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Bio-High Voltage Electron Microscope
Analytical Service Last update : 2017.08.17 Request for use
RI Category
Analytical Facilities
Keywords
Description
Bio-HVEM was installed in KBSI Ochang headquarter in 2015 and has been in operation for 3-dimensional ultrastructure analysis of biological tissues and cells and protein complexes.
Bio-HVEM has been in operation as a national co-utilization equipment in basic and applied sciences for 3-dimensional ultrastructure analysis of cell organelles, proteins, and bio-nano specimens as well as development of drug and nano materials.
- 3D modeling of cell organelles by high tilting (± 70°) and high resolution (0.15 nm)
- Enhanced high contrast imaging using high accelerating voltage / in-column energy filter
- Analysis of enhanced large-area with high resolution by limitless panorama function
- Cryo-EM analysis by rapid and continuous freezing of biological specimen
Application Area
- 3D ultrastructural analysis of cellular organelles using electron tomography
- 3D large area analysis of biological tissues using limitless panorama
- STEM analysis, STEM Electron Tomography
- Chemical analysis (Element mapping, Electron Energy Loss Spectroscopy)
Service
Specifications
1. Bio-High Voltage Electron Microscope
- Acceleration voltage: 1,000 kV
- Electron source: LaB6 filament
- Point resolution: 0.15 nm
- Lattice resolution: 0.10 nm
- STEM resolution: 2.0 nm
- Magnification range: ×200 ~ ×1,200,000 (TEM), ×20,000 ~ ×2,000,000 (STEM)
- Specimen tilt range: α = ±70°, β = ±30°
- Pole piece gap: 16 mm
- Spherical aberration: 3.7 mm (at 1,000 kV)
- Chromatic aberration: 4.2 mm (at 300 kV)
- Operating temperature: LN2 temperature ~ room temperature
- Special features:
· In-column Ω energy filter
· Cryo-electron microscopy function
· STEM and STEM tomography function
· 2 × anti-contamination device (fixed & retractable)
· Automated aperture control
- Detectors:
· 2,048 × 2,048 pixel CCD camera (Orius, GATAN)
· 3,780 × 3,710 pixel DDD camera (K2 Base, Gatan)
· Wide angle and monitor CCD
· STEM detector
- Specimen holders:
· Standard Double tilt holder (α = ±60°, β = ±30°)
· Multi-specimen holder (up to 4 specimen load, α = ±60°)
· Specimen rotation holder (α = ±70°, 180° in-plane rotation)
· Cryo-specimen holder (LN2 temperature, α = ±60°)
- Dedicated software:
· TEM Center
· TEMography package
· ShotMeister
· Digital Micrograph
Limitless Panorama analysis on brain region

Sequential limitless panorama imaging procedure for the large-area image acquisition by combination of beam shift and gonio movement

그림입니다.원본 그림의 이름: CLP000010480005.bmp원본 그림의 크기: 가로 755pixel, 세로 372pixel

3D Electron Tomography analysis on synapse region

Experimental steps for 3D electron tomography and 3D modeling; 3D model of synaptic bouton in cerebral cortex and comparison of SV number between control and RF-exposed mice

그림입니다.원본 그림의 이름: CLP0000104804dd.bmp원본 그림의 크기: 가로 3497pixel, 세로 1259pixel

Publication

Chang Joo Oh, Chae-Myeong Ha, Young-Keun Choi, Sungmi Park, Mi Sun Choe, Nam Ho Jeoung, Yang Hoon Huh, Hyo-Jeong Kim, Hee-Seok Kweon, Ji-min Lee, Sun Joo Lee, Jae-Han Jeon, Robert A. Harris, Keun-Gyu Park, In-Kyu Lee, Kidney International vol. 91, 880-895 (2017), doi: 10.1016/j.kint.2016.10.011

Clinical prescription of cisplatin, one of the most widely used chemotherapeutic agents, is limited by its side effects, particularly tubular injury–associated nephrotoxicity. Since details of the underlying mechanisms are not fully understood, we investigated the role of pyruvate dehydrogenase kinase (PDK) in cisplatin-induced acute kidney injury. Among the PDK isoforms, PDK4 mRNA and protein levels were markedly increased in the kidneys of mice treated with cisplatin, and c-Jun N-terminal kinase activation was involved in cisplatin-induced renal PDK4 expression. Treatment with the PDK inhibitor sodium dichloroacetate (DCA) or genetic knockout of PDK4 attenuated the signs of cisplatin-induced acute kidney injury, including apoptotic morphology of the kidney tubules along with numbers of TUNEL-positive cells, cleaved caspase-3, and renal tubular injury markers. Cisplatin-induced suppression of the mitochondrial membrane potential, oxygen consumption rate, expression of electron transport chain components, cytochrome c oxidase activity, and disruption of mitochondrial morphology were noticeably improved in the kidneys of DCA-treated or PDK4 knockout mice. Additionally, levels of the oxidative stress marker 4-hydroxynonenal and mitochondrial reactive oxygen species were attenuated, whereas superoxide dismutase 2 and catalase expression and glutathione synthetase and glutathione levels were recovered in DCA-treated or PDK4 knockout mice. Interestingly, lipid accumulation was considerably attenuated in DCA-treated or PDK4 knockout mice via recovered expression of peroxisome proliferator-activated receptor-α and coactivator PGC-1α, which was accompanied by recovery of mitochondrial biogenesis. Thus, PDK4 mediates cisplatin-induced acute kidney injury, suggesting that PDK4 might be a therapeutic target for attenuating cisplatin-induced acute kidney injury.

Ju Hwan Kim, Yang Hoon Huh, Hak Rim Kim, PLoS One. 2016 Apr 13;11(4):e0153308. doi: 10.1371/journal.pone.0153308

The extensive use of wireless mobile phones and associated communication devices has led to increasing public concern about potential biological health-related effects of the exposure to electromagnetic fields (EMFs). EMFs emitted by a mobile phone have been suggested to influence neuronal functions in the brain and affect behavior. However, the affects and phenotype of EMFs exposure are unclear. We applied radiofrequency (RF) of 835 MHz at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day for 4 and 12 weeks to clarify the biological effects on mouse brain. Interestingly, microarray data indicated that a variety of autophagic related genes showed fold-change within small range after 835 MHz RF exposure. qRT-PCR revealed significant up-regulation of the autophagic genes Atg5, LC3A and LC3B in the striatum and hypothalamus after a 12-week RF. In parallel, protein expression of LC3B-II was also increased in both brain regions. Autophagosomes were observed in the striatum and hypothalamus of RF-exposed mice, based on neuronal transmission electron microscopy. Taken together, the results indicate that RF exposure of the brain can induce autophagy in neuronal tissues, providing insight into the protective mechanism or adaptation to RF stress.

You‐Kyung Lee, Yong‐Woo Jun, Ha‐Eun Choi, Yang Hoon Huh, Bong‐Kiun Kaang, Deok‐Jin Jang, Jin‐A Lee, The EMBO Journal (2017) 36, 1100-1116, doi: 10.15252/embj.201696315

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP‐LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3‐interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD‐LIR‐GFP sensors harboring LIR motifs of 34 known LC3‐binding proteins, HyD‐LIR(TP)‐GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs‐positive autophagosomes. However, HyD‐LIR(TP)‐GFP preferentially localized to GABARAP/GABARAPL1‐positive autophagosomes in a LIR‐dependent manner. In contrast, HyD‐LIR(Fy)‐GFP using the LIR motif from FYCO1 specifically detected LC3A/B‐positive autophagosomes. HyD‐LIR(TP)‐GFP and HyD‐LIR(Fy)‐GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker‐positive damaged mitochondria upon mitophagy induction. HyD‐LIR(TP)‐GFP allowed live‐imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.

Ju Hwan Kim,Da-Hyeon Yu, Yang Hoon Huh, Eun Ho Lee, Hyung-Gun Kim & Hak Rim Kim, Scientific Reports 7, Article number: 41129 (2017), doi: 10.1038/srep41129

Radiofrequency electromagnetic field (RF-EMF) is used globally in conjunction with mobile communications. There are public concerns of the perceived deleterious biological consequences of RF-EMF exposure. This study assessed neuronal effects of RF-EMF on the cerebral cortex of the mouse brain as a proxy for cranial exposure during mobile phone use. C57BL/6 mice were exposed to 835 MHz RF-EMF at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day during 12 weeks. The aim was to examine activation of autophagy pathway in the cerebral cortex, a brain region that is located relatively externally. Induction of autophagy genes and production of proteins including LC3B-II and Beclin1 were increased and accumulation of autolysosome was observed in neuronal cell bodies. However, proapoptotic factor Bax was down-regulted in the cerebral cortex. Importantly, we found that RF-EMF exposure led to myelin sheath damage and mice displayed hyperactivity-like behaviour. The data suggest that autophagy may act as a protective pathway for the neuronal cell bodies in the cerebral cortex during radiofrequency exposure. The observations that neuronal cell bodies remained structurally stable but demyelination was induced in cortical neurons following prolonged RF-EMF suggests a potential cause of neurological or neurobehavioural disorders.