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

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

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).     

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 multiple-cathode ultrafast electron microscopy

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

Four-dimensional ultrafast electron microscopy 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.     

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.     

Attosecond electron pulses for 4D diffraction and microscopy

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

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.     

Single-electron pulses for ultrafast diffraction

Visualization of atomic-scale structural motion by ultrafast electron diffraction and microscopy requires electron packets of shortest duration and highest coherence. We report on the generation and application of single-electron pulses for this purpose. Photoelectric emission from metal surfaces is studied with tunable ultraviolet pulses in the femtosecond regime. The bandwidth, efficiency, coherence, and electron pulse duration are investigated in dependence on excitation wavelength, intensity, and laser bandwidth. At photon energies close to the cathode's work function, the electron pulse duration shortens significantly and approaches a threshold that is determined by interplay of the optical pulse width and the acceleration field. An optimized choice of laser wavelength and bandwidth results in sub-100-fs electron pulses. We demonstrate single-electron diffraction from polycrystalline diamond films and reveal the favorable influences of matched photon energies on the coherence volume of single-electron wave packets. We discuss the consequences of our findings for the physics of the photoelectric effect and for applications of single-electron pulses in ultrafast 4D imaging of structural dynamics.     

High numerical aperture tabletop soft x-ray diffraction microscopy with 70-nm resolution

Light microscopy has greatly advanced our understanding of nature. The achievable resolution, however, is limited by optical wavelengths to approximately 200 nm. By using imaging and labeling technologies, resolutions beyond the diffraction limit can be achieved for specialized specimens with techniques such as near-field scanning optical microscopy, stimulated emission depletion microscopy, and photoactivated localization microscopy. Here, we report a versatile soft x-ray diffraction microscope with 70- to 90-nm resolution by using two different tabletop coherent soft x-ray sources-a soft x-ray laser and a high-harmonic source. We also use field curvature correction that allows high numerical aperture imaging and near-diffraction-limited resolution of 1.5lambda. A tabletop soft x-ray diffraction microscope should find broad applications in biology, nanoscience, and materials science because of its simple optical design, high resolution, large depth of field, 3D imaging capability, scalability to shorter wavelengths, and ultrafast temporal resolution.     

From the Cover: Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs.Far-field nanoscopy (1, 2) methods discern features within subdiffraction distances by briefly forcing their molecules to two distinguishable states for the time period of detection. Typically, fluorophores are switched between a signaling “on” and a nonsignaling (i.e., dark) “off” state. Depending on the switching and fluorescence registration strategy used, these superresolution techniques can be categorized into coordinate-stochastic and coordinate-targeted approaches (2). The latter group of methods, comprising the so-called RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] (1, 3–7) approaches, have been realized using patterns of switch-off light with one or more zero-intensity points or lines, to single out target point (zero-dimensional) or line (1D) coordinates in space where the fluorophores are allowed to assume the on state. The RESOLFT idea can also be implemented in the inverse mode, by using switch-on light and confining the off state. In any case, probing the presence of molecules in new sets of points or lines at every scanning step produces images.Owing to the nature of the on and off states involved––first excited electronic and ground state––stimulated emission depletion (STED) (3) and saturated structured illumination microscopy (SSIM) (8), which both qualify as variants of the RESOLFT principle, typically apply light intensities in the range of MW/cm² and above. Especially when imaging sensitive samples where photoinduced changes must be avoided, RESOLFT is preferably realized with fluorophores which lead to the same factor of resolution improvement at much lower intensities of state-switching light. Reversibly switchable fluorescent proteins (RSFPs) are highly suitable for this purpose (4–7, 9), as transitions between their metastable on and off states require 5 orders of magnitude lower threshold intensities than STED/SSIM to guarantee switch-off. Suitable spectral properties, relatively fast millisecond switching kinetics, and high photostability of recently developed yellow-green-emitting RSFPs like rsEGFP (5), rsEGFP2 (7), and rsEGFP(N205S) (10) compared with early RSFPs have indeed enabled RESOLFT nanoscopy in living cells and tissues. To date, RSFP-based RESOLFT has achieved resolution improvements by factors of 4–5 in rsEGFP2-labeled samples (7). To further reduce the imaging time, massive parallelization of scanning has been reported (10). However, the diffraction-limited axial resolution and lack of background suppression restrict applications to thin samples.Imaging applications typically require careful tuning of imaging parameters including speed, contrast, photosensitivity, and spatial resolution, depending on the information that is sought. Light-sheet fluorescence microscopy (LSFM) (11–15) stands out by its ability to balance most of these parameters for 3D imaging of living specimens. Recently reenacted as the selective plane illumination microscope (13), this microscopy mode has sparked increasing interest notably because of its short acquisition times in 3D imaging and low phototoxicity in living specimens. It excites fluorophores only in a thin diffraction-limited slice of the sample, perpendicular to the direction of fluorescence detection. The LS is generated by a cylindrical lens which focuses an expanded laser beam in only one direction onto the specimen or into the back-focal plane of an illumination objective. Alternatively, a single beam is quickly moved as a “virtual” LS (16) across a specimen section.In such conventional LSFM imaging, the lateral resolution is determined by the numerical aperture (N.A.) of the detection objective (17), whereas axial resolution is given by the LS thickness, provided the latter is thinner than the axial extent of the point-spread function describing the imaging process from the focal plane of the detecting lens to the camera. In a previous study, the axial resolution of LSFM was pushed to the diffraction limit by using the full aperture of the illumination objective with Gaussian beams; this was carried out for practically useful combinations of N.A. (e.g., 0.8 for both illumination and detection objectives) permissible in light of the geometrical constraints given by the objective lens dimensions (18). High-N.A. illumination comes with short Rayleigh ranges of Gaussian beams, which inherently limit the field of view (FOV) along the direction of illumination. Scanned Bessel beams for diffraction-limited excitation with a virtual LS (19–21) typically offer larger FOVs (22), but side lobes broaden the scanned LS in the axial direction and contribute to phototoxicity outside of the focal plane of detection (20). A more complex approach has used Bessel-beam excitation in combination with structured illumination to obtain near-isotropic (but still diffraction-limited) resolution as measured on fluorescent beads (20), albeit at the cost of acquisition time and reduced contrast due to fluorescence generated by the side lobes. In different work, axial resolution has also been improved about fourfold by acquiring two complementary orthogonal views of the sample using two alternating LSs, followed by computationally fusing image information with a deconvolution incorporating both views (23). LS approaches have also helped suppress out-of-focus background for single-molecule imaging in biological situations (e.g., in ref. 24), including at superresolution (25–27).Slight axial resolution improvement beyond the diffraction barrier has been demonstrated by overlapping a Gaussian excitation LS with a STED LS featuring a zero-intensity plane (28). Due to scattering and possibly additional aberrations caused by the wavelength difference between excitation and STED light, the maximal achievable resolution in biological specimens was severely limited. This was the case even in fixed samples. A successful application of LS-STED to living cells or organisms has not been reported. The relatively high average STED laser power required for high resolution gains calls for developing a coordinate-targeted superresolution LS approach with low-power operation, meaning a concept that does not solely rely on changing the way the light is directed to––or collected from––the sample, but a concept that harnesses an “on–off” transition for improved feature separation.     

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.     

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

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

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

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

Femtosecond time resolution in x-ray diffraction experiments

This paper presents the theoretical background for a synthesis of femtosecond spectroscopy and x-ray diffraction. When a diffraction quality crystal with 0.1–0.3 mm overall dimensions is photoactivated by a femtosecond laser pulse (physical length = 0.3 μm), the evolution of molecules at separated points in the crystal will not be simultaneous because a finite time is required for the laser pulse to propagate through the body of the crystal. Utilizing this lack of global crystal synchronization, topographic x-ray diffraction may enable femtosecond temporal resolution to be achieved from reflection profiles in the diffraction pattern with x-ray exposures of picosecond or longer duration. Such x-ray pulses are currently available, and could be used to study femtosecond reaction dynamics at atomic resolution on crystals of both small- and macromolecules. A general treatment of excitation and diffraction geometries in relation to spatial and temporal resolution is presented.     

Macromolecular-scale resolution in biological fluorescence microscopy

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.     

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.