Applied Microscopy

Korean Society of Microscopy (한국현미경학회)
권호 표지
DOI QR코드

DOI QR Code

The Effects of Electron Beam Exposure Time on Transmission Electron Microscopy Imaging of Negatively Stained Biological Samples

  • Kim, Kyumin (Taejon Christian International School) ;
  • Chung, Jeong Min (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Lee, Sangmin (Department of Biochemistry, College of Natural Sciences, Kangwon National University) ;
  • Jung, Hyun Suk (Department of Biochemistry, College of Natural Sciences, Kangwon National University)
  • Received : 2015.08.06
  • Accepted : 2015.09.17
  • Published : 2015.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Negative staining electron microscopy facilitates the visualization of small bio-materials such as proteins; thus, many electron microscopists have used this conventional method to visualize the morphologies and structures of biological materials. To achieve sufficient contrast of the materials, a number of imaging parameters must be considered. Here, we examined the effects of one of the fundamental imaging parameters, electron beam exposure time, on electron densities generated using transmission electron microscopy. A single site of a negatively stained biological sample was illuminated with the electron beam for different times (1, 2, or 4 seconds) and sets of micrographs were collected. Computational image processing demonstrated that longer exposure times provide better electron densities at the molecular level. This report describes technical procedures for testing parameters that allow enhanced evaluations of the densities of electron microscopy images.

Keywords

References

  1. Anderson L J, Boyles J K, and Mahmood Hussain M (1989) A rapid method for staining large chylomicrons. J. Lipid Res. 30, 1819-1824.
  2. Boekema E J, Folea M, and Kouril R (2009) Single particle electron microscopy. Photosynth Res. 102, 189-196. https://doi.org/10.1007/s11120-009-9443-1
  3. Brenner S and Horne R W (1959) A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103-110. https://doi.org/10.1016/0006-3002(59)90237-9
  4. Burgess S A, Walker M L, Thirumurugan K, Trinick J, and Kinight P J (2004) Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules. J. Struct. Biol. 147, 247-258. https://doi.org/10.1016/j.jsb.2004.04.004
  5. De Carlo S and Harris J R (2011) Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron. 42, 117-131. https://doi.org/10.1016/j.micron.2010.06.003
  6. Elliott P R, Irvine A F, Jung H S, Tozawa K, Pastok M W, Picone R, Badyal S K, Basran J, Rudland P S, Barraclough R, Lian L Y, Bagshaw C R, Kriajevska M, and Barsukov I L (2012) Asymmetric mode of Ca2+-S100A4 interaction with nonmuscle myosin IIA generates nanomolar affinity required for filament remodeling. Structure 20, 654-666. https://doi.org/10.1016/j.str.2012.02.002
  7. Henderson R (1995) The potential and limitations of neutrons, electrons and x-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171-193. https://doi.org/10.1017/S003358350000305X
  8. Jaitly N, Brubaker M A, Rubinstein J L, and Lilien R H (2010) A bayesian method for 3D macromolecula r structure inference using class average images from single particle electron microscopy. Bioinformatics 26, 2406-2415. https://doi.org/10.1093/bioinformatics/btq456
  9. Jung H S, Billington N, Thirumurugan K, Salzameda B, Cremo C R, Chalovich J M, Chantler P D, and Knight P J (2011) Role of the tail in the regulated state of myosin 2. J. Mol. Biol. 408, 863-878. https://doi.org/10.1016/j.jmb.2011.03.019
  10. Jung H S, Komatsu S, Ikebe M, and Craig R (2008) Head-head and headtail interaction: a general mechanism for switching off myosin II activity in cells. Mol. Biol. Cell 19, 3234-3242. https://doi.org/10.1091/mbc.E08-02-0206
  11. Kim J, Wu S, Tomasiak T, Mergel C, Winter M B, Stiller S B, Robles-Colmanares Y, Stroud R M, Tampe R, Craik C S, and Cheng Y (2015) Subnanometer resolution cryo-EM structure of a nucleotide free heterodimeric ABC exporter. Nature 517, 396-400. https://doi.org/10.1038/nature13872
  12. Lee S, Jia B, Liu J, Pham B P, Kwak J M, Xuan Y H, and Cheong G W (2015). A 1-cys peroxiredoxin from a thermophilic archaeon moonlights as a molecular chaperone to protect protein and DNA against stressinduced damage. PLoS ONE 10, e0125325. https://doi.org/10.1371/journal.pone.0125325
  13. Massover W H (2008) Negative staining produces images of proteins by which contrast mechanisms? Microsc. Microanal. 14, 672-673. https://doi.org/10.1017/S1431927608082822
  14. Monroe J H and Brandt P M (1970) Rapid semiquantitative method for screening large numbers of virus samples by negative staining electron microscopy. Appl. Microbiol. 20, 259-262.
  15. Murata K, Liu X, Danev R, Jakana J, Khant H, Schmid M F, Nagayama K, and Chiu W (2010) Zernike phase contrast cryo-EM for biological structure determination. Microsc. Microanal. 16, 544-545. https://doi.org/10.1017/S1431927610057107
  16. Nagayama K and Danev R (2004) Phase contrast electron microscopy: development of thin-film phase plates and biological applications. Phil. Trans. R. Soc. B 363, 2153-2162.
  17. Umeki N, Jung H S, Watanabe S, Sakai T, Li X, Ikebe R, Craig R, and Ikebe M (2009) The tail binds to the head-neck domain, inhibiting ATPase activity of myosin VIIA. Proc. Natl. Acad. Sci. USA 106, 8483-8488. https://doi.org/10.1073/pnas.0812930106
  18. You D J, Jhon G J, and Jung H S (2013) Molecular assembly of thyroglobulin induced by in vitro nitric oxide treatments: implication its role in thyroid cells. Protein J. 32, 619-625. https://doi.org/10.1007/s10930-013-9524-z
  19. Zhang L, Tong H, Garewal M, and Ren G (2013) Optimized negativestaining electron microscopy for lipoprotein studies. Biochim. Biophys. Acta 1830, 2150-2159. https://doi.org/10.1016/j.bbagen.2012.09.016