Background: Field-emission scanning electron microscopy (FE-SEM) enables us to observe nano-sized objects with great depth of focus and high resolution. However, the observation of biological samples including colorectal tissues using an FE-SEM has been difficult because it requires to evacuate its inside to prevent electron scattering, therefore all organisms containing ca. 70% water are rapidly evaporated and consequently caused structural disruption and collapse. To overcome the limitations of the conventional SEM, equipment such as low-vacuum scanning electron microscopy and environmental scanning electron microscope was developed. However, they are not reliable enough to investigate living organisms or wet tissues with high resolution level. We have recently reported that a simple surface modification consisting of a thin extra layer, coined the term ‘NanoSuit®’, can keep organisms alive in the high vacuum (10−3 to 10−7 Pa) of the SEM. We now modified the technique and developed a new solution, which enables FE-SEM observations of wet tissues. In this study, we utilized this technique to observe real images of colorectal cancers and their adjacent normal mucosae at high resolution.

Materials and methods: Colorectal cancer tissues and their adjacent normal mucosa were cut with a scalpel from specimens surgically resected. All patients enrolled in this study provided written informed consent. Observations were carried out with an FE-SEM (S-4800, Hitachi or JEM-7100F, JEOL, Japan) at acceleration voltage of 1.0 kV. The vacuum level of the observation chamber was 10-3 - 10-7 Pa. The newly developed surface shield enhancer (SSE) solution was used to make NanoSuit® for wet tissue observation. To form the NanoSuit®, the specimens were dipped for 1 min into the SSE solution and blotted briefly thereafter put on dry filter paper to remove excess solution. Specimens were then introduced directly into an FE-SEM to form a NanoSuit® following irradiation of the electron beam.

Results: Fine structures of intestinal crypt and villi were observed in normal colon mucosa using FE-SEM with a NanoSuit®, whereas fixed specimens prepared with conventional method showed obvious structural damage. Comparing with the region of normal colon mucosa, colorectal cancer lesion had relatively amorphous surface, therefore the border between non-cancerous mucosa and cancer lesions in colon tissues are able to be distinguished under high magnification. In addition, fiber-like structure was observed at the border between noncancerous mucosa and cancer lesions, suggesting an invasive front of colorectal cancer.

Conclusions: We successfully observed the real mucosal surfaces and cancer lesions of colorectum with high resolution by an FE-SEM using a newly developed vacuum-proof suit, the “NanoSuit®”. This novel technique will enable us to investigate further physiopathology of GI tract including cancers.

Citation Format: Hirotoshi Kikuchi, Tomohiro Matsumoto, Takanori Hiraide, Yusuke Ozaki, Amane Hirotsu, Tomohiro Murakami, Toshiki Kawabata, Yoshihiro Hiramatsu, Kinji Kamiya, Takanori Sakaguchi, Yasuharu Takaku, Isao Ohta, Takahiro Hariyama, Hiroyuki Konno. Direct observation of colorectal cancers using field-emission scanning electron microscopy with a thin polymer membrane, the NanoSuit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1862. doi:10.1158/1538-7445.AM2017-1862