title: Cell observation method under near-living conditions by scanning electron microscopy.
abstract: Most cells of multicellular organisms have "primary cilia", which are single, non-motile, and sensory cilia. They have been reported to detect mechanical stimulation and transform it into internal cell, but the mechanisms are not still well known. Dermal papilla (DP) cells, which locate in the skin and regulate hair follicle development and hair cycle, were reported to have their primary cilia by immune-fluorescent method [1], but their detailed structure and function is unclear.For observation by scanning electron microscopy (SEM), biological specimens are conventionally fixed with glutaraldehyde and dehydrated in 30%, 50%, 70%, 90% and 100% ethanol. Then specimens are dried by butyl alcohol and coated with gold. It takes several days to prepare these specimens. Using many chemical reagent and many steps in this way may lead to destroy biological specimens structure. Here we attempted a recently proposed method using ionic liquid to prepare cell samples in near- living conditions observed the structure of DP cells (2D and clumps) with primary cilia.This time, we used ionic liquid for preparing specimens. First, cultured cells were fixed in glutaraldehyde, and immersed in ionic liquid. Next, the specimens were coated with gold and observed by SEM. Thus, it takes shorter time due to fewer step than conventional method and the process has no drying step. In a conventional way, we got the micrographs of 2D cultured DP cells and observed the cilium of DP cells (200-nm in diameter and 1.5um in length) on nucleus (15-um). In addition we could observe the clumps of DP cells and the cilia-like structure (∼12-um), but they do not attach to scaffoldings of the surface, probably due to drying. In observation using ionic liquid, we got the micrographs of 2D cultured DP cells and observed the cilium- like structure (200-nm in diameter and 2.1-um in length) on nucleus (30-um), as well. In this case, we could not find the cilia- like structure in the clumps of DP cells yet, but they well attached to the scaffoldings and kept the extending structure such as filopodia, too.We here observed DP cells and their cilia in near-living conditions. Unfortunately, we could not primary cilia in clumps of DP cells immersed in ionic liquid yet, but we could reduce damage receiving in the process of specimen's preparation, especially drying. In addition, we are challenging the observation using not only ionic liquid but also nano-suits by detergents [2] and the observation the cilia by SEM after identifying them by fluorescence microscopy, such as CLEM.
authors.affiliation: Dept. of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology 680-4 Kawazu, Iizuka, Fukuoka, Japan.
authors.forename: Misaki
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Dept. of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology 680-4 Kawazu, Iizuka, Fukuoka, Japan Saravio Cosmetics Ltd 1356-6 Oazatsurumi minoyashiki, Beppu, Oita.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Matsushima
authors.affiliation: Saravio Cosmetics Ltd 1356-6 Oazatsurumi minoyashiki, Beppu, Oita.
authors.forename: Kuniyoshi
authors.initials: K
authors.lastname: Kaseda
authors.affiliation: Dept. of Bioscience and Bioinformatics, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology 680-4 Kawazu, Iizuka, Fukuoka, Japan JST, SENTAN.
authors.forename: Takuo
authors.initials: T
authors.lastname: Yasunaga