Skip to main content Accessibility help

Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

  • Niels de Jonge (a1) (a2), Rachid Sougrat (a3), Brian M. Northan (a4) and Stephen J. Pennycook (a2)


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.


Corresponding author

Corresponding author. E-mail:


Hide All
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, 436450.
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, 38253844.
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), 30443048.
Bozzola, J.J. & Russell, L.D. (1992). Electron Microscopy. Boston, MA: Jones and Bartlett Publishers.
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), 13601369.
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, 14831487.
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, 21592164.
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. 13.113.27. Boca Raton, FL: CRC Press.
Frank, J. (2006). Three-Dimensional Electron Microscopy of Macromolecular Assemblies—Visualization of Biological Molecules in Their Native State. Oxford, UK: Oxford University Press.
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, 21122114.
Glauert, A.M. & Lewis, P.R. (1998). Biological Specimen Preparation for Transmission Electron Microscopy. London: Portland Press.
Haider, M., Uhlemann, S. & Zach, J. (2000). Upper limits for the residual aberrations of a high-resolution aberration-corrected STEM. Ultramicroscopy 81, 163175.
Hell, S.W. (2007). Far-field optical nanoscopy. Science 316, 11531158.
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), 729731.
Kremer, J.R., Mastronarde, D.N. & McIntosch, J.R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 7176.
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, 378400.
Lucic, V., Foerster, F. & Baumeister, W. (2005). Structural studies by electron tomography: From cells to molecules. Annu Rev Biochem 74, 833865.
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, 718.
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), 55655570.
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), 395403.
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, 124105-1124105-3.
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.
Pawley, J.B. (1995). Handbook of Biological Confocal Microscopy. New York: Springer.
Puetter, R.C., Gosnell, T.R. & Yahil, A. (2005). Digital image reconstruction: Deblurring and denoising. Annu Rev Astron Astrophys 43, 139194.
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, 1428.
Stahlberg, H. & Walz, T. (2008). Molecular electron microscopy: State of the art and current challenges. ACS Chem Biol 3, 268281.
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, 290303.
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), 123127.
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, 034104-1034104-3.
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, 18771881.
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, 157165.


Type Description Title

Niels de Jonge supplementary material
Supplementary movies

 Unknown (5.3 MB)
5.3 MB

Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

  • Niels de Jonge (a1) (a2), Rachid Sougrat (a3), Brian M. Northan (a4) and Stephen J. Pennycook (a2)


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed