Visualizing Nanoscale Dynamics with Time-resolved Electron Microscopy.

The large number of interactions in nanoscale systems leads to the emergence of complex behavior. Understanding such complexity requires atomic-resolution observations with a time resolution that is high enough to match the characteristic timescale of the system. Our laboratory's method of choice is time-resolved electron microscopy. In particular, we are interested in the development of novel methods and instrumentation for high-speed observations with atomic resolution. Here, we present an overview of the activities in our laboratory.

Microsolvation Structures of Protonated Glycine and l-Alanine.

The IR predissociation spectra of microsolvated glycine and l-alanine, GlyH+(H2O) n and AlaH+(H2O) n, n = 1-6, are presented. The assignments of the solvation structures are aided by H2O/D2O substitution, IR-IR double resonance spectroscopy, and computational efforts. The analysis reveals the water-amino acid as well as the water-water interactions, and the subtle effects of the methyl side chain in l-alanine on the solvation motif are also highlighted. The bare amino acids exhibit an intramolecular hydrogen bond between the protonated amine and carboxyl terminals. In the n = 1-2 clusters, the water molecules preferentially solvate the protonated amine group, and we observed differences in the relative isomer stabilities in the two amino acids due to electron donation from the methyl weakening the intramolecular hydrogen bond. The structures in the n = 3 clusters show a further preference for solvation of the carboxyl group in l-alanine. For n = 4-6 clusters, the solvation structure of the two amino acids is remarkably similar, with one dominant isomer present in each cluster size. The first solvation shell is completed at n = 4, evidenced by a lack of free NH and OH stretches on the amino acid, as well as the first observation of H2O-H2O interactions in the spectra of n = 5. Finally, we note that calculations at the density functional theory (DFT) level show excellent agreement with the experiment for the smaller clusters. However, when water-water interactions compete with water-amino acid interactions in the larger clusters, DFT results show greater disagreement with experiment when compared to MP2 results.

Probing Solvation-Induced Structural Changes in Conformationally Flexible Peptides: IR Spectroscopy of Gly3H+·(H2O).

IR predissociation spectroscopy of the Gly3H+(H2O) complex formed inside of a cryogenic ion trap reveals how the flexible model peptide structurally responds to solvation by a single water molecule. The resulting one-laser spectrum is quite congested, and the spectral analyses were assisted by both H2O/D2O substitution and IR-IR double resonance spectroscopy, revealing the presence of two contributing isomers and extensive anharmonic features. Comparisons to structures found via a systematic computational search identified the geometries of these two isomers. The major isomer, with all trans amide bonds and protonation on the terminal amine, represents ∼90% of the overall population. It noticeably differs from the unsolvated Gly3H+, which exists in two isomeric forms: one with a cis amide bond and the other with protonation on an amide C═O. These results indicate that interactions with just one water molecule can induce significant structural changes, i.e., cis- trans amide bond rotation and proton migration, even as the clustering occurs within an 80 K cryogenic ion trap. Calculations of the isomerization pathways further reveal that the binding energy of the water molecule provides sufficient internal energy to overcome the barriers for the observed structural changes, and the minor solvation isomer results from a small fraction of the ions being kinetically trapped along one of the pathways.

Vibrational Characterization of Microsolvated Electrocatalytic Water Oxidation Intermediate: [Ru(tpy)(bpy)(OH)]2+(H2O)0-4.

The infrared predissociation spectra of the mass-selected electrocatalytic water oxidation intermediate [Ru(tpy)(bpy)(OH)]2+(H2O)0-4 are reported. The [Ru(tpy)(bpy)(OH)]2+ species is generated by passing a solution of [Ru(tpy)(bpy)(H2O)](ClO4)2 through an electrochemical flow cell held at 1.2 V and is immediately introduced into the gas phase via electrospray ionization (ESI). The microsolvated clusters are formed by reconstructing the water network in a cryogenic ion trap. Details of the hydrogen bonding network in these clusters are revealed by the infrared predissociation spectra in the OH stretch region. This improved method for capturing microsolvated clusters yielded colder complexes with much better resolved IR features than previous studies. The analysis of these spectra, supported by electronic structure calculations and compared to previous results on [Ru(tpy)(bpy)(H2O)]2+(H2O)0-4 clusters, reveals the nature of the Ru-OH bond and the effect of hydrogen bonding on facilitating the subsequent oxidation to [Ru(tpy)(bpy)(O)]2+ in the proposed catalytic cycle. Particularly, the hydrogen bonding interaction in [Ru(tpy)(bpy)(OH)]2+(H2O)1 is much weaker than that in the corresponding [Ru(tpy)(bpy)(H2O)]2+(H2O)1 and thus is less effective at activating the hydroxyl ligand for further oxidation via proton coupled electron transfer (PCET). Furthermore, the results here reveal that the Ru-OH bond, though formally described as an Ru3+/OH- interaction, has more covalent bond character than ionic bond character.

Interaction between ionic liquid cation and water: infrared predissociation study of [bmim](+)·(H2O)n clusters.

The infrared predissociation spectra of [bmim](+)·(H2O)n, n = 1-8, in the 2800-3800 cm(-1) region are presented and analyzed with the help of electronic structure calculations. The results show that the water molecules solvate [bmim](+) by predominately interacting with the imidazolium C2-H moiety for the small n = 1 and 2 clusters. This is characterized by a redshifted and relatively intense C2-H stretch. For n≥ 4 clusters, hydrogen-bond interactions between the water molecules drive the formation of ring isomers which interact on top of the imidazolium ring without any direct interaction with the C2-H. The water arrangement in [bmim](+)·(H2O)n is similar to the low energy isomers of neutral water clusters up to the n = 6 cluster. This is not the case for the n = 8 cluster, which has the imidazolium ring disrupting the otherwise preferred cubic water structure. The evolution of the solvation network around [bmim](+) illustrates the competing [bmim](+)-water and water-water interactions.

Characterization of the Oxygen Binding Motif in a Ruthenium Water Oxidation Catalyst by Vibrational Spectroscopy.

For homogeneous mononuclear ruthenium water oxidation catalysts, the Ru-O2 complex plays a crucial role in the rate determining step of the catalytic cycle, but the exact nature of this complex is unclear. Herein, the infrared spectra of the [Ru(tpy)(bpy)(O2)](2+) complex (tpy=2,2':6',2''-terpyridine; bpy=2,2'-bipyridine) are presented. The complex [Ru(tpy)(bpy)(O2)](2+), formed by gas-phase reaction of [Ru(tpy)(bpy)](2+) with molecular O2, was isolated by using mass spectrometry and was directly probed by cryogenic ion IR predissociation spectroscopy. Well-resolved spectral features enable a clear identification of the O-O stretch using (18) O2 substitution. The band frequency and intensity indicate that the O2 moiety binds to the Ru center in a side-on, bidentate manner. Comparisons with DFT calculations highlight the shortcomings of the B3LYP functional in properly depicting the Ru-O2 interaction.

Coordination structure and charge transfer in microsolvated transition metal hydroxide clusters [MOH](+)(H2O)1-4.

Infrared vibrational predissociation spectra of transition metal hydroxide clusters, [MOH](+)(H2O)1-4·D2 with M = Mn, Fe, Co, Ni, Cu, and Zn, are presented and analyzed with the aid of density functional theory calculations. For the [MnOH](+), [FeOH](+), [CoOH](+) and [ZnOH](+) species, we find that the first coordination shell contains three water molecules and the four ligands are arranged in a distorted tetrahedral geometry. [CuOH](+) can have either two or three water molecules in the first shell arranged in a planar arrangement, while [NiOH](+) has an octahedral ligand geometry with the first shell likely closed with five water molecules. Upon closure of the first coordination shell, characteristic stretch frequencies of hydrogen-bonded OH in the 2500-3500 cm(-1) region are used to pinpoint the location of the water molecule in the second shell. The relative energetics of different binding sites are found to be metal dependent, dictated by the first-shell coordination geometry and the charge transfer between the hydroxide and the metal center. Finally, the frequency of the hydroxide stretch is found to be sensitive to the vibrational Stark shift induced by the charged metal center, as observed previously for the smaller [MOH](+)(H2O) species. Increasing solvation modulates this frequency by reducing the extent of the charge transfer while elongating the M-OH bond.

A dual cryogenic ion trap spectrometer for the formation and characterization of solvated ionic clusters.

A new experimental approach is presented in which two separate cryogenic ion traps are used to reproducibly form weakly bound solvent clusters around electrosprayed ions and messenger-tag them for single-photon infrared photodissociation spectroscopy. This approach thus enables the vibrational characterization of ionic clusters comprised of a solvent network around large and non-volatile ions. We demonstrate the capabilities of the instrument by clustering water, methanol, and acetone around a protonated glycylglycine peptide. For water, cluster sizes with greater than twenty solvent molecules around a single ion are readily formed. We further demonstrate that similar water clusters can be formed around ions having a shielded charge center or those that do not readily form hydrogen bonds. Finally, infrared photodissociation spectra of D2-tagged GlyGlyH(+)⋅(H2O)1-4 are presented. They display well-resolved spectral features and comparisons with calculations reveal detailed information on the solvation structures of this prototypical peptide.

Near-atomic resolution reconstructions from in situ revitrified cryo samples.

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

Microsecond melting and revitrification of cryo samples: protein structure and beam-induced motion.

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

The fragmentation mechanism of gold nanoparticles in water under femtosecond laser irradiation.

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

Microsecond melting and revitrification of cryo samples.

The dynamics of proteins that are associated with their function typically occur on the microsecond timescale, orders of magnitude faster than the time resolution of cryo-electron microscopy. We have recently introduced a novel approach to time-resolved cryo-electron microscopy that affords microsecond time resolution. It involves melting a cryo sample with a heating laser, so as to allow dynamics of the proteins to briefly occur in the liquid phase. When the laser is turned off, the sample rapidly revitrifies, trapping the particles in their transient configurations. Precise control of the temperature evolution of the sample is crucial for such an approach to succeed. Here, we provide a detailed characterization of the heat transfer occurring under laser irradiation as well as the associated phase behavior of the cryo sample. While areas close to the laser focus undergo melting and revitrification, surrounding regions crystallize. In situ observations of these phase changes therefore provide a convenient approach for assessing the temperature reached in each melting and revitrification experiment and for adjusting the heating laser power on the fly.

Real-time observation of jumping and spinning nanodroplets.

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

In Situ Observation of Coulomb Fission of Individual Plasmonic Nanoparticles.

Reshaping plasmonic nanoparticles with laser pulses has been extensively researched as a tool for tuning their properties. However, in the absence of direct observations of the processes involved, important mechanistic details have remained elusive. Here, we present an in situ electron microscopy study of one such process that involves Coulomb fission of plasmonic nanoparticles under femtosecond laser irradiation. We observe that gold nanoparticles encapsulated in a silica shell fission by emitting progeny droplets comprised of about 10-500 atoms, with ejection preferentially occurring along the laser polarization direction. Under continued irradiation, the emitted droplets coalesce into a second core within the silica shell, and the system evolves into a dual-core particle. Our findings are consistent with a mechanism in which electrons are preferentially emitted from the gold core along the laser polarization direction. The resulting anisotropic charge distribution in the silica shell then determines the direction in which progeny droplets are ejected. In addition to yielding insights into the mechanism of Coulomb fission in plasmonic nanoparticles, our experiments point toward a facile method for forming surfaces decorated with aligned dual-gold-core silica shell particles.

Accessing the Vibrational Signatures of Amino Acid Ions Embedded in Water Clusters.

We present an infrared predissociation (IRPD) study of microsolvated GlyH+(H2O) n and GlyH+(D2O) n clusters, formed inside of a cryogenic ion trap via condensation of H2O or D2O onto the protonated glycine ions. The resulting IRPD spectra, showing characteristic O-H and O-D stretches, indicate that H/D exchange reactions are quenched when the ion trap is held at 80 K, minimizing the presence of isotopomers. Comparisons of GlyH+(H2O) n and GlyH+(D2O) n spectra clearly highlight and distinguish the vibrational signatures of the water solvent molecules from those of the core GlyH+ ion, allowing for quick assessment of solvation structures. Without the aid of calculations, we can already infer solvation motifs and the presence of multiple conformations. The use of a cryogenic ion trap to cluster solvent molecules around ions of interest and control H/D exchange reactions is broadly applicable and should be extendable to studies of more complex peptidic ions in large solvated clusters.

IR-IR Conformation Specific Spectroscopy of Na+(Glucose) Adducts.

We report an IR-IR double resonance study of the structural landscape present in the Na+(glucose) complex. Our experimental approach involves minimal modifications to a typical IR predissociation setup, and can be carried out via ion-dip or isomer-burning methods, providing additional flexibility to suit different experimental needs. In the current study, the single-laser IR predissociation spectrum of Na+(glucose), which clearly indicates contributions from multiple structures, was experimentally disentangled to reveal the presence of three α-conformers and five ß-conformers. Comparisons with calculations show that these eight conformations correspond to the lowest energy gas-phase structures with distinctive Na+ coordination. Graphical Abstract ᅟ.

Mass Spectrometric and Vibrational Characterization of Reaction Intermediates in [Ru(bpy)(tpy)(H2 O)]2+ Catalyzed Water Oxidation.

Mass spectrometry coupled with an in-line electrochemical electrospray ionization source is used to capture some of the reaction intermediates formed in the [Ru(bpy)(tpy)(H2 O)]2+ (bpy=2,2'-bipyridine, tpy=2,2':6',2"-terpyridine) catalyzed water oxidation reaction. By controlling the applied electrochemical potential, we identified the parent complex, as well as the first two oxidation complexes, identified as [Ru(bpy)(tpy)(OH)]2+ and [Ru(bpy)(tpy)(O)]2+ . The structures of the parent and first oxidation complexes are probed directly in the mass spectrometer by using infrared predissociation spectroscopy of D2 -tagged ions. Comparisons between experimental vibrational spectra and density functional theory calculations confirmed the identity and structure of these two complexes. Moreover, the frequency of the O-H stretching mode in [Ru(bpy)(tpy)(OH)]2+ shows that this complex features a Ru-OH interaction that is more covalent than ionic.