Applied Microscopy

Korean Society of Microscopy (한국현미경학회)
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Observations of the Cyanobacteria Synechocystis sp. PCC 6803 using Cryo-Methods and Cryo-SEM

Cryo-Methods와 Cryo-SEM을 이용한 Cyanobacteria, Synechocystis sp. PCC 6803 미세구조 관찰

  • Lee, Eun-Ju (Division of Electron Microscopic Research, Korea Basic Science Institute) ;
  • Moon, Yoon-Jung (Proteome Research Team, Korea Basic Science Institute) ;
  • Oh, Hyun-Woo (Industrial Bio-materials Research Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Kim, Su-Jin (Division of Electron Microscopic Research, Korea Basic Science Institute) ;
  • Chung, Young-Ho (Proteome Research Team, Korea Basic Science Institute) ;
  • Kweon, Hee-Seok (Division of Electron Microscopic Research, Korea Basic Science Institute) ;
  • Kim, Youn-Joong (Division of Electron Microscopic Research, Korea Basic Science Institute)
  • 이은주 (한국기초과학지원연구원 전자현미경연구부) ;
  • 문윤정 (한국기초과학지원연구원 프로테옴연구부) ;
  • 오현우 (한국생명공학연구원 산업바이오소재 연구센터) ;
  • 김수진 (한국기초과학지원연구원 전자현미경연구부) ;
  • 정영호 (한국기초과학지원연구원 프로테옴연구부) ;
  • 권희석 (한국기초과학지원연구원 전자현미경연구부) ;
  • 김윤중 (한국기초과학지원연구원 전자현미경연구부)
  • Published : 2009.03.31
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Abstract

Cryo-SEM which enables specimens to be observed in frozen form has been used to study liquid specimens in their native states. Cryo-methods, sample preparation for cryo-SEM, are quite complex and involve several discrete but vitally interconnected steps which are rapid cooling, fracturing, sectioning, etching and coating. It is important to select practical techniques and to optimize conditions of each steps considering analytic purpose and specimen characters, viz., sample dimension, water contents. In this study, etching methods and sample preparation before freezing had been studied for observation of cyanobacteria, Synechocystis sp. PCC 6803 using cryo-SEM and their cryo-SEM images were compared to Conventional SEM (CSEM) images treated by chemical fixation. We could observe the improved morphological images of the pili of the surface and membranes of Synechocystis sp. PCC 6803 and the three-dimensional architectures of their biofilm, which were difficult to observe using chemical fixation and CSEM. These results suggest that cryo-methods/cryo-SEM are useful techniques for morphological study of biological specimen.

Cryo-SEM은 수분을 함유하거나 액상 시료를 동결된 상태로 관찰하는 방법으로 rapid cooling, fracturing, sectioning, etching, coating과 같은 다소 복잡한 여러 과정의 전처리(Cryo-methods) 로 구성된다. 각 과정에서는 분석 목적이나 시료 특성(예: 시료크기, 물 함량 등)을 고려하여 최적의 조건을 설정하여야 한다. 본 연구에서는 cryo-SEM을 이용하여 남세균 (cyanobacteria, Synechocystis sp. PCC 6803)의 표면 및 내부 구조 관찰을 위한 etching time과 시료 처리에 관하여 살펴보았고 cryo-methods로 처리한 후 cryo-SEM으로 관찰한 이미지를 화학 고정한 일반 SEM(CSEM) 결과와 비교하였다. 관찰 결과, 화학 고정한 CSEM에서는 Synechocystis sp. PCC 6803 표면에 존재하는 pili가 잘 관찰되지 않았으며 파단된 내부 구조의 이미지를 얻을 수 없었으나 cryo-methods/cryo-SEM에서는 세포 표면의 pili 및 membrane의 형태를 관찰할 수 있었다. 또한 수화 상태에 따라 구조변형이 일어나는 biofilm의 구조도 관찰할 수 있었다. 이러한 결과로부터 cryo-methods/cryo-SEM은 수분을 함유하는 의생물 시료의 형태 구조를 분석하는 유용한 방법으로 사료된다.

Keywords

References

  1. Bahya D, Bianoco NR, Bryant D, Grossman A: Type IV pilus biogenesis and motility in the cyanobacterium Synechocystic sp. PCC 6803. Mol Microbiology 37 : 941-951, 2000 https://doi.org/10.1046/j.1365-2958.2000.02068.x
  2. Bahya D: Light matters: Phototaxis and signal transduction in unicellular Cyanobacteria. Mol Microbiology 53 : 745-754, 2004 https://doi.org/10.1111/j.1365-2958.2004.04160.x
  3. Boyde A: Biological specimen preparation for the scanning electron microscope: an overview Scanning Electron Microscopy : 257, 1972
  4. Burriesci M, Bahya D: Tracking phototatic responses and modeling motility of Synechocystic sp. PCC 6803. J Photochem Photobio B: Biology 91 : 77-86, 2008 https://doi.org/10.1016/j.jphotobiol.2008.01.012
  5. Choi JS, Chung YH, Moon YJ, Kim C, Watanabe M, Song PS, Joe CO, Bogorad L, Park YM: Photomovement of the gliding cyanobacterium Synechocystic sp. PCC 6803. Photochem Photobiol 70 : 95-102, 1999 https://doi.org/10.1111/j.1751-1097.1999.tb01954.x
  6. Echlin P: Low-Temperature Microscopy and Analysis. Plenum Press, New York, pp. 349-411, 1992
  7. Handley KM, Turner SJ, Campbell KA, Mountain BW: Silicifying Biofilm Exopolymers on a Hot-Spring Microstromatolite: Templating Nanometer-Thick Laminae. Asrobiology 8 : 747-770, 2008 https://doi.org/10.1089/ast.2007.0172
  8. Kaneko T, Sato S, Kotani H, Tanaka A, Asanizu E, Nakamura T, Miyajima N, Hirosawa M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S: Sequence analysis of the genome of the unicellular cyanobacterium Synechocystic sp. strain PCC 6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res 3 : 109-136, 1996 https://doi.org/10.1093/dnares/3.3.109
  9. Liberton M, Berg RH, Heuser J, Roth R, Pakrasi HB: Ultrastrcture of membrane systems in the unicellular cyanobacterium Synechocystic sp. strain PCC 6803. Protoplasma 227 : 129-138, 2006 https://doi.org/10.1007/s00709-006-0145-7
  10. O'Toole GA, Kolter RP: Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbology 30 : 295-304, 1998 https://doi.org/10.1046/j.1365-2958.1998.01062.x
  11. Reid RP, Visscher PT, Decho AW, Stolz JF, Bebont BM, Dupraz C, Macintyre IG, Paerl HW, Pinckney JL, Prufert-Bebont L, Steppe TF, DesMarais DJ: The role of microbes in accretion, lamination and early lithification of modern marine stromatolites. Nature 406 : 989-992, 2000 https://doi.org/10.1038/35023158
  12. Richard SR, Turner RJ: A comparative study of techniques for the examination of biofilms by scanning electron microscopy. Water Res 18 : 767-773, 1984 https://doi.org/10.1016/0043-1354(84)90173-8
  13. Robards AW, Sleytr UB: Low Temperature Methods in Biological Electron Microscopy. Elsevier Science Publishers, Amsterdam, pp. 243-307, 1985
  14. Shimoni E, Muller M: On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device. J Microsc 192 : 236-247, 1998 https://doi.org/10.1046/j.1365-2818.1998.00389.x
  15. Umrath W: Calculation of freeze drying times for electron microscopic preparations. In: Robards AW, Sleytr UB, ed, Low Temperature Methods in Biological Electron Microscopy. pp. 247-261, 1985
  16. Van de Meene AML, Hohmann-Marriott MF, Vermaas WFJ, Roberson RW: The three dimensional structure of cyanobacterium Synechocystic sp. PCC 6803. Arch Microbiol 184 : 259-270, 2006 https://doi.org/10.1007/s00203-005-0027-y
  17. Yoshihara S, Geng XX, Okamoto S, Yura K, Murata T, Go M, Ohmori M, Ikeuchi M: Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystic sp. PCC 6803. Plant Cell Physiol 42 : 63-73, 2001 https://doi.org/10.1093/pcp/pce007