Abstract
Recent advances in bio-medical research, such as the production of regenerative organs from stem cells, require three-dimensional analysis of cell/tissue architectures. High-resolution imaging by electron microscopy is the best way to elucidate complex cell/tissue architectures, but the conventional method requires a skillful and time-consuming preparation. The present study developed a three-dimensional survey method for assessing cell/tissue architectures in 30-µm-thick paraffin sections by taking advantage of backscattered electron imaging in a low-vacuum scanning electron microscope. As a result, in the kidney, the podocytes and their processes were clearly observed to cover the glomerulus. The 30 µm thickness facilitated an investigation on face-side (instead of sectioned) images of the epithelium and endothelium, which are rarely seen within conventional thin sections. In the testis, differentiated spermatozoa were three-dimensionally assembled in the middle of the seminiferous tubule. Further application to vascular-injury thrombus formation revealed the distinctive networks of fibrin fibres and platelets, capturing the erythrocytes into the thrombus. The four-segmented BSE detector provided topographic bird’s-eye images that allowed a three-dimensional understanding of the cell/tissue architectures at the electron-microscopic level. Here, we describe the precise procedures of this imaging method and provide representative electron micrographs of normal rat organs, experimental thrombus formation, and three-dimensionally cultured tumour cells.
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
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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