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

Abstract

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.

Lipid Droplet Composition Varies Based on Medaka Fish Eggs Development as Revealed by NIR-, MIR-, and Raman Imaging

“…A peak at 4336 cm −1 in PC2 assigned to C-H components, shown in Figure 2, indicated the variation of interactions between water and biomolecules. Yolk and oil droplet became clearer than embryonic body by PC2 (Figure 1c), which was consistent with the previous works by Ishigaki et al [14,15]. The interesting image was constructed by PC3 and it seemed to show the component of unsaturated FA as representative peaks at 5832, 5672, 4588, 4340, and 4236 cm −1 (Figure 1d).…”

“…[8][9][10][11][12]. Despite the fact that some reports described oil droplet size and their distribution in Oryzias latipes eggs, distinct changes in lipid components in developing eggs have not been identified yet [3,[13][14][15]. Oil droplet visualization can be performed with use of these label-free spectroscopic techniques employing imaging mode.…”

“…Although NIR imaging enables measurement of a whole egg in a water environment and to determine biochemical components distribution, it is often difficult to interpret NIR spectra and to study subtle changes in chemical composition on contrary to mid-infrared (MIR) and Raman spectra that provide a fingerprint of molecules giving detailed information about the composition and molecular structure of a sample. However, illumination of a sample with MIR and laser (in Raman) light cannot penetrate sample as deeply as in NIR imaging, so to perform MIR and Raman imaging, thick samples need to be cross-sectioned [14]. Taking into account a diversity of molecular information gathered from Raman, NIR, and MIR spectra, we performed for the first time a deep analysis in macro-and micro-scale of distribution and composition of oil droplets in developing Oryzias latipes eggs (Scheme 1).…”

Near-Infrared Spectroscopy in Bio-Applications

“…NIR spectral bands of proteins carry essential information that enables monitoring complex biological processes in vivo, such as embryonic development [89,90]. In particular, NIR bands are sensitive to hydrogen bonding and thus, to the properties of such molecules in their native aqueous environment as well [3].…”

“…Fatty acids are often the major chemical constituent in a biological sample. Even when not considered as targeted molecules, due to their abundance, the NIR bands from fatty acids may obscure the target signal, e.g., from proteins [89]. Hence, understanding of the NIR spectra of fatty acids is helpful in such cases.…”