Abstract
High pressure freezing (HPF) can be fixed the fission yeast cells as vitrified bulk specimens constantly. HPF-freeze substitution method can preserve the fine structures of cell organelles such as cell membranes and cytoskeletons. We identified the presence of the autophagic pathway in fission yeast. We successfully applied the HPF ultra-low temperature low voltage scanning electron microscopy (SEM) and HPF-fracture SEM to observe the fine structures of fission yeast. As the results, the 3D structure of the yeast during cell cycle can be indicated. We also developed HPF-freeze substitution method for immuno-electron microscopy of fission yeast for molecular anatomic analysis. By using ultra-low concentration of fixative, fine structure of the cells was visualized and simultaneously antigen was detected on the same thin section. This method revealed that beta-1, 3-glucan synthase is in fact located on the cell membrane of the septum. And the electron dense area beneath the cell membrane was firstly appeared in the future site of invagination for the initiation of the septum. We showed various ultra-structural methods for fission yeast, Schizosaccharomyces pombe, using HPF.
