Microscopy, 2014, Vol. 63, No. S1
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block were cut with a diamond knife, and the newly created flat surfaces of the specimens were coated with evaporated carbon (50 Å) and observed using a SEM (Quanta 3D FEG, FEI). Results The HQ-treated specimens generated a larger amount of SEs than control specimens when subjected to irradiation with the same beam, although BSE numbers were not evidently increased by the treatment. The present results suggest that HQ treatment increases SE generation efficiency, but does not enhance the recruitment of heavy metals into specimens. HQ treatment increased the contrast-to-noise ratio of BFI for images obtained using SEs, and may reduce the total image acquisition time of recently developed 3D reconstruction methods based on SEM.
References
doi: 10.1093/jmicro/dfu078
Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy Satoko Okayama1, Keisuke Ohta1, Ryuhei Higashi2, and Kei-ichiro Nakamura1,2 1
Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine, and 2Electron microscopic Unit, Kurume University School of Medicine 830-0011 67 Asahimachi, Kurume, Fukuoka, Japan Introduction Mitochondrial 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
Materials & methods HeLa 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 & discussion We 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. Preembedding 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.
References 1. Ishihara Nat. Cell Biol. 11, 958 (2009) 2. Ishihara PNAS 110, 11863 (2013) doi: 10.1093/jmicro/dfu079
Downloaded from http://jmicro.oxfordjournals.org/ at University of Utah on November 27, 2014
1. Keisuke Ohta, et al. Micron. Vol. 5, 612–620 (2012) 2. Deerinck T, et al. Microsc. Microanal. 16, 1138–1139 3. Gen Takahashi, et al. BIOMEDICAL SEM vol 15, 42–44 (1986)
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.
i35
block were cut with a diamond knife, and the newly created flat surfaces of the specimens were coated with evaporated carbon (50 Å) and observed using a SEM (Quanta 3D FEG, FEI). Results The HQ-treated specimens generated a larger amount of SEs than control specimens when subjected to irradiation with the same beam, although BSE numbers were not evidently increased by the treatment. The present results suggest that HQ treatment increases SE generation efficiency, but does not enhance the recruitment of heavy metals into specimens. HQ treatment increased the contrast-to-noise ratio of BFI for images obtained using SEs, and may reduce the total image acquisition time of recently developed 3D reconstruction methods based on SEM.
References
doi: 10.1093/jmicro/dfu078
Correlative light and electron microscopic observation of mitochondrial DNA in mammalian cells by using focused-ion beam scanning electron microscopy Satoko Okayama1, Keisuke Ohta1, Ryuhei Higashi2, and Kei-ichiro Nakamura1,2 1
Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine, and 2Electron microscopic Unit, Kurume University School of Medicine 830-0011 67 Asahimachi, Kurume, Fukuoka, Japan Introduction Mitochondrial 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
Materials & methods HeLa 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 & discussion We 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. Preembedding 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.
References 1. Ishihara Nat. Cell Biol. 11, 958 (2009) 2. Ishihara PNAS 110, 11863 (2013) doi: 10.1093/jmicro/dfu079
Downloaded from http://jmicro.oxfordjournals.org/ at University of Utah on November 27, 2014
1. Keisuke Ohta, et al. Micron. Vol. 5, 612–620 (2012) 2. Deerinck T, et al. Microsc. Microanal. 16, 1138–1139 3. Gen Takahashi, et al. BIOMEDICAL SEM vol 15, 42–44 (1986)
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.
