Three-Dimensional Microstructural Visualization of Mitosis using Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) and 3Mv Ultra-High Voltage Electron Microscope (UHVEM) Tomography with Nanoscale Resolution at Whole Cell Level

Abstract

To better understand fundamental cellular properties, such as differentiation and division, we are developing whole single cell 3D-structure analysis technologies based on innovative electron microscopy. These new techniques are designed to reveal the dynamics and structure of intracellular material such as organelles and supramolecular proteins. Our main technologies include (1) Cryo-Tomography using Scanning-TEM and (2) FIB (Focused Ion Beam)-SEM and 3D-reconstruction. FIB-SEM is normally used to visualize metals and ceramics. We have modified it for the 3D reconstruction of an entire cell at a nanoscale resolution that lies between those of electron microscopy tomography and X-ray tomography.Last annual meeting, we described how FIB-SEM could visualize the basic 3D architecture of Cyanidioschyzon merolae (C. marolae). C. marolae is a primitive unicellular red algae whose cell division can be observed my manipulating the light/dark cycle. By synchronizing cells to a 6-h light/18-h dark cycle, we obtained > 75% S/M-phase cells at 89 hrs after synchronous culture start. Using these cells and FIB-SEM, we observed unique architectures of whole C. merolae cells during the mitotic cycle and successfully made 3D-models of individual double-membrane organelles such as the nucleus, chloroplast and mitochondria, and of single-membrane organelles such as the ER, lysosome and peroxisome using ImageJ and Amira 3D software. Using UHVEM tomography, we also observed the 3D-structure of phycobilisomes, which are essential supramolecular complexes on the surface of the thylakoid membrane in chloroplasts. Although many reports have provided structural models, we offer the first 3D-structural model of the membrane surface from specimens that were not purified using specific detergents.