Sum Frequency Generation Imaging Research Articles

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.

Direct imaging of electric field behavior in 2,7-diphenyl[1]benzothieno[3,2-b][1]benzothiophene organic field-effect transistors by sum-frequency generation imaging microscopy.

Sum-frequency generation imaging microscopy combined with compressive-sensing (CS-SFG) is a powerful micro-spectroscopic technique for probing interfaces and surfaces with a spatial resolution where contrast is based on the chemical functional groups. We reported the use of the CS-SFG technique to probe the electric field due to charge accumulation and the internal electric field in operating organic field-effect transistors (OFETs) with the aluminum oxide and octadecylphosphonic acid (ODPA) self-assembled monolayer as the gate dielectric layer and 2,7-diphenyl[1]benzothieno[3,2-b][1]benzothiophene (DPh-BTBT) as the semiconductor layer. In addition, the electric field behavior was discussed by a difference in the electric field induced SFG intensity between the open-circuit and the voltage application conditions. The SFG peak of CH stretching mode derived from methyl groups of ODPA and phenyl groups of DPh-BTBT could be observed at each interface of ODPA/DPh-BTBT or DPh-BTBT/Au, respectively. Moreover, the electric field induced SFG coming from ODPA/DPh-BTBT shows the presence of intense electric field due to charge injection and accumulation near the drain and source electrode edges under the operation of OFETs. Our studies show that the electric field-induced SFG imaging technique is useful for probing the local electric field distribution or charge accumulation behavior in OFETs under operating conditions.

Multicolor Chemical Imaging by Sum Frequency Generation Imaging Microscopy of Monolayers on Metal Surfaces

Sum frequency generation imaging microscopy (SFG-IM) is a unique surface-specific technique that can detect the spatial distributions of differing monolayer species based on chemical contrast. Here...

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.

Monitoring the morphological evolution of giant vesicles by azo dye-based sum-frequency generation (SFG) microscopy

In the present work, dye-based sum-frequency generation (SFG) imaging using sodium 4-[4-(dibutylamino)phenylazo]benzenesulfonate (butyl orange, BO) as a new non-fluorescent specific azo dye is employed to monitor the morphological evolution of giant vesicles (GVs). After loading BO to the membrane of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) single-component GVs, the outermost membranes were clearly visualized using SFG microscopy, which provided images of the distinct outer and inner faces of the lipid bilayers. In addition, SFG-active vesicles were detected also inside the GVs, depending on the dye concentrations. The dye-based SFG imaging technique provided experimental evidence that these oligolamellar vesicles containing an SFG-active interior had been formed after BO loading. The formation process of the oligolamellar vesicles with inner SFG-active vesicles was successfully monitored, and their formation mechanism was discussed.

Sum Frequency Generation Imaging Microscopy of Self-Assembled Monolayers on Metal Surfaces: Factor Analysis of Mixed Monolayers

Sum frequency generation (SFG) images of microcontact patterned self-assembled alkanethiol monolayers on metal surfaces were analyzed by factor analysis (FA) to determine the spatial distribution of the patterned monolayers over the images. Additionally, each significant abstract factor produced by FA was assessed to determine the information contained within it. These results indicate that FA of the SFG spectra is a promising method to determine the composition and identities of mixed alkanethiol systems that show different vibrational spectra and image contrast. Factor analysis has successfully been applied to SFG images obtained with low signals, which reduces the time required for full spectral SFG imaging.

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.

Multimodal Nonlinear Optical Imaging for Sensitive Detection of Multiple Pharmaceutical Solid-State Forms and Surface Transformations.

Two nonlinear imaging modalities, coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG), were successfully combined for sensitive multimodal imaging of multiple solid-state forms and their changes on drug tablet surfaces. Two imaging approaches were used and compared: (i) hyperspectral CARS combined with principal component analysis (PCA) and SFG imaging and (ii) simultaneous narrowband CARS and SFG imaging. Three different solid-state forms of indomethacin-the crystalline gamma and alpha forms, as well as the amorphous form-were clearly distinguished using both approaches. Simultaneous narrowband CARS and SFG imaging was faster, but hyperspectral CARS and SFG imaging has the potential to be applied to a wider variety of more complex samples. These methodologies were further used to follow crystallization of indomethacin on tablet surfaces under two storage conditions: 30 °C/23% RH and 30 °C/75% RH. Imaging with (sub)micron resolution showed that the approach allowed detection of very early stage surface crystallization. The surfaces progressively crystallized to predominantly (but not exclusively) the gamma form at lower humidity and the alpha form at higher humidity. Overall, this study suggests that multimodal nonlinear imaging is a highly sensitive, solid-state (and chemically) specific, rapid, and versatile imaging technique for understanding and hence controlling (surface) solid-state forms and their complex changes in pharmaceuticals.

Raman Microscopy: A Noninvasive Method to Visualize the Localizations of Biomolecules in the Cornea.

In vivo and in situ visualization of biomolecules without pretreatment will be important for diagnosis and treatment of ocular disorders in the future. Recently, multiphoton microscopy, based on the nonlinear interactions between molecules and photons, has been applied to reveal the localizations of various molecules in tissues. We aimed to use multimodal multiphoton microscopy to visualize the localizations of specific biomolecules in rat corneas. Multiphoton images of the corneas were obtained from nonlinear signals of coherent anti-Stokes Raman scattering, third-order sum frequency generation, and second-harmonic generation. The localizations of the adhesion complex-containing basement membrane and Bowman layer were clearly visible in the third-order sum frequency generation images. The fine structure of type I collagen was observed in the corneal stroma in the second-harmonic generation images. The localizations of lipids, proteins, and nucleic acids (DNA/RNA) was obtained in the coherent anti-Stokes Raman scattering images. Imaging technologies have progressed significantly and been applied in medical fields. Optical coherence tomography and confocal microscopy are widely used but do not provide information on the molecular structure of the cornea. By contrast, multiphoton microscopy provides information on the molecular structure of living tissues. Using this technique, we successfully visualized the localizations of various biomolecules including lipids, proteins, and nucleic acids in the cornea. We speculate that multiphoton microscopy will provide essential information on the physiological and pathological conditions of the cornea, as well as molecular localizations in tissues without pretreatment.

Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy

In this study, we used spectrally focused coherent anti-Stokes Raman scattering (spCARS) microscopy assisted by sum-frequency generation (SFG) to monitor the variations in the structural morphology and molecular vibrations of a live muscle of Caenorhabditis elegans. The subunits of the muscle sarcomeres, such as the M-line, myosin, dense body, and α-actinin, were alternatively observed using spCARS microscopy for different sample orientations, with the guidance of a myosin positional marker captured by SFG microscopy. Interestingly enough, the beam polarization dependence of the spCARS contrasts for two parallel subunits (dense body and myosin) showed a ~90° phase difference. The chemically sensitive spCARS spectra induced by the time-varying overlap of two pulses allowed (after a robust subtraction of the non-resonant background using a modified Kramers–Krönig transformation method) high-fidelity detection of various genetically modified muscle sarcomeres tuned to the C-H vibration (2800–3100 cm−1). Conversely, SFG image mapping assisted by phase-retrieved spCARS spectra also facilitated label-free monitoring of the changes in the muscle content of C. elegans that are associated with aging, based on the hypothesis that the C-H vibrational modes could serve as qualitative chemical markers sensitive to the amount and/or structural modulation of the muscle.

A Method For Achieving Super Resolution Vibrational Sum-Frequency Generation Microscopy By Structured Illumination

A scheme was proposed to achieve wide-field vibrational sum-frequency generation (SFG) microscopy with subdiffraction-limited resolution in one dimension. In this approach, samples are illuminated with a structured visible field and a uniform IR field to induce vibrational sum-frequency generation. With five raw images acquired at five different phases of the visible stripe pattern, a super-resolved vibrational SFG (SR-SFG) image with the resolution triple to that of the original can be reconstructed. Theoretical framework describing the coherent image formation and reconstruction scheme for the SR-SFG imaging system was derived and carried out with numerical simulations to investigate its imaging performance. With a typical imaging system, the lateral resolution was improved from 390 nm of the conventional SFG imaging system, to around 130 nm of the SR-SFG imaging system.

Surface-Induced Heterogeneity Analysis of an Alkanethiol Monolayer on Microcrystalline Copper Surface Using Sum Frequency Generation Imaging Microscopy

An octadecanethiol (ODT) self-assembled monolayer on microcrystalline copper is investigated by sum frequency generation (SFG) imaging microscopy and electron backscattering diffraction (EBSD). The strong SFG signal contrast across the domain boundary indicates the existence of grain structures on copper surface, which is further verified by EBSD measurements. The nonresonant contribution of SFG response shows anisotropy with respect to the in-plane rotation of the sample relative to the surface normal and varies in each crystal domain area. The resonant contribution of the monolayer, such as the amplitude ratio of CH3-sym/CH3-asym, is azimuthally nearly isotropic. Since the zzz tensor component of nonlinear susceptibility dominates, the resonant part of SFG spectra on the metal surface does not show any anisotropy. Further, a strong correlation between the local metal structures with the top monolayer packing behaviors is identified based on the statistical distribution analysis. Using the methyl group a...

Roles of oxygen for methanol adsorption on polycrystalline copper surface revealed by sum frequency generation imaging microscopy

The adsorption of atmospheric pressure methanol on the polycrystalline copper surface has been studied by a combination of sum frequency generation imaging microscopy (SFGIM) and temperature programmed desorption (TPD). Methoxy species can be generated by exposing the polycrystalline copper surface to methanol vapor at room temperature. SFGIM results demonstrate that oxygen promotes the surface adsorption of methanol and the increase in the amount of methoxy produced on copper surface. SFGIM orientation analysis suggests the methoxy monolayer is oriented closer to the surface normal with introduction of oxygen. Employing the image statistical analysis approach, the heterogeneities and conformation distribution of methoxy monolayers on copper surface with and without oxygen adsorption are compared. These results illustrate SFGIM indeed could provide more insight for understanding the heterogeneous metal/metal oxide surface in the molecular level.

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.

Polarization-sensitive sum-frequency generation microscopy of collagen fibers.

Point-scanning sum-frequency generation (SFG) microscopy enables the generation of images of collagen I fibers in tissues by tuning into specific vibrational resonances of the polypeptide. It is shown that when collagen-rich tissues are visualized near the 2954 cm(-1) stretching vibration of methylene groups, the SFG image contrast is higher compared to the contrast seen in nonresonant second-harmonic generation (SHG) imaging. Polarization and spectrally resolved analysis of the SFG signal as a function of fiber orientation in the CH-stretching range of the vibrational spectrum enabled a comparative characterization of the achiral tensor elements of collagen's second-order susceptibility. This analysis reveals that selected on-resonance tensor elements are enhanced over other elements, giving rise to a much stronger anisotropy ρ of the signal for SFG (ρ ≈ 15) compared to SHG (ρ ≈ 3). The improved anisotropy of the vibrationally resonant signal contributes to the higher contrast seen in the SFG tissue images.

Tip-induced deformation of a phospholipid bilayer: theoretical perspective of sum frequency generation imaging.

The paper addresses theory of Sum Frequency Generation imaging of an atomic force microscopy tip-induced deformation of a bilayer phospholipid membrane deposited over a pore: known as a nano-drum system. Image modeling employed nonlinearities of the normal modes specific to hydrocarbon terminal methyls, which are distributed about the deformed surfaces of inner and outer leaflets. The deformed profiles are according to the solutions of shape equation for Canham-Helfrich Hamiltonian accounting properties of four membranes, which differ in elasticity and adhesion. The results indicate that in continuous deformed surfaces, the difference in the curvature of the outer and inner leaflets dominates in the imaged nonlinearity. This is different comparing to the results for a perfect bilayer spherical cap system (the subject of previous study), where nonlinear image response is dominated by the mismatch of the inner and outer leaflets' surface areas (as projected to the image plane) at the edge of perfectly spherical structure. The results of theoretical studies, here, demonstrate that Sum Frequency Generation imaging in continuous and deformed bilayer surfaces are helpful to address curvature locally and anticipate mechanical properties of membrane. The articles discuss applicability and practical limitations of the approach. Combination of Atomic Force Microscopy and Sum Frequency Generation imaging under controlled tip-induced deformation provides a good opportunity to probe and test membranes physical properties with rigor of adopted theory.

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.

Monitoring Localized Initial Atmospheric Corrosion of Alkanethiol-Covered Copper Using Sum Frequency Generation Imaging Microscopy: Relation between Monolayer Properties and Cu2O Formation

In order to evaluate the interfacial monolayer order on copper surfaces in two-dimensions, the tilt angle distribution and orientation-conformation correlation maps were investigated using sum frequency generation imaging microscopy (SFGIM). In this paper, these methods of analyses were further utilized in evaluating the effect of the formation of a thin oxide film on the monolayer order of octadecanethiol (ODT) deposited on Cu. By chemically stripping the native oxide of the copper surface using amidosulfonic acid (ASA), the spontaneous atmospheric oxidation of Cu from an initial reduced state was observed as a change in the nonresonant phase response of the sum frequency generation (SFG) spectral line shape obtained from the SFGIM setup configured with a 1064 nm probe beam. Statistical calculations and contour maps for the CH-stretch modes of the terminal methyl and methylene group showed the overall decrease in the mean tilt angle and increase in the amount of alkyl gauche defects as a result of the ox...

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.

Scale Dependence of the Orientation and Conformation Distribution Analysis of a Molecular Monolayer Using Sum Frequency Generation Imaging Microscopy

A systematic approach of experimentally measuring the average orientation and distribution of octadecanethiol (ODT) monolayer on gold was performed by using sum frequency generation imaging microscopy (SFGIM). By constructing maps from region of interests (ROIs) of different length scales, measuring from 5 to 1000 μm, two-dimensional domains could be distinguished to contain less-oriented monolayers. By generating the tilt angle distributions of the scaled ROIs, SFGIM determines the experimental orientation, θ, and distribution width, σ0, as dictated by the SFGIM resolution limit. Using the same statistical methods, the heterogeneities of ODT monolayers on Au and Cu were compared. The quantitative analysis of these orientation parameters is essential for future work on characterizing monolayer integrity on more complex surfaces such as reactive metals and alloys. Composite maps that merge orientation and conformational information were demonstrated as potential tools for spectromicroscopic measurement of the reactivity of metal substrates to surface processes such as corrosion and wettability.