Structural Visualization of Mitotic Cycle by Three-Dimensional Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) with Nanoscale Resolution at Whole Cell Level

Abstract

To understand the frontier Biosciences, it is also important to observe the three-dimensional structure of biological molecules in cells and tissue. We describe a new biological application of FIB-SEM, which is normally used to visualize metals and ceramics surface, for the 3D reconstruction of an entire cell at a nanoscale resolution that lies between those of EM tomography and X-ray tomography.We used FIB-SEM to visualize the 3D architecture of Cyanidioschyzon merolae (C. marolae) , which is thinking as the primitive unicellular red algae. C. marolae is the only eukaryotic organism which can control its chloroplast, mitochondria and cell division by light/dark adjustment. Because cell division is expected a basis of life, we can know the basic mechanism of eukaryotic cells by examining the structure of C. merolae in each division process. Our system could image simple individual double-membrane organelles like nucleus, chloroplast, and mitochondria, and single-membrane organelles like the ER, Golgi, lysosome and peroxisome inside C. merolae of 2-5 μm in length. We stained C. merolae cells with Hoechst 33342 and visualized them by fluorescence microscopy, succeeding to resolve the DNA of the chloroplast, mitochondria and cell nucleus. The position of the chloroplast was determined by the autofluorescence. Cells can be classified into five types (correspond by G1, S, G2 early, G2 late, M-phase) based on differences in shape, size and distribution of the organelles during mitosis. By effective synchronizing cells to a 6-h light/18-h dark cycle according to Moriyama T. et al. (Microbiology , 156 , 1730-7, 2010) with some modifications, we obtained > 70% S/M-phase cells. We will show you 3-D surface models of the different C. merolae cell types from the obtained sequential 2D-SEM images.