystalline (12). Finally, X-ray powder diffraction, a method that has been applied to evaluate samples for the presence and concentration of NCs, requires more material than is produced in a single crystallization screening drop, and synchrotron radiation is usually required to produce measurable diffraction (13). In this study, we use UV fluorescence microscopy and DLS to detect crystallization drops containing NCs, followed by transmission electron microscopy (TEM) to identify protein NCs accurately and determine NC quality by evaluating the reciprocal lattice reflections in diffraction patterns calculated from images.     

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)–heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)–CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.

Enzymatic functions frequently involve local motions at the active site as well as large-amplitude motions of the protein, and the two are often strongly correlated. Many chemical processes at the active sites take place as a result of the interplay between atomic movement and electronic structural changes in response to external stimuli such as light, ligand binding, heat or electric field. While reaction kinetics can be predicted from thermodynamic properties, the intrinsic time scales for fundamental chemical events, such as bond breakage and formation, are often unresolved due to challenges in examining rapid electronic and atomic movements in real time. Advanced X-ray sources, especially those with intense photon bursts within the time scale of fundamental chemical events (i.e., femtoseconds), enable structural characterization in terms of the electronic and atomic motions. Combining such ultrashort X-ray pulses with laser excitation, we are able to detect the interplay of ultrafast electron and nuclear motions in the photodissociation of an axial CO ligand from the iron center in the heme site of myoglobin (Mb) (Fig. 1). The same process has been extensively studied due to numerous functions of heme or other iron porphyrins in hemoproteins, including electron transfer, catalytic oxidation or reduction of metabolites, neutralization of damaging reactive species, and famously the binding of diatomics such as dioxygen, carbon monoxide, and nitric oxide for transportation and sensing (1–6). Because the dissociation of diatomic ligands, such as CO and NO, can be synchronized through optical excitation of the porphyrin, diatomic ligand binding in hemoproteins is amenable to scrutiny by dynamic structural and electronic spectroscopies (1, 7–14). Several X-ray diffraction, solution scattering, and X-ray spectroscopy (including X-ray absorption and emission) studies have been carried out using intense X-ray pulses from synchrotron and X-ray free electron laser sources (11, 14–19). In this report, we focus on the correlations between the electronic structural change of the iron center and these nuclear motions. To investigate these correlations, we used X-ray transient absorption spectroscopy/scattering and theoretical calculations to detect and project detailed trajectories for the CO departure from Fe(II) in the heme site of bovine Mb.Open in a separate windowFig. 1.(A) Mb structure with heme in pink surrounded by helices of the protein. (B) Mb active site structural changes following CO photolysis. Upon photoexcitation, ground state MbCO (green) loses its bond to CO and adopts a square pyramidal structure with His93 (pink), resulting in the doming of the porphyrin where the Fe (red) comes out of the plane of the macrocycle. Structures are from photolysed MbCO trapped at low temperature (12) and ground state (9) MbCO, where their crystal structures are aligned by their respective porphyrin carbons.In carbonmonoxymyoglobin (MbCO), the low-spin (LS) Fe(II) center has a pseudo-octahedral coordination geometry, ligated with four nitrogens (N_p) from the heme, the nitrogen of an axial histidine (N_His, His93), and CO, a strong field ligand. Previous studies have pointed out that upon excitation of the heme Soret or Q band, photolysis occurs within ∼50 fs, although there is an ongoing debate about the mechanism of CO photodissociation and the subsequent relaxation of the heme, as well as the possible role of intermediate spin states, similar to those observed in photoexcited iron Tris(bipyridine) (20) and ferrous cytochrome c (14, 15, 21). With the loss of CO, the LS state of Fe(II) transforms to a high-spin (HS) state and adopts square-pyramidal pentacoordination with the axial histidine His93 moving ∼0.3 Å out of the porphyrin plane, perturbing the position of the alpha helix in which it sits (Fig. 1B) (4, 8, 22, 23). Protein control of this movement is critical, both because it is the first step of the mechanism of cooperativity in hemoglobin O₂ binding and because it may lead to a conformational rearrangement of the heme pocket that allows CO to escape and avoid geminate recombination (24).This CO photodissociation from MbCO, as well as the photolysis of other diatomics such as NO, and the subsequent recombination dynamics have been assessed using X-ray transient absorption (XTA) spectroscopy (Fig. 2) at synchrotron sources with ∼100-ps time resolution (19, 25), using Fe K-edge X-ray absorption near edge structure (XANES) spectral features shown in Fig. 2. The main differences between the spectral features of MbCO and Mb are an edge shift to a lower energy and a preedge conversion from two sharp peaks to one broader and weaker peak. These changes are consistent with loss of CO and a conversion of Fe(II) from LS to HS in Mb, as supported by optical and vibrational spectroscopic studies (8, 26).Open in a separate windowFig. 2.Fe K-edge XANES and difference spectra measured after CO photodissociation with 100 ps time resolution (19). Energies selected for measurement of polarization-dependent dynamics are marked in dashed vertical cyan lines: line 1, 7.112 keV, the depletion of the ground state transition of 1s → 3d_z2, 3d_x2-y2 character; line 2, 7.115 keV, the disappearance of the preedge peak associated with the CO back bonding antibonding orbital; line 3, 7.118 keV, the rising edge shoulder that appears in MbFe(II); line 4, 7.123 keV, the edge shift; and line 5, 7.172 keV, an EXAFS energy where changes are purely based on changes in the local geometry.While heme vibrational cooling was observed by time-resolved Raman techniques on the time scales of a few to tens of ps (26, 27), and optical transient absorption spectroscopy shows the development of broad excited state absorption features with lifetimes of ∼300 fs and 3 ps, there is an active discussion in the literature as to whether these features can be assigned to an excited-state evolution through a series of electronic intermediate states/species (28–31) or to an exclusively vibrational relaxation pathway (31–33). Because Fe K-edge XTA is sensitive to both the heme iron electronic configuration and the local structural geometry during heme relaxation, measurements of the XANES should distinguish between these mechanisms but only if very fast time scales are resolvable. In this regard, XTA at Linac Coherent Light Source (LCLS) provides a rare opportunity to investigate these relaxation processes with a technique with both high temporal and structural resolution.Although the photodissociation of CO from heme and the concomitant LS to HS transition and heme doming motion are well-known phenomena, many fundamental transformations in terms of electronic and nuclear motions that result in the CO departure are not well understood. The recent works on cytochtome c and NO-bound myoglobin have made progress in the timing of the spin state transitions on the femtosecond time scale and the identification of the intermediate spin state (14, 15). However, it is far from clear how the spin state change was induced electronically, how the iron spin state is related to the Fe–CO distance, and when the heme doming takes place as the Fe–CO distance elongates in dissociation processes. Understanding these correlations has important implications for other chemical and enzymatic processes involving ligand dissociation. It is therefore of great interest to link dynamic structural and electronic changes at the heme during ligand dissociation to more large-scale conformational changes, especially on the time scale of ligand departure and heme doming, which is expected to take place on tens of femtoseconds to a few picoseconds time scales.In order to address the dynamic interplay between electronic and nuclear motions that are beyond the Born–Oppenheimer approximation, we carried out combined XTA and X-ray transient scattering measurements during CO photodissociation with sub–100-fs time resolution at the X-ray Pump-Probe (XPP) facility of the LCLS. The kinetics of the spin state transition and the nuclear motion associated with doming, as well as the global motions of the protein matrix, have revealed a series of correlated events as CO departs from the heme. Although the exact trajectory of CO departure in terms of Fe–CO distance and other structural parameters is still difficult to resolve, such processes can be simulated via quantum mechanical calculations to model this process for the Fe(II) center and the ligands directly bound to the metal. The results provide the energetics of different excited states as well as their trajectories as functions of local structural changes, such as Fe–CO distance and heme relaxation, to distinguish the effect of different structural factors on the overall structural dynamics. These calculations also allow us to predict XTA features without and with the structural relaxation of the heme, providing insight into the interplays between the electronic spin state/configuration and corresponding nuclear motions as a function of the Fe–CO distance.     

A physical model of mantis shrimp for exploring the dynamics of ultrafast systems

Efficient and effective generation of high-acceleration movement in biology requires a process to control energy flow and amplify mechanical power from power density–limited muscle. Until recently, this ability was exclusive to ultrafast, small organisms, and this process was largely ascribed to the high mechanical power density of small elastic recoil mechanisms. In several ultrafast organisms, linkages suddenly initiate rotation when they overcenter and reverse torque; this process mediates the release of stored elastic energy and enhances the mechanical power output of extremely fast, spring-actuated systems. Here we report the discovery of linkage dynamics and geometric latching that reveals how organisms and synthetic systems generate extremely high-acceleration, short-duration movements. Through synergistic analyses of mantis shrimp strikes, a synthetic mantis shrimp robot, and a dynamic mathematical model, we discover that linkages can exhibit distinct dynamic phases that control energy transfer from stored elastic energy to ultrafast movement. These design principles are embodied in a 1.5-g mantis shrimp scale mechanism capable of striking velocities over 26 m s−1 in air and 5 m s−1 in water. The physical, mathematical, and biological datasets establish latching mechanics with four temporal phases and identify a nondimensional performance metric to analyze potential energy transfer. These temporal phases enable control of an extreme cascade of mechanical power amplification. Linkage dynamics and temporal phase characteristics are easily adjusted through linkage design in robotic and mathematical systems and provide a framework to understand the function of linkages and latches in biological systems.

Latch-mediated spring actuation (LaMSA) is a class of mechanisms that enable small organisms to achieve extremely high accelerations (1–5). Small organisms generate fast movements by storing elastic energy and mediating its release through latching. LaMSA mechanisms are found across the tree of life, including fungi, plants, and animals, with such iconic movements as found in trap-jaw ant mandibles, frog legs, chameleon tongue projection, fungal ballistospores, and exploding plant seeds (4–8). While the use of materials for elastic energy storage and release has been examined to some extent (9–11), the principles of how latches enable storage of elastic energy and mediate its release have only recently begun to be explored (12, 13). Indeed, even after half a century of investigation, one of the most extensively studied and impressive LaMSA systems, the mantis shrimp (Stomatopoda), uses a latch mechanism that is not yet fully understood.In recent years, robots have grown in their importance as physical models for studying the mechanics and dynamics of organisms and their behaviors (14–18). Such models can be manipulated—both at design time and at run time—in ways that natural systems cannot, thus providing tools for the study of organism functional morphology, neuroethology, and operation in different environments. Here, based on previous studies of mantis shrimp biomechanics, we develop physical and analytical models to elucidate the latch-based control of energy flow during mantis shrimp strikes and, more broadly, to establish the design principles for repeated use, extreme mechanical power amplification in small engineered devices.Mantis shrimp use a LaMSA mechanism to achieve among the fastest predatory strikes in the animal kingdom, reaching extreme accelerations with their raptorial appendages on the order of 106 rad s−2 in water. These strikes are so fast that they create cavitation bubbles and break hard molluscan shells—an impressive feat given their small size (19–22). Even the largest species, the peacock mantis shrimp (Odontodactylus scyllarus), has a striking appendage (carpus, propodus, and dactyl segments of the raptorial appendage, colored in purple in Fig. 1C) length of only 2.65 cm. Mantis shrimp store potential energy through deformation of an elastic mechanism in the merus segment which is composed of a saddle-shaped piece of the exoskeleton (the “saddle”) and another stiff yet deformable region of the exoskeleton (called the “meral-V”) (23–27); see the blue segments in Fig. 1C. These components are part of a four-bar linkage mechanism that transforms stored elastic energy into the rapid rotation of the extremely fast strikes (28, 29). Biologists have long known about two small structures, called sclerites, which are embedded in the apodemes (tendons) of the flexor muscles that release the strikes (28, 30, 31). These tiny structures brace against the interior of the merus segment and oppose the forces of the large, antagonistic extensor muscles that load the elastic mechanism. When the extensor muscles contract to load potential energy, the sclerites serve as a contact latch to prevent the rotation of the striking appendages. Then the flexor muscles release the sclerites to allow the striking appendage to rotate. Once the contact latch is released, the extensor muscle remains contracted while the elastic mechanism recoils to actuate the rotation of the striking body. The locked position of the sclerites and subsequent release are shown in SI Appendix, Fig. S13. The exact locations of the sclerites, apodemes of the flexor muscle, and meres segment in mantis shrimp can be found in figure 3 in ref. 25. A representative striking motion of a mantis shrimp can be found in Movie S5.Open in a separate windowFig. 1.An overview of biologically inspired physical models that generate extreme accelerations. (A) A diagram illustrating high acceleration within biological and synthetic LaMSA systems. From left to right, two synthetic systems, water strider-inspired robot (44) and flea-inspired robot (69), and two biological systems, flea (70) and snipefish (36, 71), are shown. A survey of more acceleration data of biological and synthetic LaMSA systems can be found in table 1 of ref. 4. Water strider–inspired robot image from ref. 69. Reprinted with permission from American Association for the Advancement of Science. Flea-inspired robot image ©2012 Institute of Electrical and Electronics Engineers; reprinted, with permission, from ref. 43. Flea image credit: CanStockPhoto/ottoflick. Snipefish image credit: Wikimedia Commons/Tony Ayling. (B) Photograph of our mantis shrimp–inspired mechanism and photograph of a peacock mantis shrimp by Roy Caldwell. The proposed mantis shrimp robot generates 104 m s−2 for striking the arm, and the mantis shrimp generates 2.5×105 m s−2 for striking the appendage (19). Photographs adjusted for contrast with background removed. Adapted with permission from ref. 28. (C) (Right) The four-bar linkage in the mantis shrimp appendage is labeled (a to d). Adapted with permission from ref. 28. The striking arm has three tightly coupled components (dactyl, propodus, and carpus), which are colored purple. Two exoskeleton elastic components are colored blue. Last, the extensor muscle, which actuates the striking motion, is colored red. (Middle) A geometric abstraction of the four-bar linkage with two rigid bodies, the arm and the body. (Left) The synthetic realization of the proposed four-bar linkage with one variable-length link. The body is highlighted orange, and the arm is purple. Flexures which allow articulation are shown in yellow. The mechanism is secured to a 3D printed base using two screws. A tendon, shown in red, is used to actuate the mechanism. A series of holes in the base allow the tendon pulling angle to be adjusted between experiments. Potential energy is stored in a torsion spring (blue).In general, after loading the potential energy in the spring, the role of the contact latch (sclerites) is to lock the system in this loaded configuration. For a typical spring loaded mechanism with a contact latch, and once the physical latch is removed, the spring would immediately begin to release the stored energy. However, analyses of the temporal sequence of loading and release of the strikes reveal a substantial time delay between release of these small latches and the onset of rotation of the appendage (20, 28, 32, 33). Therefore, biologists have hypothesized, but not tested, that while the sclerites initiate unlatching, a second, geometric latch mediates the actuation of the appendage by the recoiling elastic mechanism (5, 33, 34).Latches can be classified into three types—fluidic, contact, and geometric (4, 5)—and contact latches (e.g., the sclerites shown in SI Appendix, Fig. S13) have previously been studied and assumed to be a primary latch mechanism in mantis shrimp. Contact latches are dependent on a physical structure blocking motion, while geometric latches are based on kinematic linkage mechanisms. Ninjabot uses a contact latch, and is, to our knowledge, the only other physical model of the mantis shrimp striking appendage (35). Ninjabot’s striking arm is part of a large assembly with a hand-cranked ratchet and pawl mechanism. It was designed to emulate the speed and acceleration of mantis shrimp strikes and to characterize the fluid dynamics of the striking motion but not to emulate the linkage or latch mechanics.Four-bar linkages can function as geometric latches if they mediate a sudden directional change of rotational motion (36–39). One type of geometric latch is a torque-reversal latch that consists of an n-bar linkage (most often a four-bar) where the kinematics of the linkage admits at least one point in the configuration space such that an infinitesimal motion of a configuration variable results in an instantaneous change in the sign of the torque around one or more joints (5). A four-bar–based geometric latch is depicted in Fig. 2 A and B in which the torque reversal is achieved when the system passes through a linkage overlap. Typically, the linkage overlap condition within a four-bar mechanism is denoted as an overcentering configuration. In engineered devices, the overcentering property of four-bar linkages is frequently used. For example, a four-bar linkage has been used to design a robust aircraft landing gear (40, 41). The spring attached within the four-bar linkage provides bistability of the downlocked and uplocked positions of the landing gear, which also reduces the load on the actuator. The primary design goal for this simple example lies in the stability of the two extreme configurations, whereas we focus our study on the rapid acceleration experienced when crossing the overcentering configuration.Open in a separate windowFig. 2.A planar model for the four-bar linkage of the mantis shrimp. (A) Dimensions and inertial components of two rotating bodies composed with the four-bar links (L0,L1,L2,lt). Arm and body are shown. The arm rotates away from the body (θ2) as the spring recoils (θ1). An external force, Ft, acts on the tendon, and a torsional spring, with spring coefficient ks, is attached between the body and ground (shown here as a linear spring for convenience; a torsional spring is used in the physical system). The two generalized coordinates are θ1 and θ2. (B) Configurations before and after overcentering are shown. The tendon links, lt, for both configurations are colinear and thus overlap in this drawing. (C) Direction of the generalized constraint torque, τ, between the arm and body when in contact. The constraint torque is a reaction force which is nonzero only when the arm is in contact with the body. In our physical model, there is an offset contact angle, denoted as ϕ, between the arm and the body when they are in contact.Geometric latches have been proposed in fleas, snapping shrimp, and mantis shrimp (36, 38, 39, 42) and designed into synthetic systems, such as a flea-inspired insect-scale jumping robot (43). A more recent design, demonstrated in a water strider inspired robot (44), uses a symmetric four-bar torque reversal linkage (45). A four-bar linkage in snipefish feeding strikes causes a rapid rotational direction change, as inferred from functional morphology and micro-CT scans (36). Rotation reversal is initiated via a separate triggering muscle, and the four-bar linkage exhibits a singular overcentering configuration. This causes the linkage to rotate in the reverse direction after overcentering.Until now, the mantis shrimp four-bar linkage mechanism has been analyzed solely as a mechanical pathway to transfer energy from their elastic mechanism to the rotation of their appendages (19, 28, 29, 46–49); however, through the additional lens of a hypothesized geometric latch, previous biological analyses of the linkage mechanism may need to be revisited. The four-bar linkage in a mantis shrimp’s raptorial appendage is composed of four links and pivots (Fig. 1C) (28). The link connecting the carpus and merus is formed by contracted muscles (c–d in Fig. 1C) as also occurs in other biological linkage and lever mechanisms that operate as LaMSA systems only during configurations determined by muscle activation (50–53). In mantis shrimp, the merus extensor muscles contract during spring loading and remain contracted during unlatching and spring recoil (30, 33); therefore, the link formed by the contracted extensor muscles is shorter during the operation of the LaMSA mechanism than when it is not being used (i.e., when the extensor muscles are not contracted to load the elastic mechanism) (28). The change in the extensor muscle length reduces by 10% relative to its relaxed position while loading energy in the saddle and meral-V (28).An accurate dynamic model can allow us to explore the initiation and switching between spring loading and spring actuation phases which are crucial for control of energy flow and reducing abrupt changes that cause damage (1, 54). A previous analytical derivation of latch release dynamics for a contact-based latch model (13) was possible because the contact latch component was in contact with the projectile: the unlatched condition occurs when the latch and projectile are no longer in contact. In contrast, mathematically defining latch release for a geometric latch is challenging due to the absence of a physical component serving as a latch. Nevertheless, inspired by the fact that the mantis shrimp’s striking body (carpus, propodus, and dactyl) and the meral-V are in contact while extensor muscles load the elastic components, the latching (and latched) phase can be identified by the constraint force holding the striking body and the meral-V together. As we will demonstrate in this study, a dynamic model for switching between phases can be properly defined using constrained Lagrangian mechanics (55). A dynamic mathematical model of four-bar latch dynamics has the potential to reveal previously hidden geometric latching control in four-bar systems, which is especially likely in systems with a contractile link. Thus, inspired by the controllable link length in the mantis shrimp’s raptorial appendage, we construct mathematical and physical models of a mantis shrimp–inspired four-bar mechanism with three rigid links and one variable-length link (red) at c–d shown in Fig. 1C (akin to muscle activation control).We take a three-pronged approach to establishing the general principles of latching dynamics in LaMSA systems and specifically the geometric latch hypothesized to control mantis shrimp striking. We first present our physical model inspired by mantis shrimp LaMSA and linkage mechanics. This physical model includes multiple degrees of freedom (DoFs) and flexure-based flexible joints and uses a linear spring for potential energy storage. In parallel, we develop a dynamic mathematical model composed of multiple rigid bodies and assume linear models for the stiffness and damping at each joint. We reanalyze and incorporate a previously published dataset of mantis shrimp kinematics to revisit the linkage dynamics and incorporate the hypothesized geometric latching process. Finally, we conduct a series of experiments on the physical model in both air and water to test how latch release can be controlled with various conditions of tendon control, fluidic loading, and mechanism design.