Assessment of Embryonic Bioactivity through Changes in the Water Structure Using Near-Infrared Spectroscopy and Imaging.

We explored the influence of embryonic bioactivity on the water structure using near-infrared (NIR) spectroscopy and imaging. Four groups of Japanese medaka fish (Oryzias latipes) eggs were studied: (a) one group of eggs was activated by fertilization, and (b-d) three groups of eggs were not activated because embryogenesis was stopped or not started by (b) culturing under cold temperature, (c) instant freezing, or (d) lack of fertilization. The yolks of the activated eggs contained higher proportions of weakly hydrogen bonded water than those of nonactivated eggs. A possible factor responsible for the significant changes in the water structure was revealed to be a protein secondary structural change from an α-helix to a ß-sheet in the activated eggs. NIR images of the activated eggs successfully visualized the water structural variation in the yolk with a higher proportion of weak hydrogen bonds due to the activation of embryonic development. The embryogenic activity could be assessed through the water hydrogen bond network, which is affected by newly generated proteins with different secondary structures.

Nonstaining Blood Flow Imaging Using Optical Interference Due to Doppler Shift and Near-Infrared Imaging of Molecular Distribution in Developing Fish Egg Embryos.

In the present study, we successfully obtained nonstaining blood flow images of a developing fish egg embryo using optical interference caused by the Doppler shift. The spectral distribution of light reflected by moving objects such as the heart and red cells was found to be different from that of the incident light because of the Doppler effect. Interference between different frequency components was observed in an interferogram through heterodyne interaction using an imaging-type two-dimensional Fourier spectroscopic system, and information on the intensities of the spectral components was obtained by Fourier transformation. Beat signals with specific frequencies due to the heart beating and blood flow of the fish egg embryo were detected. When the signals were plotted in two dimensions, the heart part and vessel flows were clearly visualized without staining. In addition, near-infrared (NIR) images were produced using absorbance spectra of the molecular vibrations of O-H and C-H groups included in water, hydrocarbons, and aliphatic compounds. Obtaining nonstaining blood flow images using heterodyne optical interference and images of molecular distribution using molecular vibrational information simultaneously manifests an exciting advance in NIR imaging.

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.

Non-staining visualization of embryogenesis and energy metabolism in medaka fish eggs using near-infrared spectroscopy and imaging.

The energy metabolism and embryogenesis of fertilized Japanese medaka eggs were investigated in vivo at the molecular level using near-infrared (NIR) spectroscopy and imaging. Changes in chemical components, such as proteins and lipids, in yolk sphere and embryonic body were studied over the course of embryonic development. Metabolic changes that represent variations in the concentrations and molecular compositions of proteins and lipids in the yolk part, particularly on the 1st day after fertilization and the day just before hatching, were successfully identified in the 4900-4000 cm-1 wavenumber region. The yolk components were shown to have specific functions at the very early and final stages of the embryonic development. Proteins with α-helix- or ß-sheet-rich structures clearly showed the different variation patterns within the developing egg. Furthermore, the distribution of lipids could be selectively visualized using data from the higher wavenumber region. Detailed embryonic structures were clearly depicted in the NIR images using the data from the 6400-5500 cm-1 region in which the embryo parts had some characteristic peaks due to unsaturated fatty acids. It was made clear that yolk and embryo parts had different components especially lipid components. The present study provides new insights into material variations in the fertilized egg during its growth. NIR imaging proved to be valuable in investigating the embryogenesis in vivo at the molecular level in terms of changes in biomolecular concentrations and compositions, metabolic differentiation, and detailed information about embryonic structures without the need for staining.

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 ß-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.