Missing Piece in Colloidal Stability─Morphological Factor of Hydrophobic Nanoparticles.

Hydrophobic nanoparticles (NPs) in water were considered unstable because they lack the repulsive electrostatic interaction and steric effect to prevent aggregation. In this study, porous hydrophobic NPs of two star-shaped giant molecules, POSS-(R)8, were found to be stable in water and able to retain their kinetic stability in a wide range of temperatures, pH values, and ionic strengths. Unlike the solid hydrophobic NPs that aggregate even with the negative zeta potential (ζ) induced by surface-structured hydrogen-bonded (SHB) water, the porous morphology of POSS-(R)8 NPs reduces the entropically driven hydrophobic effect to prevent aggregation. With the porous morphology, the hydrophobic NPs are stable without the hydrophilic or charged surface functional groups and demonstrate good encapsulation capability. The morphological factor of colloids is thus one of the missing pieces in the theory of colloidal stability that extends our understanding of colloidal science.

Water-Induced Self-Assembly of Amphiphilic Discotic Molecules for Adaptive Artificial Water Channels.

Inspired by the induced-fit mechanism in nature, we developed the process of water-induced self-assembly (WISA) to make water an active substrate that regulates the self-assembly and function of amphiphilic discotic molecules (ADMs). The ADM is an isotropic liquid that self-assembles only when in contact with water. Characterization results indicate that water fits into the hydrophilic core of the ADMs and induces the formation of a hexagonal columnar phase (Colh), where each column contains a hydrated artificial water channel (AWC). The hydrated AWCs are adaptive rather than static; the dynamic incorporation/removal of water results in the reversible assembly/disassembly of the adaptive AWCs (aAWCs). Furthermore, its dynamic characteristics can enable water to act as an orientation-directional guest molecule that controls the growth direction of the aAWCs. Well-aligned aAWC arrays that showed the ability of water transport were obtained via a "directional WISA" method. In WISA, water thus governs the supramolecular chemistry and function of synthetic molecules as it does with natural materials. By making water an active component in adaptive chemistry and enabling host molecules to dynamically interact with water, this adaptive aquatic material may motivate the development of synthetic molecules further toward biomaterials.

Vibrational couplings and energy transfer pathways of water's bending mode.

Coupling between vibrational modes is essential for energy transfer and dissipation in condensed matter. For water, different O-H stretch modes are known to be very strongly coupled both within and between water molecules, leading to ultrafast dissipation and delocalization of vibrational energy. In contrast, the information on the vibrational coupling of the H-O-H bending mode of water is lacking, even though the bending mode is an essential intermediate for the energy relaxation pathway from the stretch mode to the heat bath. By combining static and femtosecond infrared, Raman, and hyper-Raman spectroscopies for isotopically diluted water with ab initio molecular dynamics simulations, we find the vibrational coupling of the bending mode differs significantly from the stretch mode: the intramode intermolecular coupling of the bending mode is very weak, in stark contrast to the stretch mode. Our results elucidate the vibrational energy transfer pathways of water. Specifically, the librational motion is essential for the vibrational energy relaxation and orientational dynamics of H-O-H bending mode.

Vesicular Membrane with Structured Interstitial Water.

Self-assembled vesicles with structured (tetrahedral order with strong hydrogen bonds) interstitial water are reported. The vesicles, known as MCsome, are assembled from metal carbonyl compounds, FpR (Fp = (Cp)Fe(PPh3)(CO)(CO-), Cp = cyclopentadiene, R = C3Bithiophene, C6Pyrene or C6) with the Fp heads exposed to water. The R groups are surrounded by the interstitial water with the hydrogen bonding strength variable depending on the hydrophobicity of R groups. The structure of the interstitial water is responsible for the integrity of the membrane and swelling behavior of the vesicles. This constructional role of water opens new concepts for the creation of aqueous assemblies with water-mediated functions.

Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies.

We demonstrate a novel bio-spectroscopic technique, "simultaneous Raman/GFP microspectroscopy". It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti-Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two-component multivariate curve resolution with alternate least squares (MCR-ALS) analysis in the anti-Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR-ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm-1 , previously reported as "Raman spectroscopic signature of life of yeast."

Phonon confinement and size effect in Raman spectra of ZnO nanoparticles.

We study Raman spectra of ZnO nanoparticles of 5-12 nm size in the whole range of the first-order phonon bands. We apply the 3D phonon confinement model (PCM) for the interpretation of the observed Raman spectra. It is found that PCM is well applicable to the acoustic modes as well as to the optical ones, despite the fact that PCM has been thought not to be suitable for acoustic phonons. We show that the asymptotic behavior of PCM for the small-size limit is more consistent with the observation than that of the elastic sphere model (ESM). Furthermore, PCM gives detailed information on the complex size-dependent shapes of the phonon bands.

A General Approach for Estimating Lamella-Thickness Distribution in Polymers with Low-Frequency Raman Spectroscopy: Application to Lamella Formation in Crystallizing Polyethylene.

A general method for estimating lamella-thickness distribution in semicrystalline polymers has been developed and applied to polyethylene (PE). The longitudinal acoustic mode (LAM) of PE appears at very low frequencies (i.e., ν˜ 8-20 cm-1 ) in the Raman spectrum. It represents a distribution of lamellae of varying thicknesses. We propose a distribution function that converts a low-frequency LAM Raman band into the corresponding lamellae-thickness distribution. By using this distribution function, we can study lamella formation in crystallizing PE to elucidate the influence of supercooling and determine critical lamella thickness, the minimum chain length at which folding occurs, and the associated thermodynamic parameters accurately. This method has a general applicability toward the examination of polymer crystallization in an accurate and straightforward manner. Understanding the molecular details of polymer crystallization has applications, particularly in polymer thin-film photovoltaics and polymer processing, beyond its fundamental academic significance.

Polarizability tensor invariants of H2, HD, and D2.

We report an exhaustive compilation of wavelength-dependent matrix elements over the mean polarizability (α¯) and polarizability anisotropy (γ) operators for the rovibrational states of the H2, HD, and D2 molecules together with an accompanying computer program for their evaluation. The matrix elements can be readily evaluated using the provided codes for rovibrational states with J = 0-15 and v = 0-4 and for any laser wavelengths in the interval 182.25-1320.6 nm corresponding to popular, commercially available lasers. The presented results substantially extend the scope of the data available in the literature, both in respect of the rovibrational transitions analyzed and the range of covered laser frequencies. The presented detailed tabulation of accurate polarizability tensor invariants is essential for successful realization of our main long-term goal: developing a universal standard for determining absolute Raman cross sections and absolute Raman intensities in experimental Rayleigh and Raman scattering studies of molecules.

Studying anti-oxidative properties of inclusion complexes of α-lipoic acid with γ-cyclodextrin in single living fission yeast by confocal Raman microspectroscopy.

α-lipoic acid (ALA) is an essential cofactor for many enzyme complexes in aerobic metabolism, especially in mitochondria of eukaryotic cells where respiration takes place. It also has excellent anti-oxidative properties. The acid has two stereo-isomers, R- and S- lipoic acid (R-LA and S-LA), but only the R-LA has biological significance and is exclusively produced in our body. A mutant strain of fission yeast, Δdps1, cannot synthesize coenzyme Q10, which is essential during yeast respiration, leading to oxidative stress. Therefore, it shows growth delay in the minimal medium. We studied anti-oxidant properties of ALA in its free form and their inclusion complexes with γ-cyclodextrin using this mutant yeast model. Both free forms R- and S-LA as well as 1:1 inclusion complexes with γ-cyclodextrin recovered growth of Δdps1 depending on the concentration and form. However, it has no effect on the growth of wild type fission yeast strain at all. Raman microspectroscopy was employed to understand the anti-oxidant property at the molecular level. A sensitive Raman band at 1602cm-1 was monitored with and without addition of ALAs. It was found that 0.5mM and 1.0mM concentrations of ALAs had similar effect in both free and inclusion forms. At 2.5mM ALAs, free forms inhibited the growth while inclusion complexes helped in recovered. 5.0mM ALA showed inhibitory effect irrespective of form. Our results suggest that the Raman band at 1602cm-1 is a good measure of oxidative stress in fission yeast.

Cylindrical micelles of a POSS amphiphilic dendrimer as nano-reactors for polymerization.

A low generation amphiphilic dendrimer, POSS-AD, which has a POSS core and eight amphiphilic arms, was synthesized and used as a nano-reactor to produce well-defined polymer nano-cylinders. Confirmed by small-angle X-ray scattering (SAXS), Raman and NMR spectrometry, monodispersed cylindrical micelles that contain a hydrophilic cavity with a diameter of 2.09 nm and a length of 4.26 nm were produced via co-assembling POSS-AD with hydrophilic liquids, such as H2O and HEMA in hydrophobic solvents. Taking the HEMA/POSS-AD cylindrical micelles as nano-reactors, polymerization of HEMA within the micelles results in polymer nano-cylinders (POSS-ADNPs) with a diameter of 2.24 nm and a length of 5.02 nm. The study confirmed that despite the inability to maintain specific shape in solution, low generation dendrimers form well-defined nano-containers or nano-reactors, which relies on co-assembling with hydrophilic guest molecules. These nano-reactors are robust enough to maintain their shape during the polymerization of the guest molecules. Polymer nano-cylinders with dimensions less than 10 nm can thus be produced from the HEMA/POSS-AD micelles. Since the chemical structure of low-generation dendrimers and the contents of the co-assembled nano-reactors can be easily adjusted, the concept holds the potential for the further developments of low-generation amphiphilic dendrimers.

Analysis of root surface properties by fluorescence/Raman intensity ratio.

The aim of this study is to evaluate the existence of residual calculus on root surfaces by determining the fluorescence/Raman intensity ratio. Thirty-two extracted human teeth, partially covered with calculus on the root surface, were evaluated by using a portable Raman spectrophotometer, and a 785-nm, 100-mW laser was applied for fluorescence/Raman excitation. The collected spectra were normalized to the hydroxyapatite Raman band intensity at 960 cm-1. Raman spectra were recorded from the same point after changing the focal distance of the laser and the target radiating angle. In seven teeth, the condition of calculus, cementum, and dentin were evaluated. In 25 teeth, we determined the fluorescence/Raman intensity ratio following three strokes of debridement. Raman spectra collected from the dentin, cementum, and calculus were different. After normalization, spectra values were constant. The fluorescence/Raman intensity ratio of calculus region showed significant differences compared to the cementum and dentin (p < 0.05). The fluorescence/Raman intensity ratio decreased with calculus debridement. For this analysis, the delta value was defined as the difference between the values before and after three strokes, with the final 2 delta values close to zero, indicating a gradual asymptotic curve and the change in intensity ratio approximating that of individual constants. Fluorescence/Raman intensity ratio was effectively used to cancel the angle- and distance-dependent fluctuations of fluorescence collection efficiency during measurement. Changes in the fluorescence/Raman intensity ratio near zero suggested that cementum or dentin was exposed, and calculus removed.

Imaging phospholipid conformational disorder and packing in giant multilamellar liposome by confocal Raman microspectroscopy.

Liposomes are closed phospholipid bilayer systems that have profound applications in fundamental cell biology, pharmaceutics and medicine. Depending on the composition (pure or mixture of phospholipids, presence of cholesterol) and preparation protocol, intra- and inter-chain molecular interactions vary leading to changes in the quality (order and packing) of liposomes. So far it is not possible to image conformational disorders and packing densities within a liposome in a straightforward manner. In this study, we utilized confocal Raman microspectroscopy to visualize structural disorders and packing efficiency within a giant multilamellar liposome model by focusing mainly on three regions in the vibrational spectrum (CC stretching, CH deformation and CH stretching). We estimated properties such as trans/gauche isomers and lateral packing probability. Interestingly, our Raman imaging studies revealed gel phase rich domains and heterogeneous lateral packing within the giant multilamellar liposome.

Tilted Two-Dimensional Array Multifocus Confocal Raman Microspectroscopy.

A simple and efficient two-dimensional multifocus confocal Raman microspectroscopy featuring the tilted-array technique is demonstrated. Raman scattering from a 4 × 4 square foci array passing through a 4 × 4 confocal pinhole array is tilted with a periscope. The tilted array of Raman scattering signals is dispersed by an imaging spectrograph onto a CCD detector, giving 16 independent Raman spectra formed as 16 bands with different heights on the sensor. Use of a state-of-the-art imaging spectrograph enables high-precision wavenumber duplicability of the 16 spectra. This high duplicability makes the simultaneously obtained spectra endurable for multivariate spectral analyses, which is demonstrated by a singular value decomposition analysis for Raman spectra of liquid indene. Although the present implementation attains only 16 measurement points, the number of points can be extended to larger than 100 without any technical leaps. Limit of parallelization depends on the interval of measurement points as well as the performance of the optical system. Criteria for finding the maximum feasible number are discussed.

On-site Direct Detection of Astaxanthin from Salmon Fillet Using Raman Spectroscopy.

A new technology employing Raman spectroscopy is attracting attention as a powerful biochemical technique for the detection of beneficial and functional food nutrients, such as carotenoids and unsaturated fatty acids. This technique allows for the dynamic characterization of food nutrient substances for the rapid determination of food quality. In this study, we attempt to detect and measure astaxanthin from salmon fillets using this technology. The Raman spectra showed specific bands corresponding to the astaxanthin present in salmon and the value of astaxanthin (Raman band, 1518 cm-1) relative to those of protein/lipid (Raman band, 1446 cm-1) in the spectra increased in a dose-dependent manner. A standard curve was constructed by the standard addition method using astaxanthin as the reference standard for its quantification by Raman spectroscopy. The calculation formula was established using the Raman bands typically observed for astaxanthin (i.e., 1518 cm-1). In addition, we examined salmon fillets of different species (Atlantic salmon, coho salmon, and sockeye salmon) and five fillets obtained from the locations (from the head to tail) of an entire Atlantic salmon. Moreover, the sockeye salmon fillet exhibited the highest astaxanthin concentration (14.2 mg/kg), while coho salmon exhibited an intermediate concentration of 7.0 mg/kg. The Raman-based astaxanthin concentration in the five locations of Atlantic salmon was more strongly detected from the fillet closer to the tail. From the results, a rapid, convenient Raman spectroscopic method was developed for the detection of astaxanthin in salmon fillets.

Estimating Percent Crystallinity of Polyethylene as a Function of Temperature by Raman Spectroscopy Multivariate Curve Resolution by Alternating Least Squares.

We have recently demonstrated a methodology to estimate the percent crystallinity (PC) of polymers directly with Raman spectroscopy and multivariate curve resolution (MCR) by alternating least-squares (ALS). In the MCR-ALS methodology, the Raman spectrum of a semicrystalline polymer is separated into two constituent components (crystalline and molten/amorphous) and their corresponding concentrations. The methodology necessitates that the Raman spectrum at any temperature be a linear combination of two MCR spectral components (one molten and one crystalline). This is true in the case of simple systems such as crystalline pendant alkyl domains in polymers (Samuel et al. Anal. Chem. 2016, 88, 4644). However, in the case of main chain polymer crystals (e.g., polyethylene), the situation can be complicated owing to several molecular changes in the lattice in addition to conformational reorganizations during melting. Under this circumstance, a simple two-state model may not be adequate and we describe the modifications required to treat such systems, keeping the basic principles of the proposed methodology unchanged. A comparative study with wide-angle X-ray scattering (WAXS) and Raman spectroscopy is also performed to substantiate our findings. In addition to estimating percent crystallinity (PC), our methodology is capable of revealing additional information, such as interchain interactions in crystal lattice, that in principle will help distinguishing polymorphic transformations, subtle changes in lamellar lattice dimensions, and other phase changes in polymers.

Microscopic solvation environments in a prototype room-temperature ionic liquid as elucidated by resonance Raman spectroscopy of iodine and bromine.

Microscopic solvation environments in a prototype ionic liquid, bmimTf2N; 1-butyl-3-methyl-imidazolium-bis(trifluoromethanesulfonyl)imide, have been studied with the use of halides, X2 and Xn- (X=I, Br; n=3,5), as molecular probes. Resonance Raman spectroscopy has been used to detect these halogen species existing in bmimTf2N as well as in reference solvents including heptane, cyclohexane, KX/H2O and benzene. In heptane and cyclohexane, only free X2 species are detected. In KX/H2O, only Xn- and, in benzene, only benzene-X2 complexes are detected. On the contrary, free X2 and Xn- are concomitantly detected in bmimTf2N, indicating that there are two distinct solvation environments in bmimTf2N, non-polar environments that solvate free X2 and polar environments that stabilize Xn-. These two distinct solvation environments are most likely to arise from microscopic structural heterogeneity of ionic liquids.

Label-free Chemical Imaging of Fungal Spore Walls by Raman Microscopy and Multivariate Curve Resolution Analysis.

Fungal cell walls are medically important since they represent a drug target site for antifungal medication. So far there is no method to directly visualize structurally similar cell wall components such as α-glucan, ß-glucan and mannan with high specificity, especially in a label-free manner. In this study, we have developed a Raman spectroscopy based molecular imaging method and combined multivariate curve resolution analysis to enable detection and visualization of multiple polysaccharide components simultaneously at the single cell level. Our results show that vegetative cell and ascus walls are made up of both α- and ß-glucans while spore wall is exclusively made of α-glucan. Co-localization studies reveal the absence of mannans in ascus wall but are distributed primarily in spores. Such detailed picture is believed to further enhance our understanding of the dynamic spore wall architecture, eventually leading to advancements in drug discovery and development in the near future.

Determination of Percent Crystallinity of Side-Chain Crystallized Alkylated-Dextran Derivatives with Raman Spectroscopy and Multivariate Curve Resolution.

We demonstrate a methodology to estimate the percent crystallinity of polymers directly with Raman spectroscopy and multivariate curve resolution (MCR) by alternating least-squares (ALS). In this methodology, the Raman spectrum of semicrystalline polymer is separated into two constituent components (crystalline and molten) and their corresponding concentrations. The percent crystallinity can be estimated as the change in area intensity of the molten spectral-component when polymer cools from a temperature above melting point to room temperature. The number of carbons in the crystalline lattice has also been estimated from the position of longitudinal acoustic (LA) Raman bands with the correlation established by Mizushima and Simanouti [ Mizushima, S.; Simanouti, T. J. Am. Chem. Soc. 1949 , 71 , 1320 ]. The new method allows direct Raman estimation of absolute percent crystallinity of polymers. Until now, Raman spectroscopic estimation of percent crystallinity was possible only in conjunction with other techniques or by using internal standards.

Automatic and objective oral cancer diagnosis by Raman spectroscopic detection of keratin with multivariate curve resolution analysis.

We have developed an automatic and objective method for detecting human oral squamous cell carcinoma (OSCC) tissues with Raman microspectroscopy. We measure 196 independent Raman spectra from 196 different points of one oral tissue sample and globally analyze these spectra using a Multivariate Curve Resolution (MCR) analysis. Discrimination of OSCC tissues is automatically and objectively made by spectral matching comparison of the MCR decomposed Raman spectra and the standard Raman spectrum of keratin, a well-established molecular marker of OSCC. We use a total of 24 tissue samples, 10 OSCC and 10 normal tissues from the same 10 patients, 3 OSCC and 1 normal tissues from different patients. Following the newly developed protocol presented here, we have been able to detect OSCC tissues with 77 to 92% sensitivity (depending on how to define positivity) and 100% specificity. The present approach lends itself to a reliable clinical diagnosis of OSCC substantiated by the "molecular fingerprint" of keratin.