title: Three-Dimensional Imaging of Plant Organs Using a Simple and Rapid Transparency Technique.
abstract: Clearing techniques eliminate factors that interfere with microscopic observation, including light scattering and absorption by pigments and cytoplasmic components. The techniques allow fluorescence-based detailed analyses of materials and characterization of the three-dimensional structure of organs. We describe a simple and rapid clearing and imaging method, termed 'TOMEI' (Transparent plant Organ MEthod for Imaging), which enables microscopic observation of intact plant organs. This method involves a clearing reagent containing 2,2'-thiodiethanol. Conveniently, transparent plant organs were prepared within only 3-6 h. We detected fluorescent stains at a depth of approximately 200 µm using confocal laser scanning microscopy and analyzed fluorescent proteins in internal tissues of transparent organs cleared using TOMEI. We adapted TOMEI for various plant organs of Arabidopsis thaliana and Oryza sativa, including leaves, flower buds, flower stalks, root and nematode-infected root-knots. We visualized whole leaves of A. thaliana from the adaxial epidermis to the abaxial epidermis as well as protoxylem and metaxylem vessels of vascular bundles embedded in spongy mesophyll cells. Inner floral organs were observed in flower buds cleared using TOMEI without the need to prepare sections or remove sepals. Multicolor imaging of fluorescent proteins and dyes, and analyses of the three-dimensional structure of plant organs based on optical sections are possible using TOMEI. We analyzed root-knots cleared using TOMEI and revealed that nematodes induce giant cell expansion in a DNA content-dependent manner. The TOMEI method is applicable to analysis of fluorescent proteins and dyes quantitatively with cell morphological characteristics in whole plant organs.
authors.affiliation: Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510 Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Hasegawa
authors.affiliation: Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510 Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sakamoto
authors.affiliation: Graduate School of Science and Technology, Kumamoto University, Kumamoto, Kumamoto, 860-8555 Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Nakagami
authors.affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192 Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Aida
authors.affiliation: Graduate School of Science and Technology, Kumamoto University, Kumamoto, Kumamoto, 860-8555 Japan.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Sawa
authors.affiliation: Department of Applied Biological Science, Graduate School of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510 Japan sachi@rs.tus.ac.jp.
authors.forename: Sachihiro
authors.initials: S
authors.lastname: Matsunaga
mesh_terms: D017360:Arabidopsis / Q000033:anatomy & histology*; D048429:Cell Size; D018744:DNA, Plant / Q000378:metabolism; D035264:Flowers / Q000033:anatomy & histology; D005453:Fluorescence; D021621:Imaging, Three-Dimensional / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009928:Organ Specificity; D012275:Oryza / Q000033:anatomy & histology*; D059828:Plant Cells / Q000378:metabolism; D018517:Plant Roots / Q000033:anatomy & histology; D013194:Staining and Labeling
chemical_list: D018744:DNA, Plant
keywords: 2,2′-Thiodiethanol; Clearing method; Deep imaging; Fluorescent protein; Transparency technique