Facilitating flip-flop: Structural tuning of molecule-membrane interactions in living bacteria.

The first barrier that a small molecule must overcome before trespassing into a living cell is the lipid bilayer surrounding the intracellular content. It is imperative, therefore, to understand how the structure of a small molecule influences its fate in this region. Through the use of second harmonic generation, we show how the differing degrees of ionic headgroups, conjugated system, and branched hydrocarbon tail disparities of a series of four styryl dye molecules influence the propensity to "flip-flop" or to be further organized in the outer leaflet by the membrane. We show here that initial adsorption experiments match previous studies on model systems; however, more complex dynamics are observed over time. Aside from probe molecule structure, these dynamics also vary between cell species and can deviate from trends reported based on model membranes. Specifically, we show here that the membrane composition is an important factor to consider for headgroup-mediated small-molecule dynamics. Overall, the findings presented here on how structural variability of small molecules impacts their initial adsorption and eventual destinations within membranes in the context of living cells could have practical applications in antibiotic and drug adjuvant design.

Publisher Correction: Reply to: On the ferroelectricity of CH3NH3PbI3 perovskites.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Total Internal Reflection Transient Absorption Microscopy: An Online Detection Method for Microfluidics.

Microreactors have garnered widespread attention for their tunability and precise control of synthetic parameters to efficiently produce target species. Despite associated advances, a lack of online detection and optimization methods has stalled the progression of microfluidic reactors. Here we employ and characterize a total internal reflection transient absorption microscopy (TIRTAM) instrument to image excited state dynamics on a continuous flow device. The experiments presented demonstrate the capability to discriminate between different chromophores as well as in differentiating the effects of local chemical environments that a chromophore experiences. This work presents the first such online transient absorption measurements and provides a new direction for the advancement and optimization of chemical reactions in microfluidic devices.

Energetics at the Surface: Direct Optical Mapping of Core and Surface Electronic Structure in CdSe Quantum Dots Using Broadband Electronic Sum Frequency Generation Microspectroscopy.

Understanding and controlling the electronic structure of nanomaterials is the key to tailoring their use in a wide range of practical applications. Despite this need, many important electronic states are invisible to conventional optical measurements and are typically identified indirectly based on their inferred impact on luminescence properties. This is especially common and important in the study of nanomaterial surfaces and their associated defects. Surface trap states play a crucial role in photophysical processes yet remain remarkably poorly understood. Here we demonstrate for the first time that broadband electronic sum frequency generation (eSFG) microspectroscopy can directly map the optically bright and dark states of nanoparticles, including the elusive below gap states. This new approach is applied to model cadmium selenide (CdSe) quantum dots (QDs), where the energies of surface trap states have eluded direct optical characterization for decades. Our eSFG measurements show clear signatures of electronic transitions both above the band gap, which we assign to previously reported one- and two-photon transitions associated with the CdSe core, as well as broad spectral signatures below the band gap that are attributed to surface states. In addition to the core states, this analysis reveals two distinct distributions of below gap states, providing the first direct optical measurement of both shallow and deep surface states on this system. Finally, chemical modification of the surfaces via oxidation results in the relative increase in the signals originating from the surface states. Overall, our eSFG experiments provide an avenue to directly map the entirety of the QD core and surface electronic structure, which is expected to open up opportunities to study how these materials are grown in situ and how surface states can be controlled to tune functionality.

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria.

Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. To capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two-photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG not only can monitor the movement of small molecules across membrane leaflets but also is sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.

Chemical nature of ferroelastic twin domains in CH3NH3PbI3 perovskite.

The extraordinary optoelectronic performance of hybrid organic-inorganic perovskites has resulted in extensive efforts to unravel their properties. Recently, observations of ferroic twin domains in methylammonium lead triiodide drew significant attention as a possible explanation for the current-voltage hysteretic behaviour in these materials. However, the properties of the twin domains, their local chemistry and the chemical impact on optoelectronic performance remain unclear. Here, using multimodal chemical and functional imaging methods, we unveil the mechanical origin of the twin domain contrast observed with piezoresponse force microscopy in methylammonium lead triiodide. By combining experimental results with first principles simulations we reveal an inherent coupling between ferroelastic twin domains and chemical segregation. These results reveal an interplay of ferroic properties and chemical segregation on the optoelectronic performance of hybrid organic-inorganic perovskites, and offer an exploratory path to improving functional devices.

Compressed supercontinuum probe for transient absorption microscopy.

Here, we combine three optical advancements to transient absorption microscopy in order to access the photodynamics in systems requiring stringent spatial and temporal resolution criteria. First, a broadband visible probe is generated by a commercial photonic crystal fiber. Second, a spatial light modulator-based pulse shaper is incorporated to reduce the pulse dispersion and improve temporal resolution. Third, 1.4 numerical aperture objectives for excitation and light collection provide optimal spatial resolution. The result of these improvements is a probe beam that spans 115 nm across the visible region yet maintains a ∼100 fs instrument response at the sample position. We demonstrate the capabilities of this microscope by imaging polystyrene beads in a solution of IR-144 dye, revealing aggregated species at the bead surfaces.

Flexible approach to vibrational sum-frequency generation using shaped near-infrared light.

We describe a new approach that expands the utility of vibrational sum-frequency generation (vSFG) spectroscopy using shaped near-infrared (NIR) laser pulses. We demonstrate that arbitrary pulse shapes can be specified to match experimental requirements without the need for changes to the optical alignment. In this way, narrowband NIR pulses as long as 5.75 ps are readily generated, with a spectral resolution of about 2.5 cm-1, an improvement of approximately a factor of 3 compared to a typical vSFG system. Moreover, the utility of having complete control over the NIR pulse characteristics is demonstrated through nonresonant background suppression from a metallic substrate by generating an etalon waveform in the pulse shaper. The flexibility afforded by switching between arbitrary NIR waveforms at the sample position with the same instrument geometry expands the type of samples that can be studied without extensive modifications to existing apparatuses or large investments in specialty optics.

Miltefosine impacts small molecule transport in Gram-positive bacteria.

Miltefosine (MLT) is an alkylphosphocholine with clinical success as an anticancer and antiparasitic drug. Although the mechanism of action of MLT is highly debated, the interaction of MLT with the membrane, specifically lipid rafts of eukaryotes, is well-documented. Recent reports suggest MLT impacts the functional membrane microdomains in bacteria - regions of the membrane structurally and functionally similar to lipid rafts. There have been conflicting reports, however, as to whether MLT impacts the overall fluidity of cellular plasma membranes. Here, we apply steady-state fluorescence techniques, generalized polarization of laurdan and anisotropy of diphenylhexatriene, to discern how MLT impacts the global ordering and lipid packing of Staphylococcus aureus membranes. Additionally, we investigate how the transport of a range of small molecules is impacted by MLT for S. aureus and Bacillus subtilis by employing time-resolved second harmonic scattering. Overall, we observe MLT does not have an influence on the overall ordering and packing of S. aureus membranes. Additionally, we show that the transport of small molecules across the membrane can be significantly altered by MLT - although this is not the case for all molecules studied. The results presented here illustrate the potential use of MLT as an adjuvant to assist in the delivery of drug molecules in bacteria.

Evidence for wavelike energy transfer through quantum coherence in photosynthetic systems.

Photosynthetic complexes are exquisitely tuned to capture solar light efficiently, and then transmit the excitation energy to reaction centres, where long term energy storage is initiated. The energy transfer mechanism is often described by semiclassical models that invoke 'hopping' of excited-state populations along discrete energy levels. Two-dimensional Fourier transform electronic spectroscopy has mapped these energy levels and their coupling in the Fenna-Matthews-Olson (FMO) bacteriochlorophyll complex, which is found in green sulphur bacteria and acts as an energy 'wire' connecting a large peripheral light-harvesting antenna, the chlorosome, to the reaction centre. The spectroscopic data clearly document the dependence of the dominant energy transport pathways on the spatial properties of the excited-state wavefunctions of the whole bacteriochlorophyll complex. But the intricate dynamics of quantum coherence, which has no classical analogue, was largely neglected in the analyses-even though electronic energy transfer involving oscillatory populations of donors and acceptors was first discussed more than 70 years ago, and electronic quantum beats arising from quantum coherence in photosynthetic complexes have been predicted and indirectly observed. Here we extend previous two-dimensional electronic spectroscopy investigations of the FMO bacteriochlorophyll complex, and obtain direct evidence for remarkably long-lived electronic quantum coherence playing an important part in energy transfer processes within this system. The quantum coherence manifests itself in characteristic, directly observable quantum beating signals among the excitons within the Chlorobium tepidum FMO complex at 77 K. This wavelike characteristic of the energy transfer within the photosynthetic complex can explain its extreme efficiency, in that it allows the complexes to sample vast areas of phase space to find the most efficient path.

Spectroscopic elucidation of uncoupled transition energies in the major photosynthetic light-harvesting complex, LHCII.

Electrostatic couplings between chromophores in photosynthetic pigment-protein complexes, and interactions of pigments with the surrounding protein environment, produce a complicated energy landscape of delocalized excited states. The resultant electronic structure absorbs light and gives rise to energy transfer steps that direct the excitation toward a site of charge separation with near unity quantum efficiency. Knowledge of the transition energies of the uncoupled chromophores is required to describe how the wave functions of the individual pigments combine to form this manifold of delocalized excited states that effectively harvests light energy. In an investigation of the major light-harvesting complex of photosystem II (LHCII), we develop a method based on polarized 2D electronic spectroscopy to experimentally access the energies of the S(0)-S(1) transitions in the chromophore site basis. Rotating the linear polarization of the incident laser pulses reveals previously hidden off-diagonal features. We exploit the polarization dependence of energy transfer peaks to find the angles between the excited state transition dipole moments. We show that these angles provide a spectroscopic method to directly inform on the relationship between the delocalized excitons and the individual chlorophylls through the site energies of the uncoupled chromophores.

Two-dimensional electronic spectroscopy reveals the dynamics of phonon-mediated excitation pathways in semiconducting single-walled carbon nanotubes.

Electronic two-dimensional Fourier transform (2D-FT) spectroscopy is applied to semiconducting single-walled carbon nanotubes and provides a spectral and time-domain map of exciton-phonon assisted excitations. Using 12 fs long pulses, we resolve side-bands above the E(22) transition that correspond with the RBM, G, G', 2G and other multiphonon modes. The appearance of 2D-FT spectral cross-peaks explicitly resolves discrete phonon assisted population transfer that scatters excitations to the E(22) (Γ-pt) state, often through a second-order exciton-phonon coupling process. All 2D-FT peaks exhibit a strong peak amplitude modulation at the G-band period (21 fs) which we show originates from an impulsive stimulated Raman process that populates a ground-state G-band vibrational coherence over a 1.3 ps phonon lifetime.

Small Molecule Sorting: A Fluorescence Study of Microemulsions.

The application of microemulsions to a wide range of industries relies on their ability to solubilize small molecules with vastly different structures. Herein, we use multiple fluorescence techniques to probe ionic (rhodamine 6g, r6g), polar (coumarin 153, c153), nonpolar (diphenylanthracene, DPA), and amphiphilic (laurdan) small molecules in a nonionic, bicontinuous microemulsion of varying hydration. All fluorophores investigated were found to associate with the surfactant region despite their different structures and properties. The hydration of the surfactant layer was found to increase linearly with water addition, but while this initially increases the fluidity of the surfactant layer, fluorescence anisotropy of c153 and r6g indicates a stiffening of the surfactant at water content >60%. This stiffening of the surfactant layer at higher water content correlates with a morphological change in the microemulsion from a bicontinuous structure to droplets. In contrast, the nonpolar DPA shows a change in partitioning as hydration changes, increasing its association with the oil domain. Overall, these studies elucidate not only the capability of these microemulsions to host a range of small molecules in the surfactant layer with tunable position but also the ability to probe the driving force of bulk structural changes in these heterogeneous fluids.

Monitoring membranes: The exploration of biological bilayers with second harmonic generation.

Nature's seemingly controlled chaos in heterogeneous two-dimensional cell membranes stands in stark contrast to the precise, often homogeneous, environment in an experimentalist's flask or carefully designed material system. Yet cell membranes can play a direct role, or serve as inspiration, in all fields of biology, chemistry, physics, and engineering. Our understanding of these ubiquitous structures continues to evolve despite over a century of study largely driven by the application of new technologies. Here, we review the insight afforded by second harmonic generation (SHG), a nonlinear optical technique. From potential measurements to adsorption and diffusion on both model and living systems, SHG complements existing techniques while presenting a large exploratory space for new discoveries.

Phosphate Ions Alter the Binding of Daptomycin to Living Bacterial Cell Surfaces.

Advancements in antibiotic drug design are often hindered by missing information on how these small molecules interact with living cells. The antibiotic, daptomycin, has found clinical success and an emerging resistance, but a comprehensive picture of its mechanism of action has remained elusive. Using a surface-specific spectroscopy technique, second harmonic generation, we are able to quantitatively assess the binding of daptomycin to living cell membranes without the addition of exogenous labels. Our results reveal similar binding affinities for both Gram-positive and Gram-negative bacteria studied, including Escherichia coli. More importantly, we show that the presence of phosphate ions influences the binding of daptomycin to the Gram-positive bacterium Enterococcus faecalis. The role of environmental phosphate has not previously been considered in any proposed mechanism, and its implications are expected to be important in vivo.

Quantum coherence and its interplay with protein environments in photosynthetic electronic energy transfer.

Recent experiments suggest that electronic energy transfer in photosynthetic pigment-protein complexes involves long-lived quantum coherence among electronic excitations of pigments. [Engel et al., Nature, 2007, 446, 782-786.] The observation has led to the suggestion that quantum coherence might play a significant role in achieving the remarkable efficiency of photosynthetic light harvesting. At the same time, the observation has raised questions regarding the role of the surrounding protein in protecting the quantum coherence. In this Perspective, we provide an overview of recent experimental and theoretical investigations of photosynthetic electronic energy transfer paying particular attention to the underlying mechanisms of long-lived quantum coherence and its non-Markovian interplay with the protein environment.

Leaving the Limits of Linearity for Light Microscopy.

Nonlinear microscopy has enabled additional modalities for chemical contrast, deep penetration into biological tissues, and the ability to collect dynamics on ultrafast timescales across heterogenous samples. The additional light fields introduced to a sample offer seemingly endless possibilities for variation to optimize and customize experimentation and the extraction of physical insight. This perspective highlights three areas of growth in this diverse field: the collection of information across multiple timescales, the selective imaging of interfacial chemistry, and the exploitation of quantum behavior for future imaging directions. Future innovations will leverage the work of the studies reviewed here as well as address the current challenges presented.

Probing ligand removal and ordering at quantum dot surfaces using vibrational sum frequency generation spectroscopy.

HYPOTHESIS: Controlling nanomaterial interfaces for emerging technologies has driven the need to understand the molecular species located there; however, challenges arise using traditional analytical techniques to directly characterize the molecular structure and local environments of these interfacial species due to their low relative populations. We hypothesized that vibrational sum frequency generation (vSFG) spectroscopy would be uniquely sensitive to the chemical modification of nanoparticle surfaces that is obscured using traditional bulk sensitive methods. EXPERIMENTS: Octadecylamine ligands were removed from model CdSe quantum dot surfaces using a common precipitation-resuspension procedure with polar protic and aprotic nonsolvents. Vibrational spectra of the ligands at the surface were collected with vSFG to directly probe the ligand ordering and coverage. Photoluminescence (PL), optical absorption, NMR, and mass spectrometry measurements were conducted for comparison. FINDINGS: vSFG was found to be sensitive to subtle changes in ligand disorder over multiple precipitation-resuspension washes, and a limit to the number of ligand molecules removed from the surface and subsequent amount of disorder introduced to their packing was clearly observed. We also find that nonsolvents do not remain associated with the surface after washing.

A new approach to vibrational sum frequency generation spectroscopy using near infrared pulse shaping.

We have developed a multipurpose vibrational sum frequency generation (vSFG) spectrometer that is uniquely capable of probing a broad range of chemical species, each requiring different experimental conditions, without optical realignment. Here, we take advantage of arbitrary near infrared (NIR) waveform generation using a 4f-pulse shaper equipped with a 2D spatial light modulator (SLM) to tailor upconversion pulses to meet sample dependent experimental requirements. This report details the experimental layout, details of the SLM calibration and implementation, and the intrinsic benefits/limitations of this new approach to vSFG spectroscopy. We have demonstrated the competency of this spectrometer by achieving an ∼3-fold increase in spectral resolution compared to conventional spectrometers by probing the model dimethyl sulfoxide/air interface. We also show the ability to suppress nonresonant background contributions from electrode interfaces using time delayed asymmetric waveforms that are generated by the NIR pulse shaper. It is expected that this advancement in instrumentation will broaden the types of samples researchers can readily study using nonlinear surface specific spectroscopies.