Imaging chirality with surface second harmonic generation microscopy

Chirality is a fundamental construct in nature which arises from an antisymmetric arrangement of atoms, molecules, or larger structures, resulting in the formation of nonsuperimposable mirror images. Bulk chiral effects can easily be measured using circular dichroism (CD) or optical rotary dispersion (ORD). However, the imaging of chirality originating from molecular surface films cannot be obtained with these linear optical methods. By using chiral second harmonic generation (C-SHG), with its inherent surface sensitivity and ability to discriminate between the symmetry of surface adsorbed species in combination with a counter-propagating optical geometry, we have developed the first nonlinear chiral microscope. In the study presented here, the intrinsic chirality of R- and S-(+)-1,1'-bi-2-naphthol (RBN, SBN) has been used to image a patterned planar supported lipid bilayer (PSLB) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using C-SHG. Spatial resolution of the patterned PSLB is visible when either RBN or SBN is intercalated into the membrane. No image is observed when a racemic mixture of RBN and SBN is present. The C-SHG images are compared with those obtained from fluorescence microscopy to verify the C-SHG imaging technique. The results presented here demonstrate that C-SHG possesses the requisite surface selectivity and sensitivity to detect interfacial chirality and provides a direct route for the visualization of chirality originating from molecular surface films.     

Deciphering second harmonic generation signals

Second harmonic generation (SHG) has emerged as one of the most powerful techniques used to selectively monitor surface dynamics and reactions for all types of interfaces as well as for imaging non-centrosymmetric structures, although the molecular origin of the SHG signal is still poorly understood. Here, we present a breakthrough approach to predict and interpret the SHG signal at the atomic level, which is freed from the hyperpolarisability concept and self-consistently considers the non-locality and the coupling with the environment. The direct ab initio method developed here shows that a bulk quadrupole contribution significantly overwhelms the interface dipole term in the purely interfacial induced second-order polarisation for water/air interfaces. The obtained simulated SHG responses are in unprecedented agreement with the experimental signal. This work not only paves the road for the prediction of SHG response from more complex interfaces of all types, but also suggests new insights in the interpretation of the SHG signal at a molecular level. In particular, it highlights the modest influence of the molecular orientation and the high significance of the bulk quadrupole contribution, which does not depend on the interface, in the total experimental response.

Second harmonic generation is one of the most powerful techniques used to selectively probe interfaces of all types. The direct ab initio method developed here allows predicting the signal and highlights the importance of local and non-local effects.     

Selective detection of protein crystals by second harmonic microscopy

The unique symmetry properties of second harmonic generation (SHG) microscopy enabled sensitive and selective imaging of protein microcrystals with negligible contributions from solvated proteins or amorphous protein aggregates. In studies of microcrystallites of green fluorescent protein (GFP) prepared in 500 pL droplets, the SHG intensities rivaled those of fluorescence, but with superb selectivity for crystalline regions. GFP in amorphous aggregates and in solution produced substantial background fluorescence, but no detectable SHG. The ratio of the forward-to-backward detected SHG provides a measure of the particle size, suggesting detection limits down to crystallites 100 nm in diameter under low magnification (10x). In addition to being sensitive and highly selective, second-order nonlinear optical imaging of chiral crystals (SONICC) is directly compatibility with virtually all common protein crystallization platforms.     

Nephila clavipes spider dragline silk microstructure studied by scanning transmission X-ray microscopy

Nephila clavipes dragline silk microstructure has been investigated by scanning transmission X-ray microscopy (STXM), a technique that allows quantitative mapping of the level of orientation of the peptide groups at high spatial resolution (<50 nm). Maps of the orientation parameter P2 have been derived for spider silk for the first time. Dragline silk presents a very fine microstructure in which small, highly oriented domains (average area of 1800 nm2, thus clearly bigger than individual beta-sheet crystallites) are dispersed in a dominant, moderately oriented matrix with several small unoriented domains. Our results also highlight the orientation of the noncrystalline fraction in silk, which has been underestimated in numerous structural models. No evidence of either a regular lamellar structure or any periodicity along the fiber was observed at this spatial resolution. The surface of fresh spider silk sections consists of a approximately 30-120 nm thick layer of highly oriented protein chains, which was found to vary with the reeling speed, where web building (0.5 cm/s) and lifeline (10 cm/s) spinning speeds were investigated. While the average level of orientation of the protein chains is unaffected by the spinning speed, STXM measurements clearly highlight microstructure differences. The slowpull fiber contains a larger fraction of highly oriented domains, while the protein chains are more homogeneously oriented in the fastpull fiber. In comparison, cocoon silk from the silkworm Bombyx mori presents a narrower orientation distribution. The strength-extensibility combination found in spider dragline silk is associated with its broad orientation distribution of highly interdigitated and unoriented domains.     

Field-free molecular alignment control by phase-shaped femtosecond laser pulse

In this paper, we theoretically show that the field-free molecular alignment can be controlled by shaping the femtosecond laser pulse with a periodic phase step modulation, involving the maximum degree and temporal structure of the molecular alignment. We show that the molecular alignment can be completely suppressed or reconstructed as that by the transform-limited laser pulse, the temporal structure of the alignment transient can be controlled with a desired shape, and the molecular alignment and antialignment for any temporal structure can be switched. Furthermore, we also show that both the degree and direction of the molecular alignment at a fix time delay can be continuously modulated.     

Phase transition of lipid-like monolayer characterized by second harmonic generation

Phase transition of a lipid-like hemicyanine compound characterized by second harmonic generation is studied carefully. The phase transition is assigned as the first order transition between solid state and liquid state. The transition temperature increases with an increase in the surface molecular concentration. A monolayer structure parameter a which is very sensitive to the phase transition is introduced.     

Polymer films on metals investigated by optical second harmonic generation

In two different types of experiments, polymer films on gold substrates were investigated by optical second harmonic generation (SHG). Thickness dependent ex situ measurements on Langmuir-Blodgett films of the octadecylammonium salt of polyamic acid (PACS) show a preferential orientation of the polymer molecules. In situ SHG experiments were performed to monitor the growth of polyamic acid films deposited from the gas phase. Pyromellitic dianhydride (PMDA), 4,4-oxydianiline (ODA), and 4,4-diaminodiphenyl disulfide (DAPS) served as monomers. Similar to the Langmuir-Blodgett films, an oriented growth is observed. The thickness dependence of the SHG signal is strongly dependent on the interfacial chemistry which is very different for the two amines used as monomers. Based on a comprehensive three layer model, the relation between the structure of the films and the SHG signal is discussed.     

Tracking oxytetracyline mobility across environmental interfaces by second harmonic generation

This work examines the binding behavior of the antibiotic oxytetracycline (OTC) to mineral oxide/water interfaces in the presence and absence of organic functional groups using the interface-specific technique second harmonic generation (SHG). Studies show that OTC binding to fused quartz, methyl ester, carboxylic acid, and alkyl interfaces is fully reversible and highly dependent on solution pH, with appreciable adsorption occurring only at pH 8. Relative surface coverage at pH 8 is highest for the polar organic-functionalized surfaces, and surface saturation occurs for the methyl ester-functionalized fused quartz/water interface at 2 x 10(-5) M. Adsorption isotherm measurements indicate that the binding process is controlled by hydrogen bonding and hydrophobic interactions, with free energies of adsorption on the order of -40 kJ/mol for all interfaces studied. The results indicate that OTC transport in the environment will depend heavily on soil pH and composition and have implications for the development of bacterial antibiotic resistance.     

Enhancement of anthracene fragmentation by circularly polarized intense femtosecond laser pulse

The authors compared circularly and linearly polarized lights in the ionization and fragmentation of anthracene, using 800 nm femtosecond laser pulses at intensities of 10(13)-10(15) W cm-2. Singly and doubly charged intact molecular ions as well as numerous fragment ions were observed in the mass spectra, which were investigated as a function of laser intensity and polarization. At comparable intensities above the saturation threshold for complete ionization, the fragmentation pathways are enhanced with a circularly polarized field compared to a linearly polarized field. Resonant excitation of the molecular cation through the 2Au<--2Bg transition is proposed to be the initial step to ion fragmentation. The circularly polarized field interacts with a larger fraction of the randomly oriented molecules than the linearly polarized field, and this is considered to be the reason for the enhanced fragmentation brought about by circularly polarized light.     

Study on second harmonic generation of 10-hydrocarbylacridones

Values of second-order polarizabilities (beta(xxx)), the composite magnitude (beta(CT microg)) of molecular hypersusceptibilities and the second harmonic generation (SHG) of microcrystal were used to characterize and evaluate the second-order nonlinear optical performance of 10-hydrocarbylacridones. Using Nd:YAG as a laser source, the SHG values of these compounds in powder as compared with urea powder were obtained, compared with the calculated values of beta(CT microg) and beta(xxx) found by solvatochromic method. The results showed that the SHG values of compounds 1, 2, and 5 are larger than that of urea. Compound 5 has a highest beta(CT microg) value among the 12 compounds. Although the different electronegativities of hydrocarbyl (R) attached to nitrogen atom should have an effect on the extent of intralmolecular charge-transfer and further influence SHG, beta(CT microg) or beta(xxx) values, the relationships between electronegativity of R and SHG, beta(CT microg) or beta(xxx) could not be found. But it is found that the bigger the donating-electron ability of R, the longer the maximum absorption wavelength of 10-hydrocarbylacridone molecules.     

Structure induced by irradiation of femtosecond laser pulse in dyed polymeric materials

We investigated the structures induced by an irradiation of a near‐infrared (NIR) femtosecond laser pulse in dye‐doped polymeric materials {poly(methyl methacrylate) (PMMA), thermoplastic epoxy resin (Epoxy), and a block copolymer of methyl methacrylate and ethyl acrylate‐butyl acrylate [p(MMA/EA‐BA) block copolymer]}. Dyes used were classified into two types—type 1 with absorption at 400 nm and type 2 with no absorption at 400 nm. The 400‐nm wavelength corresponds to the two‐photon absorption region by the irradiated NIR laser pulse at 800 nm. Type 1 dye‐doped PMMA and p(MMA/EA‐BA) block copolymer showed a peculiar dye additive effect for the structures induced by the line irradiation of a NIR femtosecond laser pulse. On the contrary, dye‐doped Epoxy did not exhibit a dye additive effect. The different results among PMMA, p(MMA/EA‐BA) block copolymer, and Epoxy matrix polymers are supposed to be related to the difference of electron‐acceptor properties. The mechanism of this type 1 dye‐additive‐effect phenomenon for PMMA and p(MMA/EA‐BA) block copolymer is discussed on the basis of two‐photon absorption of type 1 dye at 400 nm by the irradiation of a femtosecond laser pulse with 800 nm wavelength and the dissipation of the absorbed energy to the polymer matrix among various transition processes. Dyes with a low‐fluorescence quantum yield favored the formation of thicker grating structures. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 2800–2806, 2002     

Organization and orientation of amphiphilic push-pull chromophores deposited in Langmuir-Blodgett monolayers studied by second harmonic generation and atomic force microscopy

Orientation and organization of two amphiphilic push-pull chromophores mixed with two phospholipids (dipalmitoylphosphatidylcholine and dioleoylphosphatidylcholine) in Langmuir-Blodgett (LB) monolayers are investigated by second harmonic generation. The LB monolayers have also been characterized by atomic force microscopy and UV-vis spectroscopy. The effective molecular orientations and hyperpolarizabilities of the chromophores are studied as a function of the phospholipid concentrations. The experimental results are discussed within the frame of a model of orientational distribution of the chromophores which gives the orientational mean angle and bounds on the orientational disorder. The mean orientation of the chromophores is found to be within 45-55 degrees whereas their hyperpolarizability coefficients, measured with respect to quartz, are estimated to be in the range (0.3-0.7) x 10(-27) esu taking account of the maximal orientational disorder.     

Synthesis and second harmonic generation studies of nlo-functionalized polyimides

Several NLO-functionalized polyimides were synthesized through novel synthetic pathways and their NLO properties were characterized. For preventing the disadvantage in poly(amic acid) precursor route, the Mitsunobu reaction and alternative synthetic route for NLO polyimides were chosen. The second-order NLO coefficients (χ⁽²⁾) of the synthesized polyimides were between 48pm/V and 122pm/V. The thermal stability of nonlinearity characterized showed that all polyimide films retain their NLO activities at temperatures below 100°C.     

Double pulse laser-induced breakdown spectroscopy with femtosecond laser pulses

This paper presents results obtained in a study of collinear geometry double pulse femtosecond LIBS analysis of solids in ambient environment. LIBS signal enhancement of 3–10 fold, accompanied by significant improvement of signal reproducibility, in comparison with the single pulse case, has been found in different samples such as brass, iron, silicon, barium sulfate and aluminum when an optimum temporal separation between the two ablating pulses is used. The influence of the delay between pulses in the LIBS signal intensity was investigated and two intervals of interaction were established. A first transient regime from 0 to 50 ps, in which the LIBS signal increases until reaching a maximum, and a second regime that ranges from 50 to 1000 ps (maximum inter-pulse delay investigated) in which the signal enhancement remains constant. Emissions from both ionized and neutral atoms show the same pattern of enhancement with a clear tendency of lines arising from higher energy emissive states to exhibit higher enhancement factors.     

Crystallization and second harmonic generation in potassium-sodium niobiosilicate glasses

Transparent glasses having molar composition (23−x)K₂O·xNa₂O·27Nb₂O₅·50SiO₂ (x=0, 5, 10, 15 and 23) have been synthesized by the melt-quenching technique and their devitrification behaviour has been investigated by DTA and XRD. Depending on the composition, the glasses showed a glass transition temperature in the range 660-680 °C and devitrified in several steps. XRD measurements showed that the replacement of K₂O by Na₂O strongly affects the crystallization behaviour. Particularly, in the glasses with only potassium or low sodium content the first devitrification step is related to the crystallization of an unidentified phase, while in the glass containing only sodium, NaNbO₃ crystallizes. For an intermediate sodium content (x=10 and 15) a potassium sodium niobate crystalline phase, belonging to the tungsten-bronze family, is formed by bulk nucleation. This system looks promising to produce active nanostructured glasses as the tungsten-bronze type crystals have ferroelectric, electro-optical and non-linear optical properties. Preliminary measurements evidenced SHG activity in the crystallized glasses containing this phase.     

Photoionization of NO molecule in two-color femtosecond pulse laser fields

A theoretical approach used for studying the multiphoton ionization of NO molecule in two-color strong femtosecond pulse laser fields is proposed. Time- and energy-resolved photoelectron energy spectra are calculated for describing the photionization process. The NO molecule is first excited to the D²Σ⁺ Rydberg state. The pump and probe pulses then couple the D²Σ⁺ with M²Σ⁺ states via a two-photon Raman coupling and a laser-induced continuum structure state. Three channels from the D²Σ⁺ and M²Σ⁺ states to ionization state are described. The population transfers through the continuum state and the Raman coupling passage are also calculated.     

Carotenoid based bio-compatible labels for third harmonic generation microscopy

The use of carotenoids as biologically friendly labels for third harmonic generation (THG) microscopy is demonstrated. Carotenoid containing liposomes are used to label cell structures via liposome cell fusion. The THG microscopy labels, called harmonophores, were characterized by measuring the third-order nonlinear susceptibility (χ((3))) of carotenoids: violaxanthin, neoxanthin, lutein, β-carotene, zeaxanthin, canthaxanthin and astaxanthin. The THG ratio method was used, which is based on measuring the THG intensity from two interfaces using a nonlinear optical microscope. The second hyperpolarizability values of carotenoids were extracted from χ((3)) measurements taking into account the refractive index at fundamental and third harmonic wavelengths. The length dependence of the second hyperpolarizability of conjugated polyenes from 9 to 13 double bonds with varying oxygen functional groups was investigated. It appears that the presence of epoxides can have a higher influence than an additional conjugated double bond. Furthermore, labelling of both Drosophila Schneider 2 cells and Drosophila melanogaster larvae myocytes with β-carotene was achieved. This study demonstrates that THG enhancement by carotenoids can be used for nontoxic in vivo labelling of subcellular structures for third harmonic generation microscopy.     

Enhancement of second harmonic generation in helical Sc liquid crystals

In a ferroelectric liquid crystal, a special type of phase-matching for optical second harmonic generation (SHG) is possible, where two counter-propagating fundamental waves create second harmonic waves at the edge of the selective reflection band. We compute the SHG intensity in such a situation and show that, at slight detuning from exact phase-matching, useful resonance enhancement can be obtained. A considerable amount of SHG also appears when the second harmonic frequency is in the reflection band, where the SHG wave is non-propagating.     

Dynamics of a surface phase transition as monitored by in situ second harmonic generation

The dynamics of the (2 x 2)-3CO <--> dilute phase transition involving CO adsorbed on Pt(111) microfacets has been monitored in situ in a CO-saturated acidic electrolyte using potential step-second harmonic generation techniques. Apparent time constants derived strictly from the optical data were found to be orders of magnitude slower for the forward process as compared to those of the reverse process. This behavior is consistent with the activated nature of CO electrooxidation, which requires the presence of a neighboring adsorbed oxygenated species, for example, hydroxyl, for the process to ensue.