Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1966 Jun 1;29(3):435–447. doi: 10.1083/jcb.29.3.435

A SIMPLE FREEZE-FRACTURE REPLICATION METHOD FOR ELECTRON MICROSCOPY

Stanley Bullivant 1, Adelbert Ames 3rd 1
PMCID: PMC2106967  PMID: 5962938

Abstract

A simple method to achieve results similar to the freeze-etching technique of Moor et al. (1961) is described. The frozen tissue is cut under liquid nitrogen with a razor blade outside the evaporator rather than inside with a cooled microtome. The conditions of the experiment do not favor sublimation, and it is proposed that the structure of the replica be explained by local faults in the cleavage plane which leaves structures, such as membranes, standing above the ice. Micrographs of replicas of glycerol-protected frozen small intestine of mouse prepared by the method are presented and the structural details they show are discussed. The problem of vapor-deposited contamination is discussed. It is concluded that this is a practical method for obtaining electron micrographs that are relatively free of artifact, and that further improvements may be expected from the use of rapidly frozen fresh tissue and a clean vacuum system, possibly of the ion-pumped type.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BULLIVANT S. FREEZE SUBSTITUTION AND SUPPORTING TECHNIQUES. Lab Invest. 1965 Jun;14:1178–1195. [PubMed] [Google Scholar]
  2. DuPraw E. J. Macromolecular organization of nuclei and chromosomes: a folded fibre model based on whole-mount electron microscopy. Nature. 1965 Apr 24;206(982):338–343. doi: 10.1038/206338a0. [DOI] [PubMed] [Google Scholar]
  3. HAGGIS G. H. Electron microscope replicas from the surface of a fracture through frozen cells. J Biophys Biochem Cytol. 1961 Apr;9:841–852. doi: 10.1083/jcb.9.4.841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. HANZON V., HERMODSSON L. H., TOSCHI G. Ultrastructural organization of cytoplasmic nucleoprotein in the exocrine pancreas cells. J Ultrastruct Res. 1959 Dec;3:216–227. doi: 10.1016/s0022-5320(59)90016-4. [DOI] [PubMed] [Google Scholar]
  5. MOOR H. DIE GEFRIER-FIXATION LEBENDER ZELLEN UND IHRE ANWENDUNG IN DER ELEKTRONENMIKROSKOPIE. Z Zellforsch Mikrosk Anat. 1964 Apr 28;62:546–580. [PubMed] [Google Scholar]
  6. MOOR H., RUSKA C., RUSKA H. ELEKTRONENMIKROSKOPISCHE DARSTELLUNG TIERISCHER ZELLEN MIT DER GEFRIERAETZTECHNIK. Z Zellforsch Mikrosk Anat. 1964 May 15;62:581–601. [PubMed] [Google Scholar]
  7. Malhotra S. K., Van Harreveld A. Some structural features of mitochondria in tissues prepared by freeze-substitution. J Ultrastruct Res. 1965 Jun;12(5):473–487. doi: 10.1016/s0022-5320(65)80042-9. [DOI] [PubMed] [Google Scholar]
  8. REBHUN L. I. Applications of freeze-substitution to electron microscope studies of invertebrate oocytes. J Biophys Biochem Cytol. 1961 Apr;9:785–798. doi: 10.1083/jcb.9.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. SCHULTZ R. L., KARLSSON U. FIXATION OF THE CENTRAL NERVOUS SYSTEM FOR ELECTRON MICROSCOPY BY ALDEHYDE PERFUSION. II. EFFECT OF OSMOLARITY, PH OF PERFUSATE, AND FIXATIVE CONCENTRATION. J Ultrastruct Res. 1965 Feb;12:187–206. doi: 10.1016/s0022-5320(65)80015-6. [DOI] [PubMed] [Google Scholar]
  10. SJOSTRAND F. S., BAKER R. F. Fixation by freezing-drying for electron microscopy of tissue cells. J Ultrastruct Res. 1958 Apr;1(3):239–246. doi: 10.1016/s0022-5320(58)80005-2. [DOI] [PubMed] [Google Scholar]
  11. Trier J. S., Rubin C. E. Electron microscopy of the small intestine: a review. Gastroenterology. 1965 Nov;49(5):574–603. [PubMed] [Google Scholar]
  12. VANHARREVELD A., CROWELL J. ELECTRON MICROSCOPY AFTER RAPID FREEZING ON A METAL SURFACE AND SUBSTITUTION FIXATION. Anat Rec. 1964 Jul;149:381–385. doi: 10.1002/ar.1091490307. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES