Applied Microscopy

Korean Society of Microscopy (한국현미경학회)
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Serial Block-Face Imaging by Field Emission Scanning Electron Microscopy

전계방사형 주사전자현미경에 의한 연속블록면 이미징

  • Kim, Ki-Woo (School of Ecology and Environmental System, Kyungpook National University)
  • 김기우 (경북대학교 생태환경시스템학부)
  • Received : 2011.08.25
  • Accepted : 2011.09.22
  • Published : 2011.09.30
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Abstract

Backscattered electrons (BSE) are generated at the impact of the primary electron beam on the specimen. BSE imaging provides the compositional contrast to resolve chemical features of sectioned block-face. A focused ion beam (FIB) column can be combined with a field emission scanning electron microscope (FESEM) to ensure a dual (or cross)-beam system (FIB-FESEM). Due to the milling of the specimen material by 10 to 100 nm with the gallium ion beam, FIB-FESEM allows the serial block-face (SBF) imaging of plastic-embedded specimens with high z-axis resolution. After contrast inversion, BSE images are similar to transmitted electron images by transmission electron microscopy. As another means of SBF imaging, a specialized ultramirotome has been incorporated into the specimen chamber of FESEM ($3View^{(R)}$). Internal structures of plastic-embedded specimens can be serially revealed and analyzed by $3View^{(R)}$ with a large field of view to facilitate three-dimensional reconstruction. These two SBF approaches by FESEM can be employed to unravel spatial association of (sub)cellular entities for a comprehensive understanding of complex biological systems.

후방산란전자(BSE)는 입사전자빔이 시료와 충돌하면서 발생한다. BSE 이미징은 시료의 화학적 특성을 구분할 수 있는 조성대비를 제공한다. 집속이온빔장치(FIB)는 전계방사형 주사전자현미경(FESEM)과 결합할 수 있으므로 이중빔 체계(FIB-FESEM)가 구현된다. 갈륨(Ga) 이온빔으로 10~100 nm 두께로 시료를 절삭할 수 있으므로 FIB-FESEM은 플라스틱으로 포매된 블록의 면을 z축 고해상도를 유지하며 연속적으로 이미징할 수 있다. BSE이미지의 대비를 반전시키면 투과전자현미경의 이미지와 유사하다. 연속블록면 이미징의 또 다른 방안으로써 특수한 초박절편기가 FESEM 내부에 장착된 것이 $3View^{(R)}$로 상용화되어 있다. 이로써 플라스틱으로 포매된 시료의 내부 구조를 넓은 면적을 연속적으로 이미징 할 수 있으므로 3차원 재구성도 용이하게 된다. 이러한 FESEM에 기반한 두 가지 방식은 복잡한 생물계의 총체적인 이해를 위하여 세포 및 세포 수준 이하의 구조물 간의 공간적 연관성을 규명하는 데 활용될 수 있다.

Keywords

References

  1. Arenkiel BR, Ehlers MD: Molecular genetics and imaging technologies for circuit-based neuroanatomy. Nature 461 : 900-907, 2009. https://doi.org/10.1038/nature08536
  2. Bennett AE, Narayan K, Shi D, Hartnell LM, Gousset K, He H, Lowekamp BC, Yoo TS, Bliss D, Freed EO, Subramaniam S: Ion-abrasion scanning electron microscopy reveals surface-connected tubular couduits in HIV-infected macrophages. PloS Pathog 5 : e1000591, 2009. https://doi.org/10.1371/journal.ppat.1000591
  3. Bushby AJ, P'ng KMY, Young RD, Pinali C, Knupp C, Quantock AJ: Imaging three-dimensional tissue architecture by focused ion beam scanning electron microscopy. Nat Protoc 6 : 845-858, 2011. https://doi.org/10.1038/nprot.2011.332
  4. Deerinck TJ, Bushong EA, Lev-Ram V, Shu X, Tsien RY, Ellisman MH: Enhancing serial block-face scanning electron microscopy to enable high resolution 3-D nanohistology of cells and tissues. Microsc Microanal 16(Suppl 2) : 1138-1139, 2010. https://doi.org/10.1017/S1431927610055170
  5. Denk W, Horstmann H: Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11) : e329, 2004. https://doi.org/10.1371/journal.pbio.0020329
  6. Eisenstein M: Putting neurons on the map. Nature 461 : 1149-1152, 2009. https://doi.org/10.1038/4611149a
  7. Heymann JAW, Hayles M, Gestmann I, Giannuzzi LA, Lich B, Subramaniam S: Site-specific 3D imaging of cells and tissues with a dual beam microscope. J Struct Biol 155 : 63-73, 2006. https://doi.org/10.1016/j.jsb.2006.03.006
  8. Heymann JAW, Shi D, Kim S, Bliss D, Milne JLS, Subramaniam S: 3D imaging of mammalian cells with ion-abrasion scanning electron microscopy. J Struct Biol 166 : 1-7, 2009. https://doi.org/10.1016/j.jsb.2008.11.005
  9. Kim KW, Baek SG, Park BJ, Kim HW, Rhyu IJ: Applications of focused ion beam for biomedical resarch. Korean J Microsc 40 : 177-183, 2010.
  10. Kim KW, Choi SJ, Moon TW: Backscattered electron imaging for reduced charging of moisturized corn starch granules: Implications for imagery of hygroscopic powder specimens. Micron 39 : 1160-1165, 2008. https://doi.org/10.1016/j.micron.2008.05.007
  11. Kim KW, Jaksch H: Compositional contrast of uncoated fungal spores and stained section-face by low-loss backscattered electron imaging. Micron 40 : 724-729, 2009 https://doi.org/10.1016/j.micron.2009.05.001
  12. Knott G, Marchman H, Wall D, Lich B: Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28 : 2959-2964, 2008. https://doi.org/10.1523/JNEUROSCI.3189-07.2008
  13. Leighton SB: SEM images of block face, cut by a miniature microtome within the SEM-a technical note. Scan Electron Microsc 2 : 73-76, 1981.
  14. Merchan-Perez A, Rodriguez J-R, Alonso-Nanclares L, Schertel A, DeFelipe J: Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction. Front Neuroanat 3(Article 18) : 1-14, 2009.
  15. Mun JY, Jeong SY, Kim JH, Han SS, Kim IH: A low fluence Q-switched Nd:YAG laser modifies the 3D structure of melanocyte and ultrastructure of melanosome by subcellular-selective photo-thermolysis. J Elect Microsc 60 : 11-18, 2011. https://doi.org/10.1093/jmicro/dfq068
  16. Muller-Reichert T, Mancuso J, Lich B, McDonald K: Three-dimensional reconstruction methods for Caenorhabditis elegans ultrastructure. Meth Cell Biol 96 : 331-361, 2010.
  17. Schroeder-Reiter E, Perez-Willard F, Zeile U, Wanner G: Focused ion beam (FIB) combined with high resolution scanning electron microscopy: A promising tool for 3D analysis of chromosome architecture. J Struct Biol 165 : 97-106, 2009. https://doi.org/10.1016/j.jsb.2008.10.002