Frequency Generation Microscopy Research Articles

Compact oblique-incidence nonlinear widefield microscopy with paired-pixel balanced imaging.

Nonlinear (vibrational) microscopy has emerged as a successful tool for the investigation of molecular systems as it combines label-free chemical characterization with spatial resolution on the sub-micron scale. In addition to the molecular recognition, the physics of the nonlinear interactions allows in principle to obtain structural information on the molecular level such as molecular orientations. Due to technical limitations such as the relatively complex imaging geometry with the required oblique sample irradiation and insufficient sensitivity of the instrument this detailed molecular information is typically not accessible using widefield imaging. Here, we present, what we believe to be, a new microscope design that addresses both challenges. We introduce a simplified imaging geometry that enables the measurement of distortion-free widefield images with free space oblique sample irradiation achieving high spatial resolution (∼1 µm). Furthermore, we present a method based on a paired-pixel balanced detection system for sensitivity improvement. With this technique, we demonstrate a substantial enhancement of the signal-to-noise ratio of up to a factor of 10. While both experimental concepts presented in this work are very general and can, in principle, be applied to various microscopy techniques, we demonstrate their performance for the specific case of heterodyned, sum frequency generation (SFG) microscopy.

Imaging Orientation of a Single Molecular Hierarchical Self-Assembled Sheet: The Combined Power of a Vibrational Sum Frequency Generation Microscopy and Neural Network.

In this work, we determined the tilt angles of molecular units in hierarchical self-assembled materials on a single-sheet level, which were not available previously. This was achieved by developing a fast line-scanning vibrational sum frequency generation (VSFG) hyperspectral imaging technique in combination with neural network analysis. Rapid VSFG imaging enabled polarization resolved images on a single sheet level to be measured quickly, circumventing technical challenges due to long-term optical instability. The polarization resolved hyperspectral images were then used to extract the supramolecular tilt angle of a self-assembly through a set of spectra-tilt angle relationships which were solved through neural network analysis. This unique combination of both novel techniques offers a new pathway to resolve molecular level structural information on self-assembled materials. Understanding these properties can further drive self-assembly design from a bottom-up approach for applications in biomimetic and drug delivery research.

Chemical Imaging of Lipid Segregation: Determining Different Length Scales of Heterogeneity with Compressive-Sensing Sum Frequency Generation Microscopy and Brewster Angle Microscopy

Membranes of various phospholipids may separate into different domains at micrometer length scales at the air-water interface. A significant challenge is to visualize the molecular organization and obtain chemical information on this surface. Langmuir-Blodgett monolayers of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) on fused silica were investigated with compressive sensing sum frequency generation (SFG) microscopy. Before and after mixing, SFG spectra for DSPC and DLPC revealed significant differences, indicating a structural change. Brewster angle microscopy images show phase separations that directly correlate the morphology of phospholipid mixtures with SFG images. Exploiting vibrational contrast in SFG images, exchange between the two phases was discovered, and quantitative thermodynamic analysis of lipid compositions in liquid-condensed and liquid-expanded (LE) phases was provided. Local SFG spectra reveal significant differences from one another, indicating the heterogeneity resulting from domain areas with distinct molecular orientation and conformation. Further heterogeneity across the domain boundary was presented on a finer scale, revealing an effect on DLPC due to the condensed phase DSPC, and the terminal methyl of DLPC perturbs the ordering of DSPC in the LE phase. This work demonstrates the heterogeneity of a two-dimensional binary lipid system mainly due to the aliphatic chain length and transition temperature difference.

Vibrational Ground-State depletion for enhanced resolution sum frequency generation microscopy

The study of surfaces, which provides unique information on chemical and physical properties, has led to the development of specialized tools including Sum Frequency Generation (SFG) spectroscopy. To map the chemical heterogeneity across the surface plane, the SFG spectroscopic imaging has been developed. SFG imaging has proven to be a useful tool for studying surface specific species through their vibrational signatures. However, the effective resolution of the imaging is limited to micrometer length scales often by the diffraction limit of the mid-IR wavelengths deployed in vibrationally sensitive SFG imaging. Therefore, many interesting sub-micron features are out of reach for such imaging techniques. This article introduces a technique to achieve super-resolution using ground state depletion from a structured area around the focal-spot of a raster-scan imaging scheme. The proof-of-concept results are presented using hydrogen terminated silicon (Si(111)) as sample surface with results indicating a 3-fold resolution improvement.

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms.

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

Spectroscopic imaging of surfaces-Sum frequency generation microscopy (SFGM) combined with compressive sensing (CS) technique.

Surface chemistry is notoriously difficult to study, in part, due to the decreased number of molecules that contribute to the properties compared to the bulk phase but often has significant effects on the chemical activity of the material. This is especially true in topics such as corrosion, catalysis, wetting, and many others in nature and industry. Sum frequency generation (SFG) spectroscopy was developed for interface studies due to its high molecular selectivity and surface sensitivity, which is quite useful to study the effects of structural inhomogeneity in microscopy. Compressive sensing (CS) combined with SFG spectroscopy minimizes the imaging time while still producing quality images. Selected systems are presented here to demonstrate the capability of CS-SFG microscopy. CS-SFG microscopy successfully distinguished the static monolayer molecular mixtures, the orientations and adsorption of adsorbed molecules by the dip-coating technique, and the localized CO behaviors on polycrystalline Pt electrodes. Further discussion includes dynamic imaging as a future direction in CS-SFG microscopy. As materials and surfaces become more complex, imaging with chemical contrast becomes indispensable to understanding their performance and CS-SFG microscopy seems highly beneficial in this respect.

Chemical Imaging of Surfaces with Sum Frequency Generation Vibrational Spectroscopy.

Surface chemistry is a key area of study in the chemical sciences, and many system properties are dominated by the chemistry at the interface between two bulk media. While the interface may have a large influence on the system behavior, there are relatively few molecules at the interface compared to the bulk; thus, probing their unique properties has become a specialized field in physical chemistry. In addition to the heterogeneous phase chemistry, surfaces also present spatial heterogeneity (Chemistry in Two Dimensions). This 2D chemistry affects the properties as much as the heterogeneous phases. If we consider the Cartesian z-axis as defining the dimension across the interface between the two bulk phases, then the x-y plane is the 2D region of the surface. We might even consider that the majority of surface chemistry has been concerned with this z-dimension, i.e., surface structure, partition excess, thermodynamics, etc. relative to the bulk, where the 2D distribution was only considered on average. This treatment is understandable since few techniques provide the spatial and chemical resolution needed to deduce the effects of 2D heterogeneity on the surface properties. It is desirable to use an all-optical technique for interface studies because the optical methods provide the chemical specificity through spectroscopy. Also, the use of second-order spectroscopy is typically surface-sensitive without background subtractions or enhancement mechanisms that could limit the range of systems to be investigated.In this Account, the development and selected results of sum frequency generation microscopy and its contributions to the surface chemistry are presented. Sum frequency generation (SFG) provides a unique probe for studying surface chemistry in ambient conditions with surface specificity. SFG provides image contrast based on multiple-chemically important-mechanisms such as chemical functional groups, molecular orientation, surface concentration, molecular conformation, local electric fields, among others. To understand the spatial distribution of heterogeneous chemistry, multiple microscopy methods have been developed which utilize the SFG process to yield spatial information with chemical sensitivity. These spectroscopic-microscopies come with unique advantages as well as challenges. Multiple solutions have been developed in this field to overcome the challenges and improve the advantages. In this Account, some of the leading SFG surface microscopies for surface studies are introduced. Initially, direct imaging of the SFG signal onto a CCD camera provided spatially and spectrally resolved imaging of monolayers on surfaces. However, to speed up the imaging process, the technique of compressive sensing was applied to SFG imaging. Most recently the use of machine learning methods and target factor analysis have improved the quality and acquisition speed of SFG images.

Illustrating Interfacial Interaction between Honey Bee Venom Phospholipase A2 and Supported Negatively Charged Lipids with Sum Frequency Generation and Laser Scanning Confocal Microscopy.

Phospholipase A2 is an important enzyme species which can widely be found in animals, plants, bacteria, and so on. A large number of studies have shown that phospholipase A2 is highly catalytic toward the lipids. Here, sum frequency generation (SFG) vibrational spectroscopy and laser scanning confocal microscopy (LSCM) were applied to study the interaction between honey bee venom phospholipase A2 (bvPLA2) and the negatively charged DPPG bilayer. In both cases without and with the calcium ions (Ca2+), the bvPLA2 molecules were adsorbed onto the outer leaflet surface with the orientational order, and the adsorbed bvPLA2 molecules damaged the order of the packed outer leaflet. In comparison to the case without Ca2+, the addition of Ca2+ can accelerate the attaching process of bvPLA2 to the outer leaflet surface and decelerate the process of damaging the outer leaflet order. The experimental result also confirmed, with the help of the Ca2+, the DPPG molecules in the outer leaflet were hydrolyzed, with both hydrolysates, that is, lysophospholipids and fatty acids, remaining at the interface, showing a distinct difference from previous published literatures regarding neutral lipids [Phys. Chem. Chem. Phys. 2018, 20, 63-67] and PLA1 [Langmuir 2019, 35, 12831-12838].

Inhomogeneity of Cellulose Microfibril Assembly in Plant Cell Walls Revealed with Sum Frequency Generation Microscopy.

Sum frequency generation (SFG) vibrational spectroscopy can selectively detect and analyze noncentrosymmetric components interspersed in amorphous matrices; this principle has been used for studies of nanoscale structure and mesoscale assembly of cellulose in plant cell walls. However, the spectral information averaged over a large area or volume cannot provide regiospecific or tissue-specific information of different cells in plants. This study demonstrates spatially resolved SFG analysis and imaging by combining a broad-band SFG spectroscopy system with an optical microscope. The system was designed to irradiate both narrow-band 800 nm and broad-band tunable IR beams through a single reflective objective lens, but from opposite sides of the surface normal direction of the sample. The developed technique was used to reveal inhomogeneous distributions of cellulose microfibrils within single cell walls, such as cotton fibers and onion epidermis as well as among different tissues in Arabidopsis inflorescence stems and bamboo culms. SFG microscopy can be used for vibrational spectroscopic imaging of other biological systems in complement to conventional Fourier transform infrared spectroscopy and confocal Raman microscopy.

Chemical Imaging of Self-Assembled Monolayers on Copper Using Compressive Hyperspectral Sum Frequency Generation Microscopy.

Sum frequency generation microscopy is a label-free optical imaging technique with intrinsic molecular vibrational contrast for surface studies. Recent developments of compressive sensing broad-band hyperspectral SFG microscopy have demonstrated the potential application for imaging monolayer at metal surfaces with micrometer spatial resolution. Here is presented the capability of chemical imaging of spatially patterned monolayers of 1-octadecanethiol (ODT) and 16-methoxy-1-hexadecanethiol (MeOHT) molecules assembled on a copper surface. The spatial distributions of the monolayer with vibrational-spectral contrast are well-demonstrated at different frequency regions through reconstruction of the hypercube using a 3-dimensional total variation regularization algorithm (3DTV). Spatial-chemical distributions of each component are also reconstructed directly from the compressive measurements by endmember unmixing (CEU) scheme. Compared to 3DTV algorithm, the reconstruction from CEU shows spatial distribution of each component on the surfaces, and demonstrates the ability to characterize the domains of mixed-molecules on surfaces.

Nanostructuring of Au(111) during the Adsorption of an Aromatic Isocyanide from Solution.

We present a combined vibrational and morphological characterization of the self-assembly of 1,4-phenylene-diisocyanide (PDI) on Au(111) from methanol solution. Vibrational sum frequency generation (vSFG) and scanning tunneling microscopy (STM) have been applied to determine the adsorption geometry of the PDI-Au adatom complexes as well as the morphological transformations of the Au(111) substrate upon SAM formation from solutions with PDI concentrations in the μM to mM range. At low concentration/coverage, PDI adsorbs in flat adsorption geometry, with both isocyanide groups attached to Au adatoms on the Au(111) surface. Transformation to a standing-up phase is observed with increasing concentration/coverage. In contrast to findings for PDI adsorbed in ultrahigh vacuum, PDI does not form a long-range-ordered monolayer phase when adsorbed from solution. In addition, the Au(111) surface is subjected to structural modifications. Au vacancy islands and ad-islands, which are typical substrate defects formed during the self-assembly of aromatic thiols on Au(111), are also created during PDI adsorption from solution. At low PDI concentration, the Au vacancy islands and ad-islands are found at specific sites mediated by the herringbone reconstruction of the Au(111) surface, giving rise to long-range-ordered structures. These structures do not form during UHV adsorption of PDI on Au(111) nor has a similar ordering effect been observed for any related thiol-SAM system investigated so far.

Modeling of Infrared-Visible Sum Frequency Generation Microscopy Images of a Giant Liposome.

The article explores the theory of infrared-visible sum frequency generation microscopy of phospholipid envelopes with dimensions larger than the wavelength of the nonlinear emission. The main part of the study concerns derivation and accounting for the contributions of effective nonlinear responses specific to sites on the surfaces of a bilayer envelope and their dependence on polarization condition and experimental geometry. The nonlinear responses of sites are mapped onto the image plane according to their emission directions and the numerical aperture of a sampling microscope objective. According to the simulation results, we discuss possible approaches to characterize the shape of the envelope, to extract molecular hyperpolarizabilities, and to anticipate possible heterogeneity in envelope composition and anisotropy of the environment proximal to the envelope. The modeling approach offers a promising analytic facility to assist connecting microscopy observations in engineered liposomes, cellular envelopes, and subcellular organelles of relatively large dimensions to molecular properties, and hence to chemistry and structure down to available spatial resolution.

Elucidation of Compression-Induced Surface Crystallization in Amorphous Tablets Using Sum Frequency Generation (SFG) Microscopy.

To investigate the effect of compression on the crystallization behavior in amorphous tablets using sum frequency generation (SFG) microscopy imaging and more established analytical methods. Tablets containing neat amorphous griseofulvin with/without excipients (silica, hydroxypropyl methylcellulose acetate succinate (HPMCAS), microcrystalline cellulose (MCC) and polyethylene glycol (PEG)) were prepared. They were analyzed upon preparation and storage using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM) and SFG microscopy. Compression-induced crystallization occurred predominantly on the surface of the neat amorphous griseofulvin tablets, with minimal crystallinity being detected in the core of the tablets. The presence of various types of excipients was not able to mitigate the compression-induced surface crystallization of the amorphous griseofulvin tablets. However, the excipients affected the crystallization rate of amorphous griseofulvin in the core of the tablet upon compression and storage. SFG microscopy can be used in combination with ATR-FTIR spectroscopy and SEM to understand the crystallization behaviour of amorphous tablets upon compression and storage. When selecting excipients for amorphous formulations, it is important to consider the effect of the excipients on the physical stability of the amorphous formulations.

Unified Theory for Polarization Analysis in Second Harmonic and Sum Frequency Microscopy

A unified theoretical framework for the recovery of second-order nonlinear susceptibility tensors and sample orientations from polarization-dependent second harmonic generation and sum frequency generation microscopy was developed. Jones formalism was extended to nonlinear optics and was used to bridge the experimental observables and the local-frame tensor elements. Four commonly used experimental architectures were explicitly explored, including polarization rotation with no postsample optics, polarization-in polarization-out measurement, and polarization modulation with and without postsample optics. Polarization-dependent second harmonic generation measurement was performed on Z-cut quartz and the local-frame tensor elements were calculated. The recovered tensor elements agree with the expected values dictated by symmetry.

Resolving capacity of infrared–visible sum frequency generation microscopy to address discrete structural realizations of a protein at interface

In this article, we address theory and explore possible implications of infrared–visible sum frequency generation microscopy imaging for structural and orientational analysis of a single polypeptide at a phospholipid membrane interface. The suggested structural analysis is based on detection of amplitude (intensity) images specific to a single protein under a given orientation, rather than on detection of spectral dispersions of responses from orientationally averaged ensembles. To establish the principle and model implications, we perform quantum‐mechanical calculations of nonlinear responses of a pentadecapeptide helix and a penta‐pentadecapeptide helical dimer, the secondary structure of which is identical to that of gramicidin. Using properties of calculated amide I modes, we account polarization settings for the mixing fields, under the considered experimental geometry, to extract images specific to χYYY, χXYY, χXYX, χYZY, and χXYZ nonlinearities. If extracted in dependence on the angle of rotation of the sample holder, the reconstructed images provide information, which may allow identifying structure and orientation of the proteins under study. Hence, the approach may help addressing catalytic properties of proteins, whose role is to attune chemical permeability of membranes they associate with. Copyright © 2016 John Wiley & Sons, Ltd.

Grain Structures and Boundaries on Microcrystalline Copper Covered with an Octadecanethiol Monolayer Revealed by Sum Frequency Generation Microscopy.

An octadecanethiol (ODT) self-assembled monolayer on microcrystalline copper was investigated by sum frequency generation (SFG) imaging microscopy. The crystal grain and grain boundaries of the copper surface were mapped in the SFG image based on the strong brightness contrast of the SFG signal across the boundary. Local SFG spectra reveal significant difference with each other as well as the average SFG spectra, indicating the heterogeneity of the copper surface resulting from copper grains with distinct crystallographic facets and orientations. It is demonstrated that the SFG signal of crystalline domain areas contains azimuthal anisotropy with respect to the plane of incidence. In addition, the statistical orientation analyses of amplitude ratio of CH3-sym/CH3-asym and corresponding contour maps imply that the orientation of ODT molecules is affected by the underlying copper.

Sum frequency generation image reconstruction: aliphatic membrane under spherical cap geometry.

The article explores an opportunity to approach structural properties of phospholipid membranes using Sum Frequency Generation microscopy. To establish the principles of sum frequency generation image reconstruction in such systems, at first approach, we may adopt an idealistic spherical cap uniform assembly of hydrocarbon molecules. Quantum mechanical studies for decanoic acid (used here as a representative molecular system) provide necessary information on transition dipole moments and Raman tensors of the normal modes specific to methyl terminal - a typical moiety in aliphatic (and phospholipid) membranes. Relative degree of localization and frequencies of the normal modes of methyl terminals make nonlinearities of this moiety to be promising in structural analysis using Sum Frequency Generation imaging. Accordingly, the article describes derivations of relevant macroscopic nonlinearities and suggests a mapping procedure to translate amplitudes of the nonlinearities onto microscopy image plane according to geometry of spherical assembly, local molecular orientation, and optical geometry. Reconstructed images indicate a possibility to extract local curvature of bilayer envelopes of spherical character. This may have practical implications for structural extractions in membrane systems of practical relevance.

Image Contrast in Sum Frequency Generation Microscopy Based on Monolayer Order and Coverage

Sum frequency generation imaging microscopy (SFGIM) was used to study a binary system of self-assembled monolayers (SAMs) on evaporated gold. A two-step process that consists of microcontact printing (μCP) and the solution backfilling method is used to generate a sample surface with two distinct SAMs domains. The high contrast chemical identification and the homogeneity map of the SAMs for two different head groups are revealed by processing the three-dimensional images (xy-surface and infrared wavenumbers) obtained from SFGIM. For conformation order analysis, the amplitude ratios of the methylene and methyl symmetric resonance obtained by a nonlinear curve fit of the spatially and spectrally resolved SFG images are used to reconstruct spatial image maps. The conformation order map shows the distribution of gauche defects in SAMs with improved contrast across monodentate and bidentate alkanethiol monolayer regions. The results show that variation of the head group attached on the surface dominantly influences the conformation disorder and surface coverage of the molecules, while the terminal methyl group in both monodentate and bidentate SAMs shows a similar orientation angle.

Hydrogen desorption from a Si(111)1×1 surface studied by sum frequency generation spectroscopy and microscopy

We studied isothermal hydrogen desorption from a Si(111)1×1 surface at temperatures of ~711, 732, 752, and 771 K by observing sum frequency generation (SFG). Peak shift and width broadening of the Si‐H vibration occurred with increase of heating time. We also obtained SFG intensity images of monohydride species on the surface after heating for 10 s at temperatures of 592, 625, 666, 728, and 752 K. The SFG intensity images with a spatial resolution of 5 µm indicated that the hydrogen desorption was homogeneous. This result suggests that the hydrogen atoms diffused homogeneously on the Si(111)1×1 surface. Copyright © 2011 John Wiley & Sons, Ltd.

Sum Frequency Generation Microscopy Study of Cellulose Fibers

Sum frequency generation (SFG) microscopy images of cotton cellulose fibers were observed at the infrared wavenumber of ~ 2945 cm(-1) and with a spatial resolution of 2 μm. Domains of different cellulose microfibril bunches were observed and they showed different second-order nonlinear responses. The intensity of the peak of the asymmetric CH(2) stretching mode at 2945 cm(-1) depended strongly on the orientation of the electric fields of the incident visible and infrared light with respect to the cellulose fiber axis. The second-order nonlinear susceptibility arising from the chirality in the cellulose structure was found to be dominant. The SFG of the cross section of the cellulose fiber was relatively weak and showed a different spectrum from that measured from the side of the fiber axis.