title: Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy.
abstract: IntroductionMitochondrial fission and fusion events are fundamental mechanisms for quality control of mitochondrial functions. Mitochondrial DNA (mtDNA) usually divides in offspring mitochondria after fission and mtDNA dynamics are thought to be coordinated with mitochondrial turnover. Recently, several candidate mechanisms for the relationship between mtDNA division and mitochondrial fission have been suggested ([1], 2012). The dynamics of mtDNA or nucleoids can be observed using fluorescent microscopy, but the ultrastructural aspects of their coordination remain unclear. Although visualization of mtDNA at the electron microscopic level is an important step in understanding how mtDNA division and mitochondrial division are coordinated, it is quite difficult to observe using conventional electron microscopic methods. In the present study, we attempted to establish correlative light and electron microscopy (CLEM) observation to visualize the three-dimensional localization of the mtDNA /nucleoid within mitochondria at electron microscopic resolution using a combination of immuno-electron and focused-ion beam scanning electron microscopy (FIB/SEM) tomography methods. Materials & methodsHeLa cells were fixed using 4% paraformaldehyde and 0.05% glutaraldehyde in 0.1 M phosphate buffer, and then immunohistochemically labeled with anti-TFAM IgG antibody (Abnova, USA) or anti-DNA IgM antibody (Progen, Germany). The cells were then reacted with biotin-labeled secondary antibodies. The immunoreactivities were visualized using two methods: the ABC method and streptavidin Fluoro-Nanogold. Immunohistochemically labeled specimens were then observed using light microscopy. These specimens were then developed using a Gold Enhancement kit (Nanoprobe, USA) for 150 s. Specimens for electron microscopy were stained using the ROTO method, embedded in resin, and subjected to FIB/SEM tomography (Quanta 3D FEG, FEI). 3D reconstruction was performed using the software Amira (FEI). Results & discussionWe were not able to identify a nucleoid-like structure within mitochondria, even in a complete 3D reconstruction using FIB/SEM with conventional staining. In CLEM observations, immunoreaction (IR) products were correlatively observed under LM and EM. Pre-embedding immuno-electron microscopy showed DAB and gold IR in the matrix of some mitochondria. Interestingly, IR products were observed in the globular region of the mitochondrial matrix (approximately 0.4 µm in diameter), frequently localizing in the peripheral end of the mitochondrial matrix, adjacent to the inner membrane. Using the post-embedding immunogold method, gold labels were also observed in a portion of the matrix adjacent to the mitochondrial inner membrane. These immunocytochemical results were concordant with our fluorescent microscopic observations.
authors.affiliation: Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine.
authors.forename: Satoko
authors.initials: S
authors.lastname: Okayama
authors.affiliation: Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohta
authors.affiliation: Electron microscopic Unit, Kurume University School of Medicine 830-0011 67 Asahimachi, Kurume, Fukuoka, Japan.
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Higashi
authors.affiliation: Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine Electron microscopic Unit, Kurume University School of Medicine 830-0011 67 Asahimachi, Kurume, Fukuoka, Japan.
authors.forename: Kei-Ichiro
authors.initials: K
authors.lastname: Nakamura