Mika Ishigaki

Correlations between Structure and Near-Infrared Spectra of Saturated and Unsaturated Carboxylic Acids. Insight from Anharmonic Density Functional Theory Calculations

By near-infrared (NIR) spectroscopy and anharmonic density functional theory (DFT) calculations, we investigate five kinds of saturated and unsaturated carboxylic acids belonging to the group of short-chain fatty acids: propionic acid, butyric acid, acrylic acid, crotonic acid, and vinylacetic acid. The experimental NIR spectra of these five kinds of carboxylic acids are reproduced by quantum chemical calculations in a broad spectral region of 7500-4000 cm-1 and for a wide range of concentrations. By employing anharmonic GVPT2 calculations on DFT level, a detailed interpretation of experimental spectra is achieved, elucidating structure-spectra correlations of these molecules in the NIR region. We emphasize the spectral features due to saturated and unsaturated alkyl chains, the location of a C═C bond within the alkyl chain, and the dimerization of carboxylic acids. In particular, the existence of a terminal C═C bond leads to the appearance of highly specific NIR bands. These pronounced bands are located at wavenumbers where no overlapping with other structure-specific bands occurs, thus making them good structural markers. Most of the spectral differences between these two groups of molecules remain subtle, and would be difficult to reliably ascribe without quantum chemically calculated NIR spectra. Moreover, anharmonic DFT calculations provide insights on the manifestation of hydrogen bonding through distinctive spectral features corresponding to cyclic dimers. The resulting spectral baseline elevation is common for all five investigated carboxylic acids, and remains consistent with previous results on acetic acid.

Spectra-structure correlations of saturated and unsaturated medium-chain fatty acids. Near-infrared and anharmonic DFT study of hexanoic acid and sorbic acid

Quantum chemical reproduction of entire NIR spectra is a new trend, enabled by contemporary advances in the anharmonic approaches. At the same time, recent increase of the importance of NIR spectroscopy of biological samples raises high demand for gaining deeper understanding of NIR spectra of biomolecules, i.e. fatty acids. In this work we investigate saturated and unsaturated medium-chain fatty acids, hexanoic acid and sorbic acid, in the near-infrared region. By employing fully anharmonic density functional theory (DFT) calculations we reproduce the experimental NIR spectra of these systems, including the highly specific spectral features corresponding to the dimerization of fatty acids. Broad range of concentration levels from 5·10-4M in CCl4 to pure samples are investigated. The major role of cyclic dimers can be evidenced for the vast majority of these samples. A highly specific NIR feature of fatty acids, the elevation of spectral baseline around 6500-4000cm-1, is being explained by the contributions of combination bands resulting from the vibrations of hydrogen-bonded OH groups in the cyclic dimers. Based on the high agreement between the calculated and experimental NIR spectra, a detailed NIR band assignments are proposed for hexanoic acid and sorbic acid. Subsequently, the correlations between the structure and NIR spectra are elucidated, emphasizing the regions in which clear and universal traces of specific bands corresponding to saturated and unsaturated alkyl chains can be established, thus demonstrating the wavenumber regions highly valuable for structural identifications.

Near-Infrared Spectroscopy and Imaging Studies of Fertilized Fish Eggs: In Vivo Monitoring of Egg Growth at the Molecular Level

In this work, the growth of fertilized Japanese medaka (Oryzias latipes) eggs was monitored in vivo at the molecular level using near-infrared (NIR) spectroscopy and NIR imaging. NIR spectra were recorded noninvasively for three major parts of a fertilized medaka egg, the embryonic body, the oil droplets, and the yolk, from the first day after fertilization to the day before hatching. Principal component analysis (PCA) revealed that water, protein, and lipid contents in the egg yolk and oil droplets changed significantly just before hatching. The ratio of the characteristic peaks due to proteins and lipids in the second derivative spectra suggested that the relative concentration of proteins to lipids was constant in the egg yolk, while it dramatically increased just before hatching in the oil droplets. Furthermore, linear discriminant analysis (LDA) predicted the hatching possibility on the next day with 100% and 99.3% accuracy for yolk and oil droplets data, respectively. Two types of NIR images were developed in situ using the band intensities of the lipids and proteins in the second derivative spectra. The egg’s protein and lipid content was successfully visualized noninvasively. This technique should enable noninvasive quality testing of fertilized eggs in the future.

Unveiling the Aggregation of Lycopene in Vitro and in Vivo: UV–Vis, Resonance Raman, and Raman Imaging Studies

The present study investigates the structure of lycopene aggregates both in vitro and in vivo using ultraviolet-visible (UV-vis) and Raman spectroscopies. The electronic absorption bands of the J- and H-aggregates in vitro shift to lower and higher energies, respectively, compared to that of the lycopene monomer. Along with these results, the frequencies of the ν1 Raman bands were shifted to lower and higher frequencies, respectively. By plotting the frequencies of the ν1 Raman band against the S0 → S2 transition energy, a linear relationship between the data set with different aggregation conformations can be obtained. Therefore, the band positions depending on the different conformations can be explained based on the idea that the effective conjugated C═C chain lengths within lycopene molecules are different due to the environmental effect (site-shift effect) caused by the aggregation conformation. Applying this knowledge to the in vivo measurement of a tomato fruit sample, the relationship between the aggregation conformation of lycopene and the spectral patterns observed in the UV-vis as well as Raman spectra in different parts of tomato fruits was discussed in detail. The results showed that the concentration of lycopene (particularly that of the J-aggregate) specifically increased, whereas that of chlorophyll decreased, with ripening. Furthermore, Raman imaging indicated that lycopene with different aggregate conformations was distributed inhomogeneously, even within one sample. The layer formation in tomato tissues with high concentrations of J- and H-aggregates was successfully visualized. In this manner, the presence of lycopene distributions with different aggregate conformations was unveiled in vivo.

In Vivo Monitoring of the Growth of Fertilized Eggs of Medaka Fish (Oryzias latipes) by Near-Infrared Spectroscopy and Near-Infrared Imaging—A Marked Change in the Relative Content of Weakly Hydrogen-Bonded Water in Egg Yolk Just before Hatching

The present study develops further our previous study of in vivo monitoring at the molecular level of the embryonic development in Japanese medaka fish (Oryzias latipes) using near-infrared (NIR) spectroscopy and NIR imaging. NIR spectra were measured nondestructively for three major parts of fertilized medaka eggs (the embryonic body, oil droplets, and egg yolk) from the first day after fertilization to the day just before hatching (JBH). Changes in the contents of chemical components such as proteins, water, and lipids were monitored in situ during embryonic development. A marked change in the relative content of weakly hydrogen-bonded water was observed in the egg yolk JBH. Principal component analysis (PCA) was carried out using the NIR spectra data of the egg yolk and embryo on the fifth day after fertilization. The PCA clearly separates the egg yolk data from the embryo body parts. Principal component PC1 and PC2 loading plots suggest that the hydrogen bonding structure of water in the egg yolk is considerably different to those of the other parts and the fraction of weakly hydrogen-bonded water in the egg yolk is smaller than that in the embryonic body. NIR images developed from the intensities of peaks of second derivative spectra owing to water and proteins show their different distribution patterns. Images of the ratio of strongly and weakly hydrogen-bonded water confirmed that oil droplets and embryonic body parts have higher and lower ratios, respectively, of strongly hydrogen-bonded water than do the other parts. The images developed from the intensity of the peaks at 4864 and 4616 cm−1 related to the proteins indicated that the egg yolk contains a higher concentration of protein than do the other parts. The peaks at 5756 and 4530 cm−1 caused by the protein secondary structures of α-helix and β-sheet showed the configuration of the egg cell membrane. The present study might lead to new understanding at the molecular level regarding the growth of fertilized eggs and provides a new tool to visualize egg development in a nondestructive manner.

Comparison of multivariate analysis methods for extracting the paraffin component from the paraffin-embedded cancer tissue spectra for Raman imaging

This study aimed to extract the paraffin component from paraffin-embedded oral cancer tissue spectra using three multivariate analysis (MVA) methods; Independent Component Analysis (ICA), Partial Least Squares (PLS) and Independent Component - Partial Least Square (IC-PLS). The estimated paraffin components were used for removing the contribution of paraffin from the tissue spectra. These three methods were compared in terms of the efficiency of paraffin removal and the ability to retain the tissue information. It was found that ICA, PLS and IC-PLS could remove the paraffin component from the spectra at almost the same level while Principal Component Analysis (PCA) was incapable. In terms of retaining cancer tissue spectral integrity, effects of PLS and IC-PLS on the non-paraffin region were significantly less than that of ICA where cancer tissue spectral areas were deteriorated. The paraffin-removed spectra were used for constructing Raman images of oral cancer tissue and compared with Hematoxylin and Eosin (H&E) stained tissues for verification. This study has demonstrated the capability of Raman spectroscopy together with multivariate analysis methods as a diagnostic tool for the paraffin-embedded tissue section.

Discrimination of fish egg quality and viability by Raman spectroscopy

Egg or embryo quality is typically assessed by viewing the egg morphology and by observing the cleavage rate of the embryo. Assessment of egg or embryo quality based on bio-molecular information might lead to improved outcomes for aquaculture or in vitro fertilization (IVF) treatments. To assess the egg quality, we performed Raman spectroscopy on fish eggs from the Japanese medaka (Oryzias latipes). Good classification results from the yolk were obtained using principal component analysis (PCA) and linear discrimination analysis (LDA). The results show that the supply of oil energy starts immediately after fertilization and embryogenesis is initiated. The presence or absence of fertilization can be evaluated by fatty acid Raman bands with 95.7% accuracy. The Raman results show that the key factors that indicate the viability of fish eggs are amino acid production and carotenoid pigment deposition. The LDA algorithm based on the Raman bands of these substances indicates whether the development is normal or abnormal with 80.3% accuracy. These results show that Raman spectroscopy can be a powerful tool for non-invasive assessment and real time monitoring of fish egg quality and viability.

Non-destructive monitoring of mouse embryo development and its qualitative evaluation at the molecular level using Raman spectroscopy

Current research focuses on embryonic development and quality not only by considering fundamental biology, but also by aiming to improve assisted reproduction technologies, such as in vitro fertilization. In this study, we explored the development of mouse embryo and its quality based on molecular information, obtained nondestructively using Raman spectroscopy. The detailed analysis of Raman spectra measured in situ during embryonic development revealed a temporary increase in protein content after fertilization. Proteins with a β-sheet structure—present in the early stages of embryonic development—are derived from maternal oocytes, while α-helical proteins are additionally generated by switching on a gene after fertilization. The transition from maternal to embryonic control during development can be non-destructively profiled, thus facilitating the in situ assessment of structural changes and component variation in proteins generated by metabolic activity. Furthermore, it was indicated that embryos with low-grade morphology had high concentrations of lipids and hydroxyapatite. This technique could be used for embryo quality testing in the future.

High-Speed Scanning for the Quantitative Evaluation of Glycogen Concentration in Bioethanol Feedstock Synechocystis sp. PCC6803 Using a Near-Infrared Hyperspectral Imaging System with a New Near-Infrared Spectral Camera

In the present study, the high-speed quantitative evaluation of glycogen concentration accumulated in bioethanol feedstock Synechocystis sp. PCC6803 was performed using a near-infrared (NIR) imaging system with a hyperspectral NIR spectral camera named Compovision. The NIR imaging system has a feature for high-speed and wide area monitoring and the two-dimensional scanning speed is almost 100 times faster than the general NIR imaging systems for the same pixel size. For the quantitative analysis of glycogen concentration, partial least squares regression (PLSR) and moving window PLSR (MWPLSR) were performed with the information of glycogen concentration measured by high performance liquid chromatography (HPLC) and the calibration curves for the concentration within the Synechocystis sp. PCC6803 cell were constructed. The results had high accuracy for the quantitative estimation of glycogen concentration as the best squared correlation coefficient R2 was bigger than 0.99 and a root mean square error (RMSE) was less than 2.9%. The present results proved not only the potential for the applicability of NIR spectroscopy to the high-speed quantitative evaluation of glycogen concentration in the bioethanol feedstock but also the expansivity of the NIR imaging instrument to in-line or on-line product evaluation on a factory production line of bioethanol in the future.

Noninvasive, high‐speed, near‐infrared imaging of the biomolecular distribution and molecular mechanism of embryonic development in fertilized fish eggs

In this study, the distribution of biomaterials and its molecular mechanism of embryonic development in Japanese medaka fish were analyzed nondestructively and noninvasively without staining using near-infrared (NIR) imaging. The microscopic NIR imaging system used in this research was a device capable of ultra-high-speed imaging; using this system, one can acquire microscopic imaging data in a few seconds. Therefore, the medaka eggs remained alive throughout measurements and were successfully monitored in vivo. The distributions of biomolecules were examined by mapping the intensities of NIR bands resulting from lipids, proteins and water in 2 dimensions (2D). The structures of eyes, lipid bilayer membranes, micelles and water-structure differences at the interface of different substances constituting different structures on the egg were visualized. Furthermore, insights on the metabolic mechanisms of lipids and membrane functions were drawn from the biased distribution of lipoproteins and the presence of unsaturated fatty acids in the egg membrane. These results indicated the potential for NIR imaging in evaluating the biological functions and metabolic systems of cells and embryos.