Time-resolved Microscopy Research Articles

Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting complex/cyclosome (APC/C).

Substrate polyubiquitination drives a myriad of cellular processes, including the cell cycle, apoptosis and immune responses. Polyubiquitination is highly dynamic, and obtaining mechanistic insight has thus far required artificially trapped structures to stabilize specific steps along the enzymatic process. So far, how any ubiquitin ligase builds a proteasomal degradation signal, which is canonically regarded as four or more ubiquitins, remains unclear. Here we present time-resolved cryogenic electron microscopy studies of the 1.2 MDa E3 ubiquitin ligase, known as the anaphase-promoting complex/cyclosome (APC/C), and its E2 co-enzymes (UBE2C/UBCH10 and UBE2S) during substrate polyubiquitination. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the human APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unexpected interactions between multiple ubiquitins with parts of the APC/C machinery, including its coactivator CDH1. Together, we demonstrate how modification of substrates with nascent ubiquitin chains helps to potentiate processive substrate polyubiquitination, allowing us to model how a ubiquitin ligase builds a proteasomal degradation signal.

Real-time imaging of acoustic waves in bulk materials with X-ray microscopy

The dynamics of lattice vibrations govern many material processes, such as acoustic wave propagation, displacive phase transitions, and ballistic thermal transport. The maximum velocity of these processes and their effects is determined by the speed of sound, which therefore defines the temporal resolution (picoseconds) needed to resolve these phenomena on their characteristic length scales (nanometers). Here, we present an X-ray microscope capable of imaging acoustic waves with subpicosecond resolution within mm-sized crystals. We directly visualize the generation, propagation, branching, and energy dissipation of longitudinal and transverse acoustic waves in diamond, demonstrating how mechanical energy thermalizes from picosecond to microsecond timescales. Bulk characterization techniques capable of resolving this level of structural detail have previously been available on millisecond time scales-orders of magnitude too slow to capture these fundamental phenomena in solid-state physics and geoscience. As such, the reported results provide broad insights into the interaction of acoustic waves with the structure of materials, and the availability of ultrafast time-resolved dark-field X-ray microscopy opens a vista of new opportunities for 3D imaging of materials dynamics on their intrinsic submicrosecond time scales.

Ultrafast Electronic Dynamics in Anisotropic Indirect Interlayer Excitonic States of Monolayer WSe2/ReS2 Heterojunctions.

Understanding ultrafast electronic dynamics of the interlayer excitonic states in atomically thin transition metal dichalcogenides is of importance in engineering valleytronics and developing excitonic integrated circuits. In this work, we experimentally explored the ultrafast dynamics of indirect interlayer excitonic states in monolayer type II WSe2/ReS2 heterojunctions using time-resolved photoemission electron microscopy, which reveals its anisotropic behavior. The ultrafast cooling and decay of excited-state electrons exhibit significant linear dichroism. The ab initio theoretical calculations provide unambiguous evidence that this linear dichroism result is primarily associated with the anisotropic nonradiative recombination of indirect interlayer excitonic states. Measuring time-resolved photoemission energy spectra, we have further revealed the ultrafast evolution of excited-state electrons in anisotropic indirect interlayer excitonic states. The findings have important implications for controlling the interlayer moiré excitonic effects and designing anisotropic optoelectronic devices.

Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane

Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of ‘raft’-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.

(Invited) Measuring the Temporal Evolution of Surface Intermediates in Electrocatalysis

Ensemble measurements in electrocatalysis average over populations of single entities such as transient chemical intermediates and active sites. Ensemble-averaged empirical data provides general trends in reactivity but lost is a vast amount of information on how molecules interact and evolve on an electrocatalyst surface which could reveal over- or underperforming subpopulations within an ensemble. To validate and improve current theoretical predictions of reaction pathways, direct measurements of intermediates in electrocatalysis are required. Toward this goal, I will discuss our development of time-resolved surface-enhanced Raman scattering (SERS) microscopy as a tool to measure the evolution and dynamics of surface intermediates formed on metallic electrodes in CO2 reduction. Combining diffraction-limited spatial resolution with µs temporal resolution, we observe the formation of surface-bound CO ca. 1 V below the ensemble onset potential. Further, our time-resolved spectroscopy reveals the timescales of surface intermediate accumulation on and evolution from electrode surfaces, providing insight into the residence times of intermediates. Time-resolved SERS microscopy reveals the action of surface intermediates previously hidden by spatially and temporally averaged ensemble measurements, providing new mechanistic insight to the CO2 reduction reaction.

Ultrafast Electronic Relaxation Dynamics of Atomically Thin MoS2 Is Accelerated by Wrinkling.

Strain engineering is an attractive approach for tuning the local optoelectronic properties of transition metal dichalcogenides (TMDs). While strain has been shown to affect the nanosecond carrier recombination dynamics of TMDs, its influence on the sub-picosecond electronic relaxation dynamics is still unexplored. Here, we employ a combination of time-resolved photoemission electron microscopy (TR-PEEM) and nonadiabatic ab initio molecular dynamics (NAMD) to investigate the ultrafast dynamics of wrinkled multilayer (ML) MoS2 comprising 17 layers. Following 2.41 eV photoexcitation, electronic relaxation at the Γ valley occurs with a time constant of 97 ± 2 fs for wrinkled ML-MoS2 and 120 ± 2 fs for flat ML-MoS2. NAMD shows that wrinkling permits larger amplitude motions of MoS2 layers, relaxes electron-phonon coupling selection rules, perturbs chemical bonding, and increases the electronic density of states. As a result, the nonadiabatic coupling grows and electronic relaxation becomes faster compared to flat ML-MoS2. Our study suggests that the sub-picosecond electronic relaxation dynamics of TMDs is amenable to strain engineering and that applications which require long-lived hot carriers, such as hot-electron-driven light harvesting and photocatalysis, should employ wrinkle-free TMDs.

Revealing low-loss dielectric near-field modes of hexagonal boron nitride by photoemission electron microscopy

Low-loss dielectric modes are important features and functional bases of fundamental optical components in on-chip optical devices. However, dielectric near-field modes are challenging to reveal with high spatiotemporal resolution and fast direct imaging. Herein, we present a method to address this issue by applying time-resolved photoemission electron microscopy to a low-dimensional wide-bandgap semiconductor, hexagonal boron nitride (hBN). Taking a low-loss dielectric planar waveguide as a fundamental structure, static vector near-field vortices with different topological charges and the spatiotemporal evolution of waveguide modes are directly revealed. With the lowest-order vortex structure, strong nanofocusing in real space is realized, while near-vertical photoemission in momentum space and narrow spread in energy space are simultaneously observed due to the atomically flat surface of hBN and the small photoemission horizon set by the limited photon energies. Our approach provides a strategy for the realization of flat photoemission emitters.

A global sensitivity analysis of a mechanistic model of neoadjuvant chemotherapy for triple negative breast cancer constrained by in vitro and in vivo imaging data

Neoadjuvant chemotherapy (NAC) is a standard-of-care treatment for locally advanced triple negative breast cancer (TNBC) before surgery. The early assessment of TNBC response to NAC would enable an oncologist to adapt the therapeutic plan of a non-responding patient, thereby improving treatment outcomes while preventing unnecessary toxicities. To this end, a promising approach consists of obtaining in silico personalized forecasts of tumor response to NAC via computer simulation of mechanistic models constrained with patient-specific magnetic resonance imaging (MRI) data acquired early during NAC. Here, we present a new mechanistic model of TNBC growth and response to NAC, including an explicit description of drug pharmacodynamics and pharmacokinetics. As longitudinal in vivo MRI data for model calibration is limited, we perform a sensitivity analysis to identify the model mechanisms driving the response to two NAC drug combinations: doxorubicin with cyclophosphamide, and paclitaxel with carboplatin. The model parameter space is constructed by combining patient-specific MRI-based in silico parameter estimates and in vitro measurements of pharmacodynamic parameters obtained using time-resolved microscopy assays of several TNBC lines. The sensitivity analysis is run in two MRI-based scenarios corresponding to a well-perfused and a poorly perfused tumor. Out of the 15 parameters considered herein, only the baseline tumor cell net proliferation rate along with the maximum concentrations and effects of doxorubicin, carboplatin, and paclitaxel exhibit a relevant impact on model forecasts (total effect index, ST\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$S_{T}$$\\end{document}0.1). These results dramatically limit the number of parameters that require in vivo MRI-constrained calibration, thereby facilitating the clinical application of our model.

Advances in Microsecond Time-Resolved Cryo-Electron Microscopy.

Advances in Microsecond Time-Resolved Cryo-Electron Microscopy.

Visualization of Molecular Permeation into a Multi-compartment Phospholipid Vesicle.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-144-67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-144-67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

Time-resolved imaging and analysis of the electron beam-induced formation of an open-cage metallo-azafullerene.

The visualization of single-molecule reactions provides crucial insights into chemical processes, and the ability to do so has grown with the advances in high-resolution transmission electron microscopy. There is currently a limited mechanistic understanding of chemical reactions under the electron beam. However, such reactions may enable synthetic methodologies that cannot be accessed by traditional organic chemistry methods. Here we demonstrate the synthetic use of the electron beam, by in-depth single-molecule, atomic-resolution, time-resolved transmission electron microscopy studies, in inducing the formation of a doubly holed fullerene-porphyrin cage structure from a well-defined benzoporphyrin precursor deposited on graphene. Through real-time imaging, we analyse the hybrid's ability to host up to two Pb atoms, and subsequently probe the dynamics of the Pb-Pb binding motif in this exotic metallo-organic cage structure. Through simulation, we conclude that the secondary electrons, which accumulate in the periphery of the irradiated area, can also initiate chemical reactions. Consequently, designing advanced carbon nanostructures by electron-beam lithography will depend on the understanding and limitations of molecular radiation chemistry.

Comparing mechanism-based and machine learning models for predicting the effects of glucose accessibility on tumor cell proliferation

Glucose plays a central role in tumor metabolism and development and is a target for novel therapeutics. To characterize the response of cancer cells to blockade of glucose uptake, we collected time-resolved microscopy data to track the growth of MDA-MB-231 breast cancer cells. We then developed a mechanism-based, mathematical model to predict how a glucose transporter (GLUT1) inhibitor (Cytochalasin B) influences the growth of the MDA-MB-231 cells by limiting access to glucose. The model includes a parameter describing dose dependent inhibition to quantify both the total glucose level in the system and the glucose level accessible to the tumor cells. Four common machine learning models were also used to predict tumor cell growth. Both the mechanism-based and machine learning models were trained and validated, and the prediction error was evaluated by the coefficient of determination (R2). The random forest model provided the highest accuracy predicting cell dynamics (R2 = 0.92), followed by the decision tree (R2 = 0.89), k-nearest-neighbor regression (R2 = 0.84), mechanism-based (R2 = 0.77), and linear regression model (R2 = 0.69). Thus, the mechanism-based model has a predictive capability comparable to machine learning models with the added benefit of elucidating biological mechanisms.

Exciton annihilation in molecular aggregates suppressed through quantum interference.

Exciton-exciton annihilation (EEA), an important loss channel in optoelectronic devices and photosynthetic complexes, has conventionally been assumed to be an incoherent, diffusion-limited process. Here we challenge this assumption by experimentally demonstrating the ability to control EEA in molecular aggregates using the quantum phase relationships of excitons. We employed time-resolved photoluminescence microscopy to independently determine exciton diffusion constants and annihilation rates in two substituted perylene diimide aggregates featuring contrasting excitonic phase envelopes. Low-temperature EEA rates were found to differ by more than two orders of magnitude for the two compounds, despite comparable diffusion constants. Simulated rates based on a microscopic theory, in excellent agreement with experiments, rationalize this EEA behaviour based on quantum interference arising from the presence or absence of spatial phase oscillations of delocalized excitons. These results offer an approach for designing molecular materials using quantum interference where low annihilation can coexist with high exciton concentrations and mobilities.

Fluorescent/Phosphorescent Dual-Emissive Probe for Simultaneous and Independent Sensing of Intracellular Hypochlorite and pH via Time-Resolved Luminescence Imaging.

In this work, we present a dual-emissive polymeric probe capable of simultaneous and independent sensing of intracellular hypochlorite and pH. The probe integrates an anthracene-carboxamide-based fluorescent compound with a phosphorescent iridium(III) complex. The fluorescence and phosphorescence spectra are significantly overlapped at 500 to 650 nm, but their lifetimes differ substantially at 14.8 and 1113.6 ns, respectively, thereby allowing for monitoring in different time intervals through time-resolved luminescence analysis. The fluorescence is quenched upon oxidation by hypochlorite, whereas an acidic environment favors protonation of pyridyl groups on the iridium(III) complex, which causes phosphorescence quenching. During hypochlorite sensing, a linear plot of the fluorescence/phosphorescence ratio over hypochlorite concentration was obtained. During pH sensing, the pK a value was calculated to be about 4.6. The utilization of this probe for imaging cellular endogenous hypochlorite and intracellular pH values was demonstrated. The short-lived and long-lived signals were successfully extracted via time-resolved luminescence imaging microscopy, thereby allowing independent, simultaneous, and quantitative analysis of fluorescence and phosphorescence signals and their response toward hypochlorite and pH values. This study offers potential implications for the development of biosensing and bioimaging applications and paves the way for the simultaneous multiplexed detection of physiological and pathological activities within living cells.

Imaging of short-wavelength spin waves in a nanometer-thick YIG/Co bilayer

We report the imaging of short-wavelength spin waves in a continuous nanometer-thick YIG film with a Co stripe patterned on top. Dynamic dipolar coupling between the YIG film and the Co stripe lowers the spin-wave wavelength when spin waves enter the YIG/Co bilayer region from the bare YIG film, causing partial reflection at the YIG/Co edge. We use time-resolved scanning transmission x-ray microscopy to image the mode conversion process down to a wavelength of 280 nm and extract the spin-wave dispersion, decay length, and magnetic damping in the YIG/Co bilayer. We also analyze spin-wave reflection from the YIG/Co edge and its dependence on the wavelength of incoming and transmitted spin waves.

Understanding the Chemical Mechanism behind Photoinduced Enhanced Raman Spectroscopy

Photoinduced enhanced Raman spectroscopy (PIERS) is a new surface enhanced Raman spectroscopy (SERS) modality with a 680% Raman signal enhancement of adsorbed analytes over that of SERS. Despite the explosion in recent demonstrations, the PIERS mechanism remains undetermined. Using X-ray and time-resolved optical spectroscopies, electron microscopy, cyclic voltammetry, and density functional theory simulations, we elucidate the atomic-scale mechanism behind PIERS. Stable PIERS substrates were fabricated using self-organized arrays of TiO2 nanotubes with controlled oxygen vacancy doping and size-controlled silver nanoparticles. The key source of PIERS vs SERS enhancement is an increase in the Raman polarizability of the adsorbed analyte upon photoinduced charge transfer. A balance between improved crystallinity, which enhances charge transfer due to higher electron mobility but decreases light absorption, and increased oxygen vacancy defect concentration, which increases light absorption, is critical. This work enables the rational design of PIERS substrates for sensing.

Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Fluorescence Resonance Energy Transfer (FRET)-basedapproaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction-limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super-resolved FRET imaging based on single-molecule localization microscopy using an early prototype of a commercial time-resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information.

Caustic spin wave beams in soft thin films: Properties and classification

In the context of wave propagation, caustics are usually defined as the envelope of a finite-extent wavefront; folds and cusps in a caustic result in enhanced wave amplitudes. Here, we tackle a related phenomenon, namely, the existence of well-defined beams originating solely from the geometric properties of the corresponding dispersion relation. This directional emission, termed caustic beam, is enabled by a stationary group velocity direction, and has been observed first in the case of phonons. We propose an overview of this ``focusing'' effect in the context of spin waves excited in soft, thin ferromagnetic films. Based on an analytical dispersion relation, we provide tools for a systematic survey of caustic spin wave beams. Our theoretical approach is validated by time-resolved microscopy experiments using the magneto-optical Kerr effect. Then, we identify two cases of particular interest both from fundamental and applicative perspectives. Indeed, both of them enable broadband excitations (in terms of wave vectors) to result in narrow-band beams of low divergence.

Nanoscale imaging of quantum dot dimers using time-resolved super-resolution microscopy combined with scanning electron microscopy

Time-resolved super-resolution microscopy was used in conjunction with scanning electron microscopy to image individual colloidal CdSe/CdS semiconductor quantum dots (QD) and QD dimers. The photoluminescence (PL) lifetimes, intensities, and structural parameters were acquired with nanometer scale spatial resolution and sub-nanosecond time resolution. The combination of these two techniques was more powerful than either alone, enabling us to resolve the PL properties of individual QDs within QD dimers as they blinked on and off, measure interparticle distances, and identify QDs that may be participating in energy transfer. The localization precision of our optical imaging technique was ∼3 nm, low enough that the emission from individual QDs within the dimers could be spatially resolved. While the majority of QDs within dimers acted as independent emitters, at least one pair of QDs in our study exhibited lifetime and intensity behaviors consistent with resonance energy transfer from a shorter lifetime and lower intensity donor QD to a longer lifetime and higher intensity acceptor QD. For this case, we demonstrate how the combined super-resolution optical imaging and scanning electron microscopy data can be used to characterize the energy transfer rate.

D-Band Hole Dynamics in Gold Nanoparticles Measured with Time-Resolved Emission Upconversion Microscopy.

The performance of photocatalysts and photovoltaic devices can be enhanced by energetic charge carriers produced from plasmon decay, and the lifetime of these energetic carriers greatly affects overall efficiencies. Although hot electron lifetimes in plasmonic gold nanoparticles have been investigated, hot hole lifetimes have not been as thoroughly studied in plasmonic systems. Here, we demonstrate time-resolved emission upconversion microscopy and use it to resolve the lifetime and energy-dependent cooling of d-band holes formed in gold nanoparticles by plasmon excitation and by following plasmon decay into interband and then intraband electron-hole pairs.