Use of ionic liquid for X-ray micro-CT specimen preparation of imbibed seeds

Microscopy, 2019, 92–97 doi: 10.1093/jmicro/dfy130 Advance Access Publication Date: 13 November 2018 Technical Report Use of ionic liquid for X-ray micro-CT specimen preparation of imbibed seeds Daisuke Yamauchi1, Aki Fukuda1, Tomonori Nakai1, Ichirou Karahara2, Miyuki Takeuchi1,3, Daisuke Tamaoki1,2, Tetsuya Tsuda4, Katsuhiko Tsunashima5, Susumu Kuwabata4, Masato Hoshino6, Kentaro Uesugi6, Akihisa Takeuchi6, Yoshio Suzuki6,7, and Yoshinobu Mineyuki1,* 1 Graduate School of Life Science, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2280, Japan, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama, Toyama 930-8555, Japan, 3Graduate School of Agricultural and Life Sciences, The university of Tokyo, 1-1-1 Yayoi, Bunkyou-ku, Tokyo 113-8657, Japan, 4Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, 5National Institute of Technology, Wakayama College, 77 Noshima, Nada-cho, Gobo, Wakayama 644-0023, Japan, 6Japan Synchrotron Radiation Research Institute, 1-1-1 Kouto, Sayo, Sayo-gun, Hyogo 679-5198, Japan, and 7Present address: Graduate School of Frontier Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8561, Japan 2 * To whom correspondence should be addressed. E-mail: Received 30 August 2018; Editorial Decision 18 October 2018; Accepted 29 October 2018 Abstract X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained. Key words: Arabidopsis imbibed seed, ionic liquid, SPring-8, 3D tissue imaging, cell structure in seeds, X-ray micro-CT Plants are composed of various types of cells. The way that these cells contribute to global body shaping is an important question in the study of plant development. Plant cells are surrounded by rigid cell walls and newly formed cells are not able to move easily because the geometrical relationships among neighboring cells are already firmly established during plant development [1]. Seed germination is one of the key processes in plant morphogenesis. Generally, seed germination is initiated with rapid water uptake. The cell volume increase caused by the water uptake contributes to the morphological changes in imbibing seeds, before the cells begin to divide [2]. Although cell and organ shapes © The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: 92 Microscopy, 2019, Vol. 68, No. 1 (Heraeus Chemicals, Port Elizabeth, South Africa) diluted in distilled water at 4°C for 1 day. After washing with distilled water three times, the samples were (1) placed in a well of an opened 4-well dish covered with a dry Kimwipe and kept at RT for 3 days (air-dried (AD), Fig. 1c), (2) freezedried (FD), or (3) treated with various concentrations of ILs as described below. To prepare the FD specimen, the OsO4-fixed imbibed seeds were washed with distilled water three times, dehydrated in an ethanol series (10%, 30%, 50%, 60%, 70%, 80%, 90%, 100% and 100% for 10 min at RT), and then kept in 100% ethanol for 1 day. Later, the ethanol was gradually replaced by t-butanol (25%, 50%, 75%, 100% and 100% for 15 min at 40°C). After removing the excess t-butanol, the samples were frozen for 15 min at 0°C and degassed at 0°C for 1 h. The samples were kept at RT for more than 3 days before observation. To prepare IL-treated specimens, the fixed seeds were immersed in various concentrations of ILs dissolved in water in 2-ml microtubes at 4°C for 1 day. Seeds were then transferred into a well of a 4-well dish. The excess amount of IL solution around the seeds was wiped off lightly using a piece of filter paper. The dish was covered with a dry Kimwipe and was kept at RT for 3 days. To examine the effect of long-term immersion in ILs, a few seeds were immersed in 100% IL for 4 days at 4°C. To prepare a step-by-step increase in the concentration of ILs, the fixed seeds were incubated through an IL series (15%, 30% and 60% in distilled water for 25 min at 4°C) and immersed in 100% IL for 1 day at 4°C. The seeds were then transferred into a well of a 4-well dish. The dish was covered with a dry Kimwipe and kept at RT for 3 days before observation. Images of dry seeds (Fig. 1a), 1 h-imbibed seeds (Fig. 1b), and seeds after drying (Fig. 1c) or after treatments were taken using a stereomicroscope (MZ16 F, Leica Microsystems, Weiziar, Germany). Seed size was compared in terms of seed area in the images. Area was determined using the software Fiji (http://fiji.sc/Fiji). Following the method described in Yamauchi et al. [5], we obtained a series of images using X-ray micro-CT equipment at BL20B2 (10 keV, 2.76 μm/pixel) [9], BL20XU (8 keV, 0.50 μm/pixel) [10] or BL47XU (8 keV, 0.522 μm/pixel) [3] of the SPring-8 synchrotron radiation facility. The convolution back projection method was used for tomographic reconstruction [11]. Tomographic slices were acquired using IMOD software [12]. To determine the best treatment method for preventing shrinkage during treatments, we recorded images of dry seeds (Fig. 1a), 1 h-imbibed seeds (Fig. 1b), and seeds after drying (Fig. 1c) by stereomicroscopy. To estimate changes in seed size during experimental procedures quantitatively, the area in the 2D image of each seed was measured. The change during increase in cell volume, the mechanism by which shaping of individual cells contributes to organ shape remains unclear. X-ray computed tomography (CT) is a tool that enables the examination of the complex 3D organization of cells within tissues. Some X-ray CT methods dedicated to a microscopic application called X-ray micro-CT, have achieved resolutions at the sub-micron level [3,4]. Previously, we reported the observation of dry Arabidopsis seeds using refraction contrast X-ray CT equipment available at the SPring-8 synchrotron radiation facility and certain cell geometrical studies are in progress [5]. However, there are difficulties in the examination of imbibed seeds in vivo because immersion in water and the subsequent rapid seed swelling decrease the image quality of seed tissues. In addition, the vaporization of water during image acquisition causes deformation of the specimens. Sample preparation methods for scanning electron microscopy (SEM), such as dehydration after chemical fixation, are also applicable to X-ray micro-CT observation [6]. However, shrinkag (...truncated)