Photoactive Yellow Protein Adsorption at Hydrated Polyethyleneimine and Poly-l-Glutamic Acid Interfaces.

Chiral and achiral vibrational sum-frequency generation (VSFG) spectroscopy was performed in the 1400-1700 and 2800-3800 cm-1 range to study the interfacial structure of photoactive yellow protein (PYP) adsorbed on polyethyleneimine (PEI) and poly-l-glutamic acid (PGA) surfaces. Nanometer-thick polyelectrolyte layers served as the substrate for PYP adsorption, with 6.5-pair layers providing the most homogeneous surfaces. When the topmost material was PGA, it acquired a random coil structure with a small number of ß2-fibrils. Upon adsorption on oppositely charged surfaces, PYP yielded similar achiral spectra. However, the VSFG signal intensity increased for PGA surfaces with a concomitant redshift of the chiral Cα-H and N-H stretching bands, suggesting increased adsorption for PGA compared to PEI. At low wavenumbers, both the backbone and the side chains of PYP induced drastic changes to all measured chiral and achiral VSFG spectra. Decreasing ambient humidity led to the loss of tertiary structure with a re-orientation of α-helixes, evidenced by a strongly blue-shifted chiral amide I band of the ß-sheet structure with a shoulder at 1654 cm-1. Our observations indicate that chiral VSFG spectroscopy is not only capable of determining the main type of secondary structure of PYP, i.e., ß-scaffold, but is also sensitive to tertiary protein structure.

Efficient generation of few-cycle pulses beyond 10 µm from an optical parametric amplifier pumped by a 1-µm laser system.

Nonlinear vibrational spectroscopy profits from broadband sources emitting in the molecular fingerprint region. Yet, broadband lasers operating at wavelengths above 7 µm have been lacking, while traditional cascaded parametric frequency down-conversion schemes suffer from exceedingly low conversion efficiencies. Here we present efficient, direct frequency down-conversion of femtosecond 100-kHz, 1.03-µm pulses to the mid-infrared from 7.5 to 13.3 µm in a supercontinuum-seeded, tunable, single-stage optical parametric amplifier based on the wide-bandgap material Cd0.65Hg0.35Ga2S4. The amplifier delivers near transform-limited, few-cycle pulses with an average power > 30 mW at center wavelengths between 8.8 and 10.6 µm, at conversion efficiencies far surpassing that of optical parametric amplification followed by difference-frequency generation or intrapulse difference-frequency generation. The pulse duration at 10.6 µm is 101 fs corresponding to 2.9 optical cycles with a spectral coverage of 760-1160 cm-1. CdxHg1-xGa2S4 is an attractive alternative to LiGaS2 and BaGa4S7 in small-scale, Yb-laser-pumped, few-cycle mid-infrared optical parametric amplifiers and offers a much higher nonlinear figure of merit compared to those materials. Leveraging the inherent spatial variation of composition in CdxHg1-xGa2S4, an approach is proposed to give access to a significant fraction of the molecular fingerprint region using a single crystal at a fixed phase matching angle.

Analytical challenges of glycosaminoglycans at biological interfaces.

The analysis of glycosaminoglycans (GAGs) is a challenging task due to their high structural heterogeneity, which results in diverse GAG chains with similar chemical properties. Simultaneously, it is of high importance to understand their role and behavior in biological systems. It has been known for decades now that GAGs can interact with lipid molecules and thus contribute to the onset of atherosclerosis, but their interactions at and with biological interfaces, such as the cell membrane, are yet to be revealed. Here, analytical approaches that could yield important knowledge on the GAG-cell membrane interactions as well as the synthetic and analytical advances that make their study possible are discussed. Due to recent developments in laser technology, we particularly focus on nonlinear spectroscopic methods, especially vibrational sum-frequency generation spectroscopy, which has the potential to unravel the structural complexity of heterogeneous biological interfaces in contact with GAGs, in situ and in real time.

All-Optical Switching Demonstrated with Photoactive Yellow Protein Films.

Integrated optics (IO) is a field of photonics which focuses on manufacturing circuits similar to those in integrated electronics, but that work on an optical basis to establish means of faster data transfer and processing. Currently, the biggest task in IO is finding or manufacturing materials with the proper nonlinear optical characteristics to implement as active components in IO circuits. Using biological materials in IO has recently been proposed, the first material to be investigated for this purpose being the protein bacteriorhodopsin; however, since then, other proteins have also been considered, such as the photoactive yellow protein (PYP). In our current work, we directly demonstrate the all-optical switching capabilities of PYP films combined with an IO Mach-Zehnder interferometer (MZI) for the first time. By exploiting photoreactions in the reaction cycle of PYP, we also show how a combination of exciting light beams can introduce an extra degree of freedom to control the operation of the device. Based on our results, we discuss how the special advantages of PYP can be utilized in future IO applications.

The interaction of chondroitin sulfate with a lipid monolayer observed by using nonlinear vibrational spectroscopy.

The first vibrational sum-frequency generation (VSFG) spectra of chondroitin sulfate (CS) interacting with dipalmitoyl phosphatidylcholine (DPPC) at air-liquid interface are reported here, collected at a laser repetition rate of 100 kHz. By studying the VSFG spectra in the regions of 1050-1450 cm-1, 2750-3180 cm-1, and 3200-3825 cm-1, it was concluded that in the presence of Ca2+ ions, the head groups together with the head-group-bound water molecules in the DPPC monolayer are strongly influenced by the interaction with CS, while the organization of the phospholipid tails remains mostly unchanged. The interactions were observed at a CS concentration below 200 nM, which exemplifies the potential of VSFG in studying biomolecular interactions at low physiological concentrations. The VSFG spectra recorded in the O-H stretching region at chiral polarization combination imply that CS molecules are organized into ordered macromolecular superstructures with a chiral secondary structure.

Multivariate Raman mapping for phenotypic characterization in plant tissue sections.

Identifying and characterizing the biochemical variation in plant tissues is an important task in many research fields. Small spectral differences of the plant cell wall that are caused by genetic or environmental influences may be superimposed by individual variation as well as by a microscopic heterogeneity in molecular composition and structure of different histological substructures. A set of 56 samples from Cucumis sativus (cucumber) plants, comprising a total of ~168,000 spectra from tissue sections of leaf, stem, and roots was investigated by Raman microspectroscopic mapping excited at 532 nm. A multivariate analysis was carried out in order to assess the variation of the spectra with respect to origin of the tissue, the histological (cell wall) substructures, and the possibility to discriminate the spectra obtained from different individuals that had been subjected to two different conditions during growth. Combining the results of principal component analysis (PCA) based classification with the original spatial information in the maps of 23 sections of leaf xylem, variation in cell wall composition is found for four different individuals that also includes a discrimination of tissue grown in the presence and absence of additional silicic acid in the irrigation water of the plants. The spectral data point to differences in a contribution by carotenoids, as well as by hydroxycinnamic acids to the spectra. The results give new insight into the chemical heterogeneity of plant tissues and may be useful for elucidating biochemical processes associated with biomineralization by vibrational spectroscopy.

Nonlinear Optical Investigation of Microbial Chromoproteins.

Membrane-bound or cytosolic light-sensitive proteins, playing a crucial role in energy- and signal-transduction processes of various photosynthetic microorganisms, have been optimized for sensing or harvesting light by myriads of years of evolution. Upon absorption of a photon, they undergo a usually cyclic reaction series of conformations, and the accompanying spectro-kinetic events assign robust nonlinear optical (NLO) properties for these chromoproteins. During recent years, they have attracted a considerable interest among researchers of the applied optics community as well, where finding the appropriate NLO material for a particular application is a pivotal task. Potential applications have emerged in various branches of photonics, including optical information storage and processing, higher-harmonic and white-light continuum generation, or biosensorics. In our earlier work, we also raised the possibility of using chromoproteins, such as bacteriorhodopsin (bR), as building blocks for the active elements of integrated optical (IO) circuits, where several organic and inorganic photonic materials have been considered as active components, but so far none of them has been deemed ideal for the purpose. In the current study, we investigate the linear and NLO properties of biofilms made of photoactive yellow protein (PYP) and bR. The kinetics of the photoreactions are monitored by time-resolved absorption experiments, while the refractive index of the films and its light-induced changes are measured using the Optical Waveguide Lightmode Spectroscopy (OWLS) and Z-scan techniques, respectively. The nonlinear refractive index and the refractive index change of both protein films were determined in the green spectral range in a wide range of intensities and at various laser repetition rates. The nonlinear refractive index and refractive index change of PYP were compared to those of bR, with respect to photonics applications. Our results imply that the NLO properties of these proteins make them promising candidates for utilization in applied photonics, and they should be considered as valid alternatives for active components of IO circuits.

Efficient, sub-4-cycle, 1-µm-pumped optical parametric amplifier at 10 µm based on BaGa4S7.

We report on a microjoule-scale mid-infrared optical parametric amplifier (OPA) based on the recently developed wide-bandgap orthorhombic crystal, BaGa4S7 (BGS) and directly compare its performance to that of LiGaS2 (LGS) in the same OPA setup. The source is based on a single OPA stage amplifying supercontinuum seed pulses with a quantum efficiency of 29% at an idler wavelength of 10 µm, featuring nominally carrier-envelope phase-stable pulses. As a result of pumping the OPA directly at 1 µm, the overall conversion efficiency far exceeds that of traditional schemes based on OPAs followed by difference frequency generation. Chirp compensation using bulk germanium resulted in 126 fs pulses covering the 7.6-11.5 µm spectral range. BGS holds great promise for power scaling due to its availability in larger single-crystal sizes than LGS.

Surface-Enhanced Hyper Raman Spectra of Aromatic Thiols on Gold and Silver Nanoparticles.

We report the two-photon excited nonresonant surface-enhanced hyper Raman scattering (SEHRS) spectra of six aromatic thiol molecules during their interaction with gold and silver nanostructures. SEHRS spectra were obtained from thiophenol, benzyl mercaptan, and phenylethyl mercaptan and from the three isomers 2-aminothiophenol (2-ATP), 3-aminothiophenol (3-ATP), and 4-aminothiophenol (4-ATP). All SEHRS spectra were excited off-resonance at a wavelength of 1064 nm and compared to surface-enhanced Raman scattering (SERS) spectra excited at 785 nm or at 633 nm. The SEHRS spectra show a different interaction of thiophenol, benzyl mercaptan, and phenylethyl mercaptan with silver and gold nanostructures. Density functional theory calculations were used to support band assignments, in particular, for the unknown SERS spectrum of 3-ATP, and identify a band of phenylethyl mercaptan as a vibrational mode unique to the SEHRS spectrum and very weak in the Raman and infrared spectra. 2-ATP, 3-ATP, and 4-ATP show a different interaction with gold nanostructures that was found to depend on pH. Bands in the SEHRS spectrum of 2-ATP could be assigned to 2,2'-dimercaptoazobenzene, suggested to be obtained in a plasmon-assisted reaction that occurred during the SEHRS experiment. The results provide the basis for a better characterization of organic thiols at surfaces in a variety of fields, including surface functionalization and plasmonic catalysis.

Broadband vibrational sum-frequency generation spectrometer at 100 kHz in the 950-1750 cm-1 spectral range utilizing a LiGaS2 optical parametric amplifier.

We present a 100 kHz broadband vibrational sum-frequency generation (VSFG) spectrometer operating in the 5.7-10.5 µm (950-1750 cm-1) wavelength range. The mid-infrared beam of the system is obtained from a collinear, type-I LiGaS2-crystal-based optical parametric amplifier seeded by a supercontinuum and pumped directly by 180 fs, ~32 µJ, 1.03 µm pulses from an Yb:KGd(WO4)2 laser system. Up to 0.5 µJ mid-infrared pulses with durations below 100 fs were obtained after dispersion compensation utilizing bulk materials. We demonstrate the utility of the spectrometer by recording high-resolution, low-noise vibrational spectra of Langmuir-Blodgett supported lipid monolayers on CaF2. The presented VSFG spectrometer scheme offers superior signal-to-noise ratios and constitutes a high-efficiency, low-cost, easy-to-use alternative to traditional schemes relying on optical parametric amplification followed by difference frequency generation.

High-resolution and high-repetition-rate vibrational sum-frequency generation spectroscopy of one- and two-component phosphatidylcholine monolayers.

We present broadband vibrational sum-frequency generation (VSFG) spectra of Langmuir-Blodgett monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and different mixtures of them as model systems of pulmonary surfactants. The systematic study explored the dependence of the vibrational spectra as a function of surface tension and mixture ratio in various polarization combinations. The extremely short acquisition time and the high spectral resolution of our recently developed spectrometer helped minimize sample degradation under ambient conditions throughout the duration of the measurement and allowed the detection of previously unseen vibrational bands with unprecedented signal-to-noise ratio. The dramatically improved capability to record reliable vibrational spectra together with the label-free nature of the VSFG method provides direct access to native lipid structure and dynamics directly in the monolayer. The resulting data deliver quantitative information for structural analysis of multi-component phospholipid monolayers and may aid in the development of new synthetic pulmonary surfactants.

43 W, 1.55 µm and 12.5 W, 3.1 µm dual-beam, sub-10 cycle, 100 kHz optical parametric chirped pulse amplifier.

We present a 100 kHz optical parametric chirped pulse amplifier (OPCPA) developed for strong-field attosecond physics and soft-x-ray transient absorption experiments. The system relies on noncollinear potassium titanyl arsenate booster OPCPAs and is pumped by a 244 W, 1.1 ps Yb:YAG Innoslab chirped pulse laser amplifier. Two optically synchronized infrared output beams are simultaneously available: a 430 µJ, 51 fs, carrier-envelope phase stable beam at 1.55 µm and an angular-dispersion-compensated, 125 µJ, 73 fs beam at 3.1 µm.

100-kHz, dual-beam OPA delivering high-quality, 5-cycle angular-dispersion-compensated mid-infrared idler pulses at 3.1 µm.

We demonstrate a dual-beam infrared optical parametric source featuring a noncollinear KTA booster amplifier and straightforward angular dispersion compensation of the idler beam. Through careful beam and pulse characterization, and high-harmonic generation in a crystalline solid, we show that the corrected idler beam is diffraction-limited, astigmatism-free, and compressible to its transform-limited, 5-cycle pulse duration. Pumped by only 40-µJ pulses at 1.03 µm, the parametric source delivers 7.8-µJ, 38-fs, 1.53-µm and 2.3-µJ, 53-fs, CEP-stable, 3.1-µm pulses at a repetition rate of 100 kHz. The scheme provides a promising route to scale the pulse energy and average power beyond PPLN- or KTA-based collinear OPA architectures.

Vibrational sum-frequency generation spectroscopy of lipid bilayers at repetition rates up to 100 kHz.

Broadband vibrational sum-frequency generation (BB-VSFG) spectroscopy has become a well-established surface analytical tool capable of identifying the orientation and structure of molecular layers. A straightforward way to boost the sensitivity of the technique could be to increase the laser repetition rate beyond that of standard BB-VSFG spectrometers, which rely on Ti:sapphire lasers operating at repetition rates of 1-5 kHz. Nevertheless, possible thermally induced artifacts in the vibrational spectra due to higher laser average powers are unexplored. Here, we discuss laser power induced temperature accumulation effects that distort the BB-VSFG spectra of 1,2-diacyl-sn-glycero-3-phosphocholine at an interface between two transparent phases at repetition rates of 5, 10, 50, and 100 kHz at constant pulse energy. No heat-induced distortions were found in the spectra, suggesting that the increase in the laser repetition rate provides a feasible route to an improved signal-to-noise ratio or shorter data acquisition times in BB-VSFG spectroscopy for thin films on transparent substrates. The results have implications for future BB-VSFG spectrometers pushing the detection limit for molecular layers with low surface coverage.

Raman Imaging of Plant Cell Walls in Sections of Cucumis sativus.

Raman microspectra combine information on chemical composition of plant tissues with spatial information. The contributions from the building blocks of the cell walls in the Raman spectra of plant tissues can vary in the microscopic sub-structures of the tissue. Here, we discuss the analysis of 55 Raman maps of root, stem, and leaf tissues of Cucumis sativus, using different spectral contributions from cellulose and lignin in both univariate and multivariate imaging methods. Imaging based on hierarchical cluster analysis (HCA) and principal component analysis (PCA) indicates different substructures in the xylem cell walls of the different tissues. Using specific signals from the cell wall spectra, analysis of the whole set of different tissue sections based on the Raman images reveals differences in xylem tissue morphology. Due to the specifics of excitation of the Raman spectra in the visible wavelength range (532 nm), which is, e.g., in resonance with carotenoid species, effects of photobleaching and the possibility of exploiting depletion difference spectra for molecular characterization in Raman imaging of plants are discussed. The reported results provide both, specific information on the molecular composition of cucumber tissue Raman spectra, and general directions for future imaging studies in plant tissues.

Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging.

Spontaneous Raman scattering microspectroscopy, second harmonic generation (SHG) and 2-photon excited fluorescence (2PF) were used in combination to characterize the morphology together with the chemical composition of the cell wall in native plant tissues. As the data obtained with unstained sections of Sorghum bicolor root and leaf tissues illustrate, nonresonant as well as pre-resonant Raman microscopy in combination with hyperspectral analysis reveals details about the distribution and composition of the major cell wall constituents. Multivariate analysis of the Raman data allows separation of different tissue regions, specifically the endodermis, xylem and lumen. The orientation of cellulose microfibrils is obtained from polarization-resolved SHG signals. Furthermore, 2-photon autofluorescence images can be used to image lignification. The combined compositional, morphological and orientational information in the proposed coupling of SHG, Raman imaging and 2PF presents an extension of existing vibrational microspectroscopic imaging and multiphoton microscopic approaches not only for plant tissues.

Kinetics of Light-Induced Intramolecular Energy Transfer in Different Conformational States of NADH.

When bound to a protein, the coenzyme NAD+/NADH typically exists in an extended conformation, while in aqueous solutions it can be characterized by an equilibrium of folded and unfolded structures. It was recognized long ago that in the folded conformation light absorption at the adenine ring initiates an effective energy transfer (ET) toward the nicotinamide group, but the mechanism of this process is still unexplored. Here we apply ultrafast transient absorption measurements on NADH combined with compartmental model analysis for following the kinetics of the ET. We find that the actual ET is extremely rapid (∼70 fs). The high rate can be well described by a Förster-type mechanism, promoted by both the special photophysical properties of adenine and the subnanometer inter-ring distance. The rapid ET creates a vibrationally hot excited state on nicotinamide, the vibrational and electronic relaxation of which is characterized by 1.7 and 650 ps, respectively.

Surface-enhanced hyper Raman hyperspectral imaging and probing in animal cells.

Hyper Raman scattering, that is, spontaneous, two-photon excited Raman scattering, of organic molecules becomes strong when it occurs as surface-enhanced hyper Raman scattering (SEHRS), in the proximity of plasmonic nanostructures. Its advantages over one-photon excited surface-enhanced Raman scattering (SERS) include complementary vibrational information resulting from different selection rules, probing of very small focal volumes, and beneficial excitation with long wavelengths. Here, imaging of macrophage cells by SEHRS is demonstrated, using SEHRS labels consisting of silver nanoparticles and two different molecules, 2-naphthalenethiol and para-mercaptobenzoic acid, that are excited off-resonance. The vibrational signatures of the molecules are discriminated using hyperspectral analysis and provide information about the subcellular localization of the SEHRS probes. The SEHRS based hyperspectral imaging approach presented here uses principal component analysis (PCA) to localize the reporter molecules inside the cells and is augmented by hierarchical cluster analysis (HCA). The high sensitivity of SEHRS spectra with respect to small environmental changes can be utilized for mapping of physiological parameters in the endosomal system of the cells. This is illustrated by discussing the spatial distribution of endosomes of varying pH inside the cytosol.

Surface enhanced hyper Raman scattering (SEHRS) and its applications.

Surface enhanced hyper Raman scattering (SEHRS) is the spontaneous, two-photon excited Raman scattering that occurs for molecules residing in high local optical fields of plasmonic nanostructures. Being regarded as a non-linear analogue of surface enhanced Raman scattering (SERS), SEHRS shares most of its properties, but also has additional characteristics. They include complementary spectroscopic information resulting from different selection rules and a stronger enhancement due to the non-linearity in excitation. In practical spectroscopy, this can translate to advantages, which include a high selectivity when probing molecule-surface interactions, the possibility of probing molecules at low concentrations due to the strong enhancement, and the advantages that come with excitation in the near-infrared. In this review, we give examples of the wealth of vibrational spectroscopic information that can be obtained by SEHRS and discuss work that has contributed to understanding the effect and that therefore provides directions for SEHRS spectroscopy. Future applications could range from biophotonics to materials research.

Templated green synthesis of plasmonic silver nanoparticles in onion epidermal cells suitable for surface-enhanced Raman and hyper-Raman scattering.

We report fast and simple green synthesis of plasmonic silver nanoparticles in the epidermal cells of onions after incubation with AgNO3 solution. The biological environment supports the generation of silver nanostructures in two ways. The plant tissue delivers reducing chemicals for the initial formation of small silver clusters and their following conversion to plasmonic particles. Additionally, the natural morphological structures of the onion layers, in particular the extracellular matrix provides a biological template for the growth of plasmonic nanostructures. This is indicated by red glowing images of extracellular spaces in dark field microscopy of onion layers a few hours after AgNO3 exposure due to the formation of silver nanoparticles. Silver nanostructures generated in the extracellular space of onion layers and within the epidermal cell walls can serve as enhancing plasmonic structures for one- and two-photon-excited spectroscopy such as surface enhanced Raman scattering (SERS) and surface enhanced hyper-Raman scattering (SEHRS). Our studies demonstrate a templated green preparation of enhancing plasmonic nanoparticles and suggest a new route to deliver silver nanoparticles as basic building blocks of plasmonic nanosensors to plants by the uptake of solutions of metal salts.