Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy: Applicability to intraoperative cancer diagnosis

Memtily,Nassirhadjy; Okada,Tomoko; Ebihara,Tatsuhiko; Sato,Mari; Kurabayashi,Atsushi; Furihata,Mutsuo; Suga,Mitsuo; Nishiyama,Hidetoshi; Mio,Kazuhiro; Sato,Chikara

doi:10.3892/ijo.2015.2905

Observation of tissues in open aqueous solution by atmospheric scanning electron microscopy: Applicability to intraoperative cancer diagnosis

Authors:
- Nassirhadjy Memtily
- Tomoko Okada
- Tatsuhiko Ebihara
- Mari Sato
- Atsushi Kurabayashi
- Mutsuo Furihata
- Mitsuo Suga
- Hidetoshi Nishiyama
- Kazuhiro Mio
- Chikara Sato
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Affiliations: Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8568, Japan, Department of Pathology, Kochi Medical School, University of Kochi, Nankoku, Kochi 783-8505, Japan, Advanced Technology Division, JEOL Ltd., Akishima, Tokyo 196‑8558, Japan
Published online on: February 24, 2015 https://doi.org/10.3892/ijo.2015.2905
Pages: 1872-1882
Copyright: © Memtily et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

In the atmospheric scanning electron microscope (ASEM), a 2- to 3-µm layer of the sample resting on a silicon nitride-film window in the base of an open sample dish is imaged, in liquid, at atmospheric pressure, from below by an inverted SEM. Thus, the time-consuming pretreatments generally required for biological samples to withstand the vacuum of a standard electron microscope are avoided. In the present study, various mouse tissues (brain, spinal cord, muscle, heart, lung, liver, kidney, spleen and stomach) were fixed, stained with heavy metals, and visualized in radical scavenger D-glucose solution using the ASEM. While some stains made the nuclei of cells very prominent (platinum-blue, phosphotungstic acid), others also emphasized cell organelles and membranous structures (uranium acetate or the NCMIR method). Notably, symbiotic bacteria were sometimes observed on stomach mucosa. Furthermore, kidney tissue could be stained and successfully imaged in <30 min. Lung and spinal cord tissue from normal mice and mice metastasized with breast cancer cells was also examined. Cancer cells present in lung alveoli and in parts of the spine tissue clearly had larger nuclei than normal cells. The results indicate that the ASEM has the potential to accelerate intraoperative cancer diagnosis, the diagnosis of kidney diseases and pathogen detection. Importantly, in the course of the present study it was possible to increase the observable tissue area by using a new multi-windowed ASEM sample dish and sliding the tissue across its eight windows.

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