Informative Three‐dimensional Survey of Cell/tissue Architectures in Thick Paraffin Sections by Simple Low‐vacuum Scanning Electron Microscopy

Takeshi Kamimura,Nobuyasu Takahashi,Akira Sawaguchi,Yujiro Asada,Kaori Ichikawa,Fimiyo Aoyama,Atsushi Yamashita

doi:10.1096/fasebj.2018.32.1_supplement.642.5

Takeshi Kamimura, Nobuyasu Takahashi + Show 5 more

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https://doi.org/10.1096/fasebj.2018.32.1_supplement.642.5

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Abstract

Recent advances in bio-medical researches, such as the production of regenerative organ from stem cells, require three-dimensional analysis in those cell/tissue architectures. High-resolution imaging by electron microscopy remains superior to elucidate complex cell/tissue architectures, but the conventional method requires skillful and time-consuming preparation. The present study developed a three-dimensional survey of the cell/tissue architectures in 30 μm-thick paraffin sections, by taking the advantages of backscattered electron imaging in low-vacuum scanning electron microscope. As results, in the kidney, the podocytes and their processes were clearly observed covering the glomerulus. The 30 μm-thickness facilitated investigation on a face-side, instead of sectioned, image of the epithelium and endothelium that is rarely seen within conventional thin section. In the testis, differentiated sperms were three-dimensionally assembled in the middle of seminiferous tubule. Further application to vascular injury thrombus formation revealed the distinctive networks of fibrin fibers and platelets, capturing the erythrocytes into the thrombus. The four segmented BSE detector provided topographic bird's-eye images for three-dimensional understanding of the cell/tissue architectures at electron microscopic level. Here we describe the precise procedures accompanied by representative electron micrographs of normal rat organs, experimental thrombus formation, and three-dimensionally cultured tumor cells. Support or Funding Information Ministry of Education, Culture, Sports, Science and Technology(MEXT), Japan Three-dimensional survey of cell/tissue architectures in thick paraffin sections by simple low-vacuum scanning electron microscopy This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Highlights

One of the major goals of biological microscopy is to elucidate the structural evidence with which we can correlate functional activity
The application provided a simple three-dimensional survey method of the cell/tissue architectures embedded in the thick paraffin section, by which the missing third dimension could be viewed
The sections were floated in a water bath (PS-125WH, Sakura Finetek Japan, Tokyo, Japan) at 40 °C to be mounted onto New Silane II-coated microscope slides (Fig. 1b), and they were extended on a slide warmer (PS-53, Sakura Finetek Japan) heated at 50 °C for 10 seconds (Fig. 1c)

Summary

Materials and Methods

The excised femoral arteries were further fixed in 4% paraformaldehyde for 12 hours at 4 °C and dehydrated specimens were longitudinally embedded in paraffin to be cut into 5 or 30 μm in thickness as described above. Prior to the cultivation, 1 ml of collagen gel was poured into each well of a Cell Culture Insert (6-well format, pore size 3.0 μm: Falcon, New York, USA) combined with a microplate (Iwaki, Shizuoka, Japan), and solidified within an incubator at 37 °C for 30 min. The culture medium was changed in every other day, and approximately eight weeks later, the cultivated SUIT-58 cells among the collagen gel layers were fixed with 4% paraformaldehyde in 0.1 M PB for 1 hour and processed for paraffin embedment.

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