High-resolution infrared spectroscopy of jet cooled cyclobutyl in the α-CH stretch region: large-amplitude puckering dynamics in a 4-membered ring radical.

Gas-phase cyclobutyl radical (c-C4H7) is generated at a rotational temperature of Trot = 26(1) K in a slit-jet discharge mixture of 70% Ne/30% He and 0.5-0.6% cyclobromobutane (c-C4H7Br). A fully rovibrationally resolved absorption spectrum of the α-CH stretch fundamental band between 3062.9 cm-1 to 3075.7 cm-1 is obtained and analyzed, yielding first precision structural and dynamical information for this novel radical species. The α-CH stretch band origin is determined to be 3068.7887(4) cm-1, which implies only a modest (≈0.8 cm-1) blue shift from rotationally unresolved infrared spectroscopic studies of cyclobutyl radicals in liquid He droplets [A. R. Brown, P. R. Franke and G. E. Douberly, J. Phys. Chem. A, 2017, 121, 7576-7587]. Of particular dynamical interest, a one-dimensional potential energy surface with respect to the ring puckering coordinate is computed at CCSD(T)/ANO2 level of theory and reveals a double minimum Cs puckered geometry, separated by an exceedingly shallow planar C2v transition state barrier (Ebarr ≈ 1 cm-1). Numerical solutions on this double minimum potential yield a zero-point energy for the ground state (Ezero-point ≈ 27 cm-1) greatly in excess of the interconversion barrier. This is indicative of highly delocalized, large amplitude motion of the four-membered ring structure, for which proper vibrationally averaging of the moment of inertia tensor reproduces the experimentally determined inertial defect remarkably well. Finally, intensity alternation in the experimental spectrum due to nuclear spin statistics upon exchange of three indistinguishable H atom pairs (IH = ½) matches Ka + Kc = even : odd = 36 : 28 predictions, implying that the unpaired electron in the radical center lies in an out-of-plane pπ orbital. Thus, high-resolution infrared spectroscopy provides first experimental confirmation of a shallow double minimum ring puckering potential with a highly delocalized ground state wave function peaked at a planar C2v transition state geometry consistent with a cyclobutyl π radical.

Ergodicity breaking in rapidly rotating C60 fullerenes.

Ergodicity, the central tenet of statistical mechanics, requires an isolated system to explore all available phase space constrained by energy and symmetry. Mechanisms for violating ergodicity are of interest for probing nonequilibrium matter and protecting quantum coherence in complex systems. Polyatomic molecules have long served as a platform for probing ergodicity breaking in vibrational energy transport. Here, we report the observation of rotational ergodicity breaking in an unprecedentedly large molecule, 12C60, determined from its icosahedral rovibrational fine structure. The ergodicity breaking occurs well below the vibrational ergodicity threshold and exhibits multiple transitions between ergodic and nonergodic regimes with increasing angular momentum. These peculiar dynamics result from the molecule's distinctive combination of symmetry, size, and rigidity, highlighting its relevance to emergent phenomena in mesoscopic quantum systems.

Kinetic and Thermodynamic Control of G-Quadruplex Polymorphism by Na+ and K+ Cations.

G-Quadruplexes (G4s) are ubiquitous nucleic acid folding motifs that exhibit structural diversity that is dependent on cationic conditions. In this work, we exploit temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) to elucidate the kinetic and thermodynamic mechanisms by which monovalent cations (K+ and Na+) impact folding topologies for a simple G-quadruplex sequence (5'-GGG-(TAAGGG)3-3') with a three-state folding equilibrium. Kinetic measurements indicate that Na+ and K+ influence G4 formation in two distinctly different ways: the presence of Na+ modestly enhances an antiparallel G4 topology through an induced fit (IF) mechanism with a low affinity (Kd = 228 ± 26 mM), while K+ drives G4 into a parallel/hybrid topology via a conformational selection (CS) mechanism with much higher affinity (Kd = 1.9 ± 0.2 mM). Additionally, temperature-dependent studies of folding rate constants and equilibrium ratios reveal distinctly different thermodynamic driving forces behind G4 binding to K+ (ΔH°bind > 0, ΔS°bind > 0) versus Na+ (ΔH°bind < 0, ΔS°bind < 0), which further illuminates the diversity of the possible pathways for monovalent facilitation of G-quadruplex folding.

Energy-Resolved Femtosecond Hot Electron Dynamics in Single Plasmonic Nanoparticles.

Efficient excitation and harvesting of hot carriers from nanoscale metals is central to many emerging photochemical, photovoltaic, and ultrafast optoelectronic applications. Nevertheless, direct experimental evidence of the energy-dependent femtosecond dynamics in ubiquitous tens-of-nanometer gold structures remains elusive, despite the potentially rich interplay between interfacial and internal plasmonic fields, excitation distributions, and scattering processes. To explore the effects of nanoscale structure on these dynamics, we employ simultaneous time-, angle-, and energy-resolved photoemission spectroscopy of single plasmonic nanoparticles. Photoelectron velocity and electric field distributions reveal bulk-like ballistic hot electron transport in different geometries, lacking any signatures of surface effects. Energy-resolved dynamics are measured in the 1-2 eV range and extrapolated to lower energies via Boltzmann theory, providing a detailed view of hot electron lifetimes within nanoscale gold. We find that particles with relevant dimensions as small as 10 nm serve as exemplary platforms for studying intrinsic metal dynamics.

Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection.

Rapid testing is essential to fighting pandemics such as coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Exhaled human breath contains multiple volatile molecules providing powerful potential for non-invasive diagnosis of diverse medical conditions. We investigated breath detection of SARS-CoV-2 infection using cavity-enhanced direct frequency comb spectroscopy (CE-DFCS), a state-of-the-art laser spectroscopic technique capable of a real-time massive collection of broadband molecular absorption features at ro-vibrational quantum state resolution and at parts-per-trillion volume detection sensitivity. Using a total of 170 individual breath samples (83 positive and 87 negative with SARS-CoV-2 based on reverse transcription polymerase chain reaction tests), we report excellent discrimination capability for SARS-CoV-2 infection with an area under the receiver-operating-characteristics curve of 0.849(4). Our results support the development of CE-DFCS as an alternative, rapid, non-invasive test for COVID-19 and highlight its remarkable potential for optical diagnoses of diverse biological conditions and disease states.

Non-equilibrium dynamics at the gas-liquid interface: State-resolved studies of NO evaporation from a benzyl alcohol liquid microjet.

First measurements of internal quantum-state distributions for nitric oxide (NO) evaporating from liquid benzyl alcohol are presented over a broad range of temperatures, performed by liquid-microjet techniques in an essentially collision-free regime, with rotational/spin-orbit populations in the 2Π1/2,3/2 manifolds measured by laser-induced fluorescence. The observed rotational distributions exhibit highly linear (i.e., thermal) Boltzmann plots but notably reflect rotational temperatures (Trot) as much as 30 K lower than the liquid temperature (Tjet). A comparable lack of equilibrium behavior is also noted in the electronic degrees of freedom but with populations corresponding to spin-orbit temperatures (TSO) consistently higher than Trot by ∼15 K. These results unambiguously demonstrate evaporation into a non-equilibrium distribution, which, by detailed-balance considerations, predict quantum-state-dependent sticking coefficients for incident collisions of NO at the gas-liquid interface. Comparison and parallels with previous experimental studies of NO thermal desorption and molecular-beam scattering in other systems are discussed, which suggests the emergence of a self-consistent picture for the non-equilibrium dynamics.

Ionic Cooperativity between Lysine and Potassium in the Lysine Riboswitch: Single-Molecule Kinetic and Thermodynamic Studies.

Functionality in many biological systems, including proteins and nucleic acid structures, including protein and nucleic acid riboswitch structures, can depend on cooperative kinetic behavior between multiple small molecule ligands. In this work, single-molecule FRET data on the Bacillus subtilis lysine riboswitch reveals that affinity for the cognate lysine ligand increases significantly with K+, providing evidence for synergism between lysine/K+ binding to the aptamer and successful folding of the riboswitch. To describe/interpret this more complex kinetic scenario, we explore the conventional 4-state ("square") model for aptamer binding as a function of K+. Extension into this additional dimension generates a novel "cube" model for riboswitch folding dynamics with respect to lysine/K+ binding, revealing that riboswitch folding (kfold) and unfolding (kunfold) rate constants increase and decrease dramatically with K+, respectively. Furthermore, temperature-dependent single-molecule kinetic studies indicate that the presence of K+ entropically enhances the transition state barrier to folding but partially compensates for this by increasing the overall exothermicity for lysine binding. We rationalize this behavior as evidence that K+ facilitates hydrogen bonding between the negatively charged carboxyl group of lysine and the RNA, increasing structural rigidity and lowering entropy in the binding pocket. Finally, we explore the effects of cation size with Na+ and Cs+ studies to demonstrate that K+ is optimally suited for bridging interactions between lysine and the riboswitch aptamer domain. Regulation of lysine production and transport, dictated by the riboswitch's ability to recognize and bind lysine, is therefore intimately tied to the presence of K+ in the binding pocket and is strongly modulated by local cation conditions. The results suggest an increase in lysine riboswitch functionality by sensitivity to additional species in the cellular riboswitch environment.

Nonequilibrium Scattering/Evaporation Dynamics at the Gas-Liquid Interface: Wetted Wheels, Self-Assembled Monolayers, and Liquid Microjets.

ConspectusWe often teach or are taught in our freshman courses that there are three phases of matter─gas, liquid and solid─where the ordering reflects increasing complexity and strength of interaction between the molecular constituents. But arguably there is also a fascinating additional "phase" of matter associated with the microscopically thin interface (<10 molecules thick) between the gas and liquid, which is still poorly understood and yet plays a crucial role in fields ranging from chemistry of the marine boundary layer and atmospheric chemistry of aerosols to the passage of O2 and CO2 through alveolar sacs in our lungs. The work in this Account provides insights into three challenging new directions for the field, each embracing a rovibronically quantum-state-resolved perspective. Specifically, we exploit the powerful tools of chemical physics and laser spectroscopy to pose two fundamental questions. (i) At the microscopic level, do molecules in all internal quantum-states (e.g., vibrational, rotational, electronic) colliding with the interface "stick" with unit probability? (ii) Can reactive, scattering, and/or evaporating molecules at the gas-liquid interface avoid collisions with other species and thereby be observed in a truly "nascent" collision-free distribution of internal degrees of freedom? To help address these questions, we present studies in three different areas: (i) reactive scattering dynamics of F atoms with wetted-wheel gas-liquid interfaces, (ii) inelastic scattering of HCl from self-assembled monolayers (SAMs) via resonance-enhanced photoionization (REMPI)/velocity map imaging (VMI) methods, and (iii) quantum-state-resolved evaporation dynamics of NO at the gas-water interface. As a recurring theme, we find that molecular projectiles reactively, inelastically, or evaporatively scatter from the gas-liquid interface into internal quantum-state distributions substantially out of equilibrium with respect to the bulk liquid temperatures (TS). By detailed balance considerations, the data unambiguously indicate that even simple molecules exhibit rovibronic state dependences to how they "stick" to and eventually solvate into the gas-liquid interface. Such results serve to underscore the importance of quantum mechanics and nonequilibrium thermodynamics in energy transfer and chemical reactions at the gas-liquid interface. This nonequilibrium behavior may well make this rapidly emergent field of chemical dynamics at gas-liquid interfaces more complicated but even more interesting targets for further experimental/theoretical exploration.

Ligand-Dependent Volumetric Characterization of Manganese Riboswitch Folding: A High-Pressure Single-Molecule Kinetic Study.

Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch. Detailed pressure-dependent analysis of the conformational kinetics allows one to extract precision changes (σ ≲ 4-8 Å3) in free volumes not only between the fully folded/unfolded conformations but also with respect to the folding transition state of the manganese riboswitch. This permits first extraction of a novel "reversible work" free energy (PΔV) landscape, which reveals a monotonic increase in manganese riboswitch volume along the folding coordinate. Furthermore, such a tool permits exploration of pressure-dependent effects on both Mn2+ binding and riboswitch folding, which demonstrate that ligand attachment stabilizes the riboswitch under pressure by decreasing the volume increase upon folding (ΔΔV < 0). Such competition between ligand binding and pressure-induced denaturation dynamics could be of significant evolutionary advantage, compensating for a weakening in riboswitch tertiary structure with pressure-mediated ligand binding and promotion of folding response.

Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy.

Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under "crowded" in vivo cellular conditions and thus incompletely characterized by in vitro studies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of the B. subtilis LysC lysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprising increase in affinity for the cognate ligand. This is furthermore confirmed by temperature-dependent studies, which reveal that PEG crowding is not purely entropic and instead significantly impacts both enthalpic and entropic contributions to the free energy landscape for folding. The results indicate that PEG molecular crowding/stabilization of the lysine riboswitch is more mechanistically complex and requires extension beyond the conventional picture of purely repulsive solvent-solute steric interactions arising from excluded volume and entropy. Instead, the current experimental FRET data support an alternative multistep mechanism, whereby PEG first entropically crowds the unfolded riboswitch into a "pre-folded" conformation, which in turn greatly increases the ligand binding affinity and thereby enhances the overall equilibrium for riboswitch folding.

High-resolution CH stretch spectroscopy of jet-cooled cyclopentyl radical: First insights into equilibrium structure, out-of-plane puckering, and IVR dynamics.

First, high-resolution sub-Doppler infrared spectroscopic results for cyclopentyl radical (C5H9) are reported on the α-CH stretch fundamental with suppression of spectral congestion achieved by adiabatic cooling to Trot ≈ 19(4) K in a slit jet expansion. Surprisingly, cyclopentyl radical exhibits a rotationally assignable infrared spectrum, despite 3N - 6 = 36 vibrational modes and an upper vibrational state density (ρ ≈ 40-90 #/cm-1) in the critical regime (ρ ≈ 100 #/cm-1) necessary for onset of intramolecular vibrational relaxation (IVR) dynamics. Such high-resolution data for cyclopentyl radical permit detailed fits to a rigid-rotor asymmetric top Hamiltonian, initial structural information for ground and vibrationally excited states, and opportunities for detailed comparison with theoretical predictions. Specifically, high level ab initio calculations at the coupled-cluster singles, doubles, and perturbative triples (CCSD(T))/ANO0, 1 level are used to calculate an out-of-plane bending potential, which reveals a C2 symmetry double minimum 1D energy surface over a C2v transition state. The inversion barrier [Vbarrier ≈ 3.7(1) kcal/mol] is much larger than the effective moment of inertia for out-of-plane bending, resulting in localization of the cyclopentyl wavefunction near its C2 symmetry equilibrium geometry and tunneling splittings for the ground state too small (<1 MHz) to be resolved under sub-Doppler slit jet conditions. The persistence of fully resolved high-resolution infrared spectroscopy for such large cyclic polyatomic radicals at high vibrational state densities suggests a "deceleration" of IVR for a cycloalkane ring topology, much as low frequency torsion/methyl rotation degrees of freedom have demonstrated a corresponding "acceleration" of IVR processes in linear hydrocarbons.

Formation and detection of metastable formic acid in a supersonic expansion: High resolution infrared spectroscopy of the jet-cooled cis-HCOOH conformer.

High-resolution direct absorption infrared spectra of metastable cis-formic acid (HCOOH) trapped in a cis-well resonance behind a 15 kcal/mol barrier are reported for the first time, with the energetically unstable conformer produced in a supersonic slit plasma expansion of trans-formic acid/H2 mixtures. We present a detailed high-resolution rovibrational analysis for cis-formic acid species in the OH stretch (ν1) fundamental, providing first precision vibrational band origin, rotational constants, and term values, which in conjunction with ab initio calculations at the couple-cluster with single, double, and perturbative triple [CCSD(T)]/ANOn (n = 0, 1, 2) level support the experimental assignments and establish critical points on the potential energy surface for internal rotor trans-to-cis isomerization. Relative intensities for a- and b-type transitions observed in the spectra permit the transition dipole moment components to be determined in the body fixed frame and prove to be in good agreement with ab initio CCSD(T) theoretical estimates but in poor agreement with simple bond-dipole predictions. The observed signal dependence on H2 in the discharge suggests the presence of a novel H atom radical chemical mechanism for strongly endothermic "up-hill" internal rotor isomerization between trans- and cis-formic acid conformers.

High-resolution infrared spectroscopy of supersonically cooled singlet carbenes: Bromomethylene (HCBr) in the CH stretch region.

First high-resolution spectra of cold (∼35 K) singlet bromomethylene HCBr in the CH stretching (v1) region from 2770 to 2850 cm-1 are reported using near quantum shot-noise limited laser absorption methods in a slit jet supersonic discharge expansion source. Three rovibrational bands are identified at high S/N (20:1-40:1) and rotationally assigned to (i) the CH stretch fundamental (v1) band X̃1,0,0←X̃0,0,0 and (ii) vibrational hot bands [X̃(1,1,0)←X̃(0,1,0) and X̃(1,0,1)←X̃(0,0,1)] arising from vibrationally excited HCBr populated in the discharge with single quanta in either the H-C-Br bend (v2) or C-Br stretch (v3) modes. Precision rotational constants are reported for a total of six states, with an experimentally determined CH stretch vibrational frequency (2799.38 cm-1) in good agreement with previous low-resolution fluorescence studies [M. Deselnicu et al., J. Chem. Phys. 124(13), 134302 (2006)]. Detailed analysis of the fundamental v1 band highlights the presence of perturbations in the X̃1,0,0 level, which we tentatively attribute to arise from the nearby triplet state ã(0,0,1) through spin-orbit interaction or the multiple quanta X̃0,2,1 singlet state via c-type Coriolis coupling. Reduced-Doppler resolution (60 MHz) in the slit-jet IR spectrometer permits for clear observation of a nuclear spin hyperfine structure, with experimental line shapes well reproduced by nuclear quadrupole/spin-rotation coupling constants from microwave studies [C. Duan et al., J. Mol. Spectrosc. 220(1), 113-121 (2003)]. Finally, the a-type to b-type transition intensity ratio for the fundamental CH stretch band is notably larger than that predicted by using a bond-dipole model, which from high level ab initio quantum calculations [CCSD(T)/PVQZ] can be attributed to vibrationally induced "charge-sloshing" of electron density along the polar C-Br bond.

Regulating and Directionally Controlling Electron Emission from Gold Nanorods with Silica Coatings.

Dielectric coatings offer a versatile means of manipulating hot carrier emission from nanoplasmonic systems for emerging nanocatalysis and photocathode applications, with uniform coatings acting as regulators and nonuniform coatings providing directional photocurrent control. However, the mechanisms for electron emission through dense and mesoporous silica (SiO2) coatings require further examination. Here, we present a systematic investigation of photoemission from single gold nanorods as a function of dense versus mesoporous silica coating thicknesses. Studies with dense coatings on gold nanostructures clarify the short (∼1 nm) attenuation length responsible for severely reduced transmission through the silica conduction band. By contrast, mesoporous silica is much more transmissive, and a simple geometric model quantitatively recapitulates the electron escape probability through nanoscopic porous channels. Finally, photoelectron velocity map imaging (VMI) studies of nanorods with coating defects verify that photoemission occurs preferentially through the thinner regions, illustrating new opportunities for designing photocurrent distributions on the nanoscale.

Lysine-Dependent Entropy Effects in the B. subtilis Lysine Riboswitch: Insights from Single-Molecule Thermodynamic Studies.

Riboswitches play an important role in RNA-based sensing/gene regulation control for many bacteria. In particular, the accessibility of multiple conformational states at physiological temperatures allows riboswitches to selectively bind a cognate ligand in the aptamer domain, which triggers secondary structural changes in the expression platform, and thereby "switching" between on or off transcriptional or translational states for the downstream RNA. The present work exploits temperature-controlled, single-molecule total internal reflection fluorescence (TIRF) microscopy to study the thermodynamic landscape of such ligand binding/folding processes, specifically for the Bacillus subtilis lysine riboswitch. The results confirm that the riboswitch folds via an induced-fit (IF) mechanism, in which cognate lysine ligand first binds to the riboswitch before structural rearrangement takes place. The transition state to folding is found to be enthalpically favored (ΔHfold‡ < 0), yet with a free-energy barrier that is predominantly entropic (-TΔSfold‡ > 0), which results in folding (unfolding) rate constants strongly dependent (independent) of lysine concentration. Analysis of the single-molecule kinetic "trajectories" reveals this rate constant dependence of kfold on lysine to be predominantly entropic in nature, with the additional lysine conferring preferential advantage to the folding process by the presence of ligands correctly oriented with respect to the riboswitch platform. By way of contrast, van't Hoff analysis reveals enthalpic contributions to the overall folding thermodynamics (ΔH0) to be surprisingly constant and robustly independent of lysine concentration. The results demonstrate the crucial role of hydrogen bonding between the ligand and riboswitch platform but with only a relatively modest fraction (45%) of the overall enthalpy change needed to access the transition state and initiate transcriptional switching.

Effects of Molecular Crowders on Single-Molecule Nucleic Acid Folding: Temperature-Dependent Studies Reveal True Crowding vs Enthalpic Interactions.

Biomolecular folding in cells can be strongly influenced by spatial overlap/excluded volume interactions (i.e., "crowding") with intracellular solutes. As a result, traditional in vitro experiments with dilute buffers may not accurately recapitulate biomolecule folding behavior in vivo. In order to account for such ubiquitous excluded volume effects, biologically inert polyethylene glycol (PEG) and polysaccharides (dextran and Ficoll) are often used as in vitro crowding agents to mimic in vivo crowding conditions, with a common observation that high concentrations of these polymers stabilize the more compact biomolecule conformation. However, such an analysis can be distorted by differences in polymer interactions with the folded vs unfolded conformers, requiring temperature-dependent analysis of the thermodynamics to reliably assess competing enthalpic vs entropic contributions and thus the explicit role of excluded volume. In this work, temperature-controlled single-molecule fluorescence resonance energy transfer (smFRET) is used to characterize the thermodynamic interaction between nucleic acids and common polymer crowders PEG, dextran, and Ficoll. The results reveal that PEG promotes secondary and tertiary nucleic acid folding by simultaneously increasing the folding rate while decreasing the unfolding rate, with temperature-dependent studies confirming that the source of PEG stabilization is predominantly entropic and consistent with a true excluded volume crowding mechanism. By way of contrast, neither dextran nor Ficoll induces any significant concentration-dependent change in nucleic acid folding stability at room temperature, but instead, stabilization effects gradually appear with a temperature increase. Such a thermal response indicates that both folding enthalpies and entropies are impacted by dextran and Ficoll. A detailed thermodynamic analysis of the kinetics suggests that, instead of true entropic molecular crowding, dextran and Ficoll associate preferentially with the unfolded vs folded nucleic acid conformer as a result of larger solvent accessible surface area, thereby skewing the free energy landscapes through both significant entropic/enthalpic contributions that compete and fortuitously cancel near room temperature.

Ultrasensitive multispecies spectroscopic breath analysis for real-time health monitoring and diagnostics.

Breath analysis enables rapid, noninvasive diagnostics, as well as long-term monitoring of human health, through the identification and quantification of exhaled biomarkers. Here, we demonstrate the remarkable capabilities of mid-infrared (mid-IR) cavity-enhanced direct-frequency comb spectroscopy (CE-DFCS) applied to breath analysis. We simultaneously detect and monitor as a function of time four breath biomarkers-[Formula: see text]OH, [Formula: see text], [Formula: see text]O, and HDO-as well as illustrate the feasibility of detecting at least six more ([Formula: see text]CO, [Formula: see text], OCS, [Formula: see text], [Formula: see text], and [Formula: see text]) without modifications to the experimental apparatus. We achieve ultrahigh detection sensitivity at the parts-per-trillion level. This is made possible by the combination of the broadband spectral coverage of a frequency comb, the high spectral resolution afforded by the individual comb teeth, and the sensitivity enhancement resulting from a high-finesse cavity. Exploiting recent advances in frequency comb, optical coating, and photodetector technologies, we can access a large variety of biomarkers with strong carbon-hydrogen-bond spectral signatures in the mid-IR.

Measuring Excess Heat Capacities of Deoxyribonucleic Acid (DNA) Folding at the Single-Molecule Level.

Measurements of the thermodynamic properties of biomolecular folding (ΔG°, ΔH°, ΔS°, etc.) provide a wealth of information on the folding process and have long played a central role in biophysical investigation. In particular, the excess heat capacity of folding (ΔCP) is crucial, as typically measured in bulk ensemble studies by differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Here, we report the first measurements of ΔCP at the single-molecule level using the single-molecule fluorescence resonance energy transfer (smFRET) as well as the very first measurements of the heat capacity change associated with achieving the transition state (ΔC‡P) for nucleic acid folding. The deoxyribonucleic acid (DNA) hairpin used in these studies exhibits an excess heat capacity for hybridization (ΔCP = -340 ± 60 J/mol/K per base pair) consistent with the range of literature expectations (ΔCP = -100 to -420 J/mol/K per base pair). Furthermore, the measured activation heat capacities (ΔC‡P) for such hairpin unfolding are consistent with a folding transition state containing few fully formed base pairs, in agreement with prevailing models of DNA hybridization.

Pushing Camera-Based Single-Molecule Kinetic Measurements to the Frame Acquisition Limit with Stroboscopic smFRET.

Single-molecule fluorescence resonance energy transfer (smFRET) experiments permit detailed examination of microscopic dynamics. However, kinetic rate constants determined by smFRET are susceptible to systematic underestimation when the rate constants are comparable to the data acquisition rate. We demonstrate how such systematic errors in camera-based total internal reflection fluorescence microscopy experiments can be greatly reduced by using stroboscopic illumination/detection, allowing accurate rate constant determination up to the data sampling rate and yielding an order of magnitude increase in the dynamic range. Implementation of these stroboscopic smFRET ideas is straightforward, and the stroboscopically obtained data are compatible with multiple trajectory analysis methods, including dwell-time analysis and hidden Markov modeling. Such stroboscopic methods therefore offer a remarkably simple yet valuable addition to the smFRET toolkit, requiring only relatively modest modification to the normal data collection and analysis procedures.

Controlling the Spatial and Momentum Distributions of Plasmonic Carriers: Volume vs Surface Effects.

Spatial and momentum distributions of excited charge carriers in nanoplasmonic systems depend sensitively on optical excitation parameters and nanoscale geometry, which therefore control the efficiency and functionality of plasmon-enhanced catalysts, photovoltaics, and nanocathodes. Growing appreciation over the past decade for the different roles of volume- vs surface-mediated excitation in such systems has underscored the need for explicit separation and quantification of these pathways. Toward these ends, we utilize angle-resolved photoelectron velocity map imaging to distinguish these processes in gold nanorods of different aspect ratios down to the spherical limit. Despite coupling to the longitudinal surface plasmon, we find that resonantly excited nanorods always exhibit transverse (sideways) multiphoton photoemission distributions due to photoexcitation within volume field enhancement regions rather than at the tip hot spots. This behavior is accurately reproduced via ballistic Monte Carlo modeling, establishing that volume-excited electrons primarily escape through the nanorod sides. Furthermore, we demonstrate optical control over the photoelectron angular distributions via a screening-induced transition from volume (transverse/side) to surface (longitudinal/tip) photoemission with red detuning of the excitation laser. Frequency-dependent cross sections are separately quantified for these mechanisms by comparison with theoretical calculations, combining volume and surface velocity-resolved photoemission modeling. Based on these results, we identify nanomaterial-specific contributions to the photoemission cross sections and offer general nanoplasmonic design principles for controlling photoexcitation/emission distributions via geometry- and frequency-dependent tuning of the volume vs surface fields.