Hostname: page-component-586b7cd67f-t7fkt Total loading time: 0 Render date: 2024-11-24T15:12:16.977Z Has data issue: false hasContentIssue false
  • English
  • Français

Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

Published online by Cambridge University Press:  18 January 2010

Niels de Jonge ,
Rachid Sougrat ,
Brian M. Northan  and
Stephen J. Pennycook
Niels de Jonge*
Affiliation:
Vanderbilt University Medical Center, Department of Molecular Physiology and Biophysics, Light Hall 702, Nashville, TN 37232-0615, USA Oak Ridge National Laboratory, Materials Science and Technology Division, 1 Bethel Valley Rd., Oak Ridge, TN 37831-6064, USA
Rachid Sougrat
Affiliation:
Cell Biology and Metabolism Branch, NICHD, National Institute of Health, 18 Library Drive, Bethesda, MD 20892-5430, USA
Brian M. Northan
Affiliation:
Media Cybernetics Inc., 4340 East-West Hwy, Suite 400, Bethesda, MD 20814-4411, USA
Stephen J. Pennycook
Affiliation:
Oak Ridge National Laboratory, Materials Science and Technology Division, 1 Bethel Valley Rd., Oak Ridge, TN 37831-6064, USA
*
Corresponding author. E-mail: [email protected]
Get access

Abstract

A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Keywords

three-dimensional electron microscopyaberration-corrected STEMbiological electron microscopythin sectionscytoskeletonclathrin-coated pitdeconvolutionnanoparticles
Type
Biological Imaging: Techniques Development and Applications
Information
Microscopy and Microanalysis , Volume 16 , Issue 1 , February 2010 , pp. 54 - 63
Copyright
Copyright © Microscopy Society of America 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Bartesaghi, A., Sprechmann, P., Liu, J., Randall, G., Sapiro, G. & Subramaniam, S. (2008). Classification and 3D averaging with missing wedge correction in biological electron tomography. J Struct Biol 162, 436–450.CrossRefGoogle ScholarPubMed
Behan, G., Cosgriff, E.C., Kirkland, A.I. & Nellist, P.D. (2009). Electron microscopein the aberration-corrected scanning transmission three-dimensional imaging by optical sectioning. Phil Trans R Soc A 367, 3825–3844.CrossRefGoogle ScholarPubMed
Borisevich, A.Y., Lupini, A.R. & Pennycook, S.J. (2006). Depth sectioning with the aberration-corrected scanning transmission electron microscope. Proc Natl Acad Sci 103(9), 3044–3048.CrossRefGoogle ScholarPubMed
Bozzola, J.J. & Russell, L.D. (1992). Electron Microscopy. Boston, MA: Jones and Bartlett Publishers.Google Scholar
Burnette, D.T., Schaefer, A.W., Ji, L., Danuser, G. & Forscher, P. (2007). Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones. Nat Cell Biol 9(12), 1360–1369.CrossRefGoogle Scholar
Carrington, W.A., Lynch, R.M., Moore, E.D.W., Isenberg, G., Fogarty, K.E. & Fay, F.S. (1995). Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science 268, 1483–1487.CrossRefGoogle Scholar
de Jonge, N., Peckys, D.B., Kremers, G.J. & Piston, D.W. (2009). Electron microscopy of whole cells in liquid with nanometer resolution. Proc Natl Acad Sci 106, 2159–2164.CrossRefGoogle ScholarPubMed
de Jonge, N., Sougrat, R., Peckys, D.B., Lupini, A.R. & Pennycook, S.J. (2007). 3-dimensional aberration corrected scanning transmission electron microscopy for biology. In Nanotechnology in Biology and Medicine-Methods, Devices and Applications, Vo-Dinh, T. (Ed.), pp. 13.1–13.27. Boca Raton, FL: CRC Press.Google Scholar
Frank, J. (2006). Three-Dimensional Electron Microscopy of Macromolecular Assemblies—Visualization of Biological Molecules in Their Native State. Oxford, UK: Oxford University Press.CrossRefGoogle Scholar
Frigo, S.P., Levine, Z.H. & Zaluzec, N.J. (2002). Submicron imaging of buried integrated circuit structures using scanning confocal electron microscopy. Appl Phys Lett 81, 2112–2114.CrossRefGoogle Scholar
Glauert, A.M. & Lewis, P.R. (1998). Biological Specimen Preparation for Transmission Electron Microscopy. London: Portland Press.CrossRefGoogle Scholar
Haider, M., Uhlemann, S. & Zach, J. (2000). Upper limits for the residual aberrations of a high-resolution aberration-corrected STEM. Ultramicroscopy 81, 163–175.CrossRefGoogle ScholarPubMed
Hell, S.W. (2007). Far-field optical nanoscopy. Science 316, 1153–1158.CrossRefGoogle ScholarPubMed
Hohmann-Marriott, M.F., Sousa, A.A., Azari, A.A., Glushakova, S., Zhang, G., Zimmerberg, J. & Leapman, R.D. (2009). Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nat Methods 6(10), 729–731.CrossRefGoogle ScholarPubMed
Kremer, J.R., Mastronarde, D.N. & McIntosch, J.R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 71–76.CrossRefGoogle ScholarPubMed
Kuebel, C., Voigt, A., Schoenmakers, R., Otten, M., Su, D., Lee, T.C., Carlsson, A. & Bradley, J. (2005). Recent advances in electron tomography: TEM and HAADF-STEM tomography for materials science and semiconductor applications. Microsc Microanal 11, 378–400.CrossRefGoogle Scholar
Lucic, V., Foerster, F. & Baumeister, W. (2005). Structural studies by electron tomography: From cells to molecules. Annu Rev Biochem 74, 833–865.CrossRefGoogle ScholarPubMed
Luther, P.K., Lawrence, M.C. & Crowther, R.A. (1988). A method for monitoring the collapse of plastic sections as a function of electron dose. Ultramicroscopy 24, 7–18.CrossRefGoogle ScholarPubMed
Marsh, B.J., Volkmann, N., McIntosh, J.R. & Howell, K.E. (2004). Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells. Proc Natl Acad Sci USA 101(15), 5565–5570.CrossRefGoogle ScholarPubMed
Meyer-Ilse, W., Hamamoto, D., Nair, A., Lelievre, S.A., Denbeaux, G., Johnson, L., Pearson, A.L., Yager, D., Legros, M.A. & Larabell, C.A. (2001). High resolution protein localization using soft X-ray microscopy. J Micros 201(3), 395–403.CrossRefGoogle ScholarPubMed
Nellist, P.D., Behan, G., Kirkland, A.I. & Hetherington, C.J.D. (2006). Confocal operation of a transmission electron microscope with two aberration correctors. Appl Phys Lett 89, 124105-1–124105-3.CrossRefGoogle Scholar
Nellist, P.D., Chisholm, M.F., Dellby, N., Krivanek, O.L., Murfitt, M.F., Szilagyi, Z.S., Lupini, A.R., Borisevich, A., Sides, W.H. & Pennycook, S.J. (2004). Direct sub-angstrom imaging of a crystal lattice. Science 305, 1741.CrossRefGoogle ScholarPubMed
Pawley, J.B. (1995). Handbook of Biological Confocal Microscopy. New York: Springer.CrossRefGoogle Scholar
Puetter, R.C., Gosnell, T.R. & Yahil, A. (2005). Digital image reconstruction: Deblurring and denoising. Annu Rev Astron Astrophys 43, 139–194.CrossRefGoogle Scholar
Sousa, A.A., Hohmann-Marriott, M., Aronova, M.A., Zhang, G. & Leapman, R.D. (2008). Determination of quantitative distributions of heavy-metal stain in biological specimens by annular dark-field STEM. J Struct Biol 162, 14–28.CrossRefGoogle ScholarPubMed
Stahlberg, H. & Walz, T. (2008). Molecular electron microscopy: State of the art and current challenges. ACS Chem Biol 3, 268–281.CrossRefGoogle ScholarPubMed
Svitkina, T.M., Verkhovsky, A.B. & Borisy, G.G. (1995). Improved procedures for electron microscopic visualization of the cytoskeleton of cultured cells. J Struct Biol 115, 290–303.CrossRefGoogle ScholarPubMed
Takeguchi, M., Hashimoto, A., Shimojo, M., Mitsuishi, K. & Furuya, K. (2008). Development of a stage-scanning system for high-resolution confocal STEM. J Electron Microsc (Tokyo) 57(4), 123–127.CrossRefGoogle ScholarPubMed
van Benthem, K., Lupini, A.R., Kim, M., Baik, H.S., Doh, S.J., Lee, J.H., Oxley, M.P., Findlay, S.D., Allen, L.J. & Pennycook, S.J. (2005). Three-dimensional imaging of individual hafnium atoms inside a semiconductor device. Appl Phys Lett 87, 034104-1–034104-3.CrossRefGoogle Scholar
Xiao, Y., Patolsky, F., Katz, E., Hainfeld, J.F. & Willner, I. (2003). “Plugging into enzymes”: Nanowiring of redox enzymes by a gold nanoparticle. Science 299, 1877–1881.CrossRefGoogle ScholarPubMed
Xin, H.L. & Muller, D.A. (2009). Aberration-corrected ADF-STEM depth sectioning and prospects for reliable 3D imaging in S/TEM. J Electron Microsc 58, 157–165.CrossRefGoogle ScholarPubMed
Supplementary material: File

Niels de Jonge supplementary material

Supplementary movies

Download Niels de Jonge supplementary material(File)
File 5.3 MB