Good Ultrastructural Preservation of Human Tissues and Cultured Cells by Glutaraldehyde Fixation, Sandwich Freezing, and Freeze-Substitution

Abstract

Sandwich freezing (freezing the specimen rapidly with liquid propane by placing it between two copper disks) and freeze-substitution of living yeast cells has been used for observing exquisite close-to-native ultrastructure of cells. Glutaraldehyde fixation, sandwich freezing, and freeze substitution of bacteria and other microorganisms also yield close-to-native ultrastructure. Here, we have used glutaraldehyde fixation, sandwich freezing, and freeze substitution to observe human cells and tissues. We obtained clear and natural cell images of tissues sliced to 0.2 mm thickness. This is a remarkable result because, in the past, tissues as thick as 0.2 mm could only be frozen by high-pressure freezing. The present study has shown that it is possible to observe clear and natural cell structures in animal and human tissues anytime because glutaraldehyde-fixed tissues can be stored at 4 C for several months before freezing, and a sandwich-freezing device will soon become commercially available. Also, natural ultrastructure of cultured cells in suspension was found to be observed more clearly by glutaraldehyde-fixation, sandwich freezing, and freeze-substitution than sandwich freezing and freeze substitution of living cells. The present method should be used as a standard method to observe the close-to-native ultrastructure of animal and human tissues.

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

“…Glutaraldehyde fixation was performed for animal or human tissues before sandwich freezing to obtain ice-crystal-free ultrastructure. The micrographs obtained by this method show exquisitely clear images similar to the ones obtained by the rapid freezing of living tissues (Figure 14) 1 . The studies on yeast cells, deep-sea microorganisms, and cultured cells show that the deformation of ultrastructure is mainly due to osmium tetroxide fixation and dehydration by ethanol at room temperature 1 , 17 , 18 , 28 .…”

“…Figure 13 shows ultrathin sections of cultured cells (Figure 13A-C) 1 , 28 and isolated cells from mouse abdominal cavity (Figure 13D) 28 . Figure 14 shows ultrathin sections of human skin (Figure 14A-D) and buffy coat (Figure 14E) 1 . Note that the images are also very clear and show natural morphology.…”

“…The close-to-native ultrastructure of cells is best observed by rapid freezing and freeze-substitution of living cells. Ice crystal formation with the SFD can be avoided by limiting the thickness of cells to ≤30 µm 1 . Fixing tissues with glutaraldehyde often yields better preservation of the cell structure for observing suspension-cultured cells because glutaraldehyde fixation makes the cell structure more rigid and prevents the possible ultrastructural changes during collection and centrifugation of living cells 1 .…”

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

“…by rapid freezing and freeze-substitution of living cells. Ice crystal formation with the SFD can be avoided by limiting the thickness of cells to ≤30 µm1 . Fixing tissues with glutaraldehyde often yields better preservation of the cell structure for observing suspension-cultured cells because glutaraldehyde fixation makes the cell structure more rigid and prevents the possible ultrastructural changes during collection and centrifugation of living cells1 .…”

“…Ice crystal formation with the SFD can be avoided by limiting the thickness of cells to ≤30 µm1 . Fixing tissues with glutaraldehyde often yields better preservation of the cell structure for observing suspension-cultured cells because glutaraldehyde fixation makes the cell structure more rigid and prevents the possible ultrastructural changes during collection and centrifugation of living cells1 . Glutaraldehyde fixation also allows the extension of the freezing depth to as much as 0.2 mm 1 , similar to that achieved by the high-pressure freezing (HPF) method.…”