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      A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy.

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      Journal of electron microscopy

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          Abstract

          Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.

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          Author and article information

          Journal
          J Electron Microsc (Tokyo)
          Journal of electron microscopy
          1477-9986
          0022-0744
          Apr 2012
          : 61
          : 2
          Affiliations
          [1 ] Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan. dk1224@med.niigata-u.ac.jp
          Article
          dfr098
          10.1093/jmicro/dfr098
          22257590
          13f6af71-ad5a-49a4-ae9a-902c4c590b6a
          History

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