Optical Sum Frequency Generation Image of Rice Grains

We observed optical sum frequency generation (SFG) images of cross-sections of glutinous rice grains, in order to test a possibility of the SFG microscopy as a tool for monitoring polysaccharide species in rice grains. The SFG response in the CH vibration range was the most intense in the crush cell layer at the edge of the endosperm adjacent to the embryo probably due to optical reflection and scattering effectby the rugged dielectric structure of the crush cell layer. The SFG spectra as a function of the infrared wavelength depended on the measurement position in the endosperm. The SFG results were compared with those by Raman and infrared spectroscopies for the same samples.

• Optical Sum Frequency Generation Microscopy; <i>Oryzaglutinosa</i> (Rice); Amylopectin; Starch; Crush Cell Layer; Endosperm

1. Introduction

Vibrational spectroscopy is a useful tool for characterizing biomaterials. The most frequently used are infrared (IR) absorption and Raman spectroscopies, and they give different information from each other because of their different symmetry selection rules [1-4]. Second-order nonlinear optical response has been also proposed as a viable tool for analyzing biological samples. By optical second harmonic generation (SHG) microscopy one gets images of non-centrosymmetric parts in biological samples without their pretreatment and damage [5,6]. By optical sum frequency generation (SFG) spectroscopy one can generally distinguish between different biomaterial species.

In this study we analyze SFG from the endosperm of glutinous rice grains in the CH vibrational range in order to check if the SFG can be a tool for observing the polysaccharide distribution in them. In a SFG phenomenon two light beams with different photon energies and irradiate the medium and another beam with their sum photon energy is emitted. SFG occurs in a medium without inversion symmetry and it detects chirality in the bulk medium. By using the resonance characteristics of one IR beam with photon energy to molecular vibrations in the medium, one can distinguish between different molecular species. An optical sum frequency microscopic image is obtained by mapping the distribution of the molecular vibration.

The demonstration of the SFG microscopy was performed for the first time by Flörsheimer and his coworkers for Langmuir-Blodget films in 1999 [7]. Only a few number of SFG image observations of living organisms have been done afterwards. As for a botanical system, Miyauchi and his coworkers were the first to carry out the SFG microscopy [8]. Their sample was a water plant Charafibrosa. Inoue and his coworkers reported an SFG microscopy observation of an onion root cell [9]. In order to develop the application range of this microscopy, we further attempted to monitor the polysaccharide distribution in the cross-sections of rice grains in this study.

Our another trigger for observing rice grains was the fact that we observed intense SHG and SFG from starch from living Chara in our previous studies [8,10]. The structure of α-D-glucopyranose constituting the polysaccharides in rice has chirality and lacks inversion symmetry. In the starch crystalline domains, amylopectin chains have orderly structures and have intense SHG and SFG response [8]. Starch consists of amylopectin and amylose. The starch of non-glutinous rice consists of 80% amylopectin and 20% amylose, while that of the glutinous rice is almost 100% amylopectin [11]. Miyauchi and his coworkers proposed that SFG in Chara originates from amylopectin [8]. Zhuo and his coworkers [6] proved SHG occurs in amylopectin in rice. We chose glutinous rice as our sample, since stronger SFG is expected from it than from non-glutinous rice.

Monitoring the distribution of polysaccharides in rice grains during their growth should be a very important research topic in order to improve the breed of rice. After the rice flower gets fertilized, the endosperm nucleus in the ovary grows into a full endosperm and then the rice ear grows. Enzymes make small polysaccharide clusters grow into groups of amylopectin and amylose and then starch granules. Here it is a hot research topic to control the functions of the relevant enzymes using biotechnology and produce various kinds of useful starches [12,13]. Understanding the growth or digestion process of starch in the growing or germinating ovary can contribute to the controlling technology of the starch structure and the breed improvement of rice against cold or bad environment. SFG response is sensitive to the higher order structure of polysaccharide [8,9], so the attempt to analyze rice grains by SFG microscopy can be a good step to contribute to rice science and technology.

The purpose of this study is to check the capability of the SFG microscopy of observing the distribution of density and compositions of polysaccharides in the cross sections of glutinous rice grains. From this viewpoint we have also performed Raman and IR spectroscopies of the same samples, in order to check the difference between the information provided by SFG and these other vibrational spectroscopies. As a future potential topic of research we are also interested in studying the transformation of starch in their growth and digestion.

2. Materials and Methods

Figure 1 shows the setup of the optical sum frequency generation confocal microscope [14]. As a visible light source of wavelength 532 nm (photon energy 2.33 eV) we used a doubled frequency output from a mode-locked cavity-dumped Nd³⁺: YAG laser operating at a repetition rate of 10 Hz. As a light source of wavelength-tunable IR light we used an output with wavelength ~3.4 µm (wave number ~2950 cm^–1) from an optical parametric generator and amplifier system (OPG/OPA) driven by the same Nd³⁺: YAG laser. In the following we use eV and cm^–1 units to indicate the photon energy of the visible light and the wave number of the IR light, respectively. The band width of the IR light was narrower than 6 cm^–1. The visible light of photon energy 2.33 eV passed through a

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