Sum Frequency Generation Microscopy Research Articles

Spiral packing and chiral selectivity in model membranes probed by phase-resolved sum-frequency generation microscopy

Since the lipid raft model was developed at the end of the last century, it became clear that the specific molecular arrangements of phospholipid assemblies within a membrane have profound implications in a vast range of physiological functions. Studies of such condensed lipid islands in model systems using fluorescence and Brewster angle microscopies have shown a wide range of sizes and morphologies, with suggestions of substantial in-plane molecular anisotropy and mesoscopic structural chirality. Whilst these variations can significantly alter many membrane properties including its fluidity, permeability and molecular recognition, the details of the in-plane molecular orientations underlying these traits remain largely unknown. Here, we use phase-resolved sum-frequency generation microscopy on model membranes of mixed chirality phospholipid monolayers to fully determine the three-dimensional molecular structure of the constituent micron-scale condensed domains. We find that the domains possess curved molecular directionality with spiralling mesoscopic packing, where both the molecular and spiral turning directions depend on the lipid chirality, but form structures clearly deviating from mirror symmetry for different enantiomeric mixtures. This demonstrates strong enantioselectivity in the domain growth process and indicates fundamental thermodynamic differences between homo- and heterochiral membranes, which may be relevant in the evolution of homochirality in all living organisms.

High spatial resolution collinear chiral sum-frequency generation microscopy

High spatial resolution collinear chiral sum-frequency generation microscopy

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue 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.

Revealing the Molecular Physics of Lattice Self-Assembly by Vibrational Hyperspectral Imaging.

Lattice self-assemblies (LSAs), which mimic protein assemblies, were studied using a new nonlinear vibrational imaging technique called vibrational sum-frequency generation (VSFG) microscopy. This technique successfully mapped out the mesoscopic morphology, microscopic geometry, symmetry, and ultrafast dynamics of an LSA formed by β-cyclodextrin (β-CD) and sodium dodecyl sulfate (SDS). The spatial imaging also revealed correlations between these different physical properties. Such knowledge shed light on the functions and mechanical properties of LSAs. In this Feature Article, we briefly introduce the fundamental principles of the VSFG microscope and then discuss the in-depth molecular physics of the LSAs revealed by this imaging technique. The application of the VSFG microscope to the artificial LSAs also paved the way for an alternative approach to studying the structure-dynamic-function relationships of protein assemblies, which were essential for life and difficult to study because of their various and complicated interactions. We expect that the hyperspectral VSFG microscope could be broadly applied to many noncentrosymmetric soft materials.

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.

Phase-Sensitive Vibrationally Resonant Sum-Frequency Generation Microscopy in Multiplex Configuration at 80 MHz Repetition Rate.

Vibrationally resonant sum-frequency generation (VR SFG) microscopy is an advanced imaging technique that can map out the intensity contrast of infrared and Raman active vibrational modes with micron to submicron lateral resolution. To broaden its applications and to obtain a molecular level of understanding, further technical advancement is needed to enable high-speed measurements of VR SFG microspectra at every pixel. In this study, we demonstrate a new VR SFG hyperspectral imaging platform combined with an ultrafast laser system operated at a repetition rate of 80 MHz. The multiplex configuration with broadband mid-infrared pulses makes it possible to measure a single microspectrum of CH/CH2 stretching modes in biological samples, such as starch granules and type I collagen tissue, with an exposure time of hundreds of milliseconds. Switching from the homodyne- to heterodyne-detected VR SFG hyperspectral imaging can be achieved by inserting a pair of optics into the beam path for local oscillator generation and delay time adjustment, which enables self-phase-stabilized spectral interferometry. We investigate the relationship between phase images of several different C-H modes and the relative orientation of collagen triple-helix in fibril bundles. The results show that the new multiplex VR SFG microscope operated at a high repetition rate is a powerful approach to probe the structural features and spatial arrangements of biological systems in detail.

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.

Vibrational Sum-Frequency Generation Hyperspectral Microscopy for Molecular Self-Assembled Systems.

In this review, we discuss the recent developments and applications of vibrational sum-frequency generation (VSFG) microscopy. This hyperspectral imaging technique can resolve systems without inversion symmetry, such as surfaces, interfaces and noncentrosymmetric self-assembled materials, in the spatial, temporal, and spectral domains. We discuss two common VSFG microscopy geometries: wide-field and confocal point-scanning. We then introduce the principle of VSFG and the relationships between hyperspectral imaging with traditional spectroscopy, microscopy, and time-resolved measurements. We further highlight crucial applications of VSFG microscopy in self-assembled monolayers, cellulose in plants, collagen fibers, and lattice self-assembled biomimetic materials. In these systems, VSFG microscopy reveals relationships between physical properties that would otherwise be hidden without being spectrally, spatially, and temporally resolved. Lastly, we discuss the recent development of ultrafast transient VSFG microscopy, which can spatially measure the ultrafast vibrational dynamics of self-assembled materials. The review ends with an outlook on the technical challenges of and scientific potential for VSFG microscopy.

Distinguishing Mesoscale Polar Order (Unidirectional vs Bidirectional) of Cellulose Microfibrils in Plant Cell Walls Using Sum Frequency Generation Spectroscopy.

Cellulose in plant cell walls are synthesized as crystalline microfibrils with diameters of 3-4 nm and lengths of around 1-10 μm. These microfibrils are known to be the backbone of cell walls, and their multiscale three-dimensional organization plays a critical role in cell wall functions including plant growth and recalcitrance to degradation. The mesoscale organization of microfibrils over a 1-100 nm range in cell walls is challenging to resolve because most characterization techniques investigating this length scale suffer from low spatial resolution, sample preparation artifacts, or inaccessibility of specific cell types. Here, we report a sum frequency generation (SFG) study determining the mesoscale polarity of cellulose microfibrils in intact plant cell walls. SFG is a nonlinear optical spectroscopy technique sensitive to the molecular-to-mesoscale order of noncentrosymmetric domains in amorphous matrices. However, the quantitative theoretical model to unravel the effect of polarity in packing of noncentrosymmetric domains on SFG spectral features has remained unresolved. In this work, we show how the phase synchronization principle of the SFG process is used to predict the relative intensities of vibrational modes with different polar angles from the noncentrosymmetric domain axis. Applying this model calculation for the first time and employing SFG microscopy, we found that cellulose microfibrils in certain xylem cell walls are deposited unidirectionally (or biased in one direction) instead of the bidirectional polarity which was believed to be dominant in plant cell walls from volume-averaged characterizations of macroscopic samples. With this advancement in SFG analysis, one can now determine the relative polarity of noncentrosymmetric domains such as crystalline biopolymers interspersed in amorphous polymer matrices, which will open opportunities to study new questions that have not been conceived in the past.

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.

Multimodal spectral focusing CARS and SFG microscopy with a tailored coherent continuum from a microstructured fiber

We report a technologically novel microscopy system for bioimaging based on a 100 fs titanium:sapphire (Ti:Sa) laser pumped coherent continuum from a tailored, 9-cm long, all normal dispersion (ANDi) fiber, enabling concurrent image contrast with (a) spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) (spanning 900–3200 cm−1) and (b) sum frequency generation (SFG). Both modalities were efficiently excited with power levels at the microscope focus compatible with biological samples. Moreover, using the continuum, images were recorded in the back-scattering (epi-detection) geometry, without the necessity for an expensive, computer-controlled, spatial light modulator (SLM), clearly demonstrating the strong signal levels achieved. Image contrast from the multiple modalities provided greater chemical and structural insights than imaging with any single technique in isolation. Numerical simulations supported these developments in regard to both the optimum fiber length for SC generation and the achievement of high spectral resolution in SF-CARS via careful group delay dispersion matching across the pump and Stokes pulses using just an inexpensive sequence of short glass blocks inserted into the Stokes beam. We show bio-images of mouse tissue recorded concurrently via label/stain-free contrast from multiple modalities: CARS, two-photon auto-fluorescence (TPaF) and second harmonic/sum frequency generation (SHG/SFG). Overall, our approach delivers optimum performance in back-scattered (epi-) detection configuration, suited for thick samples, at reduced complexity and cost. The addition of this simple fiber add-on to lasers already widely used for TPF microscopy can thus extend the capabilities of a significant number of existing microscopy laboratories.

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.

Surface Phonon Polariton Resonance Imaging Using Long-Wave Infrared-Visible Sum-Frequency Generation Microscopy

We experimentally demonstrate long-wave infrared-visible sum-frequency generation microscopy for imaging polaritonic resonances of infrared (IR) nanophotonic structures. This nonlinear-optical appr...

Nonlinear optical microscopy with achromatic lenses extending from the visible to the mid-infrared

With the advent of near-infrared broadband sources stretching into the mid-infrared (MIR) region, there is a growing demand for optical components with utility over an increasingly broad spectral range. For refractive lenses, color correction over such broad bandwidths can be a challenge. In this work, we discuss and demonstrate a two-element lens design with achromaticity spanning the visible to the mid-infrared. The air-spaced doublet designed from commercially available materials shows a significant reduction in spot size and chromatic shift compared to single lens alternatives. We have tested these new broad bandwidth achromats for the purpose of laser-scanning sum-frequency generation microscopy, confirming their improved performance for nonlinear optical imaging applications. The super broadband achromatic lenses represent an attractive alternative to reflective components in ultrabroadband applications, as they enable compact transmission-based optical designs and good focusing performance at off-axis field angles.

Study of the Wetting of Paraffin Films on the Metal Surface in the Dynamic Dip-Coating Process Using Compressive-Sensing Sum-Frequency Generation Microscopy

A compressive-sensing sum-frequency generation microscope (CS-SFGM) is used to investigate the adsorption of the interfacial dotriacontane (n-C32H66) molecules on polycrystalline gold surfaces in t...

High-resolution infrared imaging of biological samples with third-order sum-frequency generation microscopy

We studied the use of vibrationally resonant, third-order sum-frequency generation (TSFG) for imaging of biological samples. We found that laser-scanning TSFG provides vibrationally sensitive imaging capabilities of lipid droplets and structures in sectioned tissue samples. Although the contrast is based on the infrared-activity of molecular modes, TSFG images exhibit a high lateral resolution of 0.5 µm or better. We observed that the imaging properties of TSFG resemble the imaging properties of coherent anti-Stokes Raman scattering (CARS) microscopy, offering a nonlinear infrared alternative to coherent Raman methods. TSFG microscopy holds promise as a high-resolution imaging technique in the fingerprint region where coherent Raman techniques often provide insufficient sensitivity.

Advances in nonlinear optical microscopy for biophotonics

This article reviews recent advances in nonlinear optical microscopy for biophotonics, including stimulated Raman scattering (SRS), coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), second harmonic generation (SHG), and sum frequency generation (SFG). We first introduce the principles and applications of SRS, CARS, TPEF, SHG, and SFG and their individual applications for biophotonics. We then introduce the combination of SRS, CARS, TPEF, SHG, and SFG microscopy for biophotonics. Our review not only summarizes the recent advances in nonlinear optical microscopy but also can deepen the understanding of the combination of these types of nonlinear optical microscopy for biophotonics.

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.