Sample Shrinkage and Radiation Damage of Plastic Sections

  • Pradeep K. Luther


Just as fossil insects embalmed in amber are extraordinarily preserved, so are biological samples that have been embedded in plastic for electron microscopy. The success of embedding samples in plastic lies in the astounding resilience of the sections in the electron microscope, albeit after initial changes. The electron microscope image results from projection of the sample density in the direction of the beam, i.e. through the depth of the section, and therefore is independent of physical changes in this direction. In contrast, the basis of electron tomography is the constancy of the physical state of the whole section during the time that different views at incremental tilt angle steps are recorded.


Gold Particle Radiation Damage Electron Tomography Section Thickness Rapid Freezing 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Amos, L. A., Henderson, R. and Unwin, P. N. (1982). Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog. Biophys. Mol. Biol. 39: 183–231.PubMedCrossRefGoogle Scholar
  2. Bedi, K. S. (1987). A simple method of measuring the thickness of semi-thin and ultra-thin sections. J. Microsc. 148:107–111.PubMedGoogle Scholar
  3. Bennett, P. M. (1974). Decrease in section thickness on exposure to the electron beam; the use of tilted sections in estimating the amount of shrinkage. J. Cell Sci. 15:693–701.PubMedGoogle Scholar
  4. Berriman, J., Bryan, R. K., Freeman, R. and Leonard, K.R. (1984). Methods for specimen thickness determination in electron microscopy. Ultramicroscopy 13:351–364.PubMedCrossRefGoogle Scholar
  5. Berriman, J. and Leonard, K.R. (1986). Methods for specimen thickness determination in electron microscopy. II. Changes in thickness with dose. Ultramicroscopy 19:349–366.PubMedCrossRefGoogle Scholar
  6. Bouwer, J. C., Mackey, M. R., Lawrence, A., Deerinck, T. J., Jones, Y. Z., Terada, M., Martone, M. E., Peltier, S. and Ellisman, M. H. (2004). Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement. J. Struct. Biol. 148:297–306.PubMedCrossRefGoogle Scholar
  7. Braunfeld, M. B., Koster, A. J., Sedat, J.W. and Agard, D. A. (1994). Cryo automated electron tomography: towards high-resolution reconstructions of plastic-embedded structures. J. Microsc. 174:75–84.PubMedGoogle Scholar
  8. Cosslett, A. (1960). The effect of the electron beam on thin sections. In Proceedings of the 1st European Conference on Electron Microscopy. Delft, Vol. 2, pp. 678–681.Google Scholar
  9. Craig, R., Alamo, L. and Padron, R. (1992). Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy. J. Mol. Biol. 228:474–487.PubMedCrossRefGoogle Scholar
  10. Deng, Y., Marko, M., Buttle, K. F., Leith, A., Mieczkowski, M. and Mannella, C.A. (1999). Cubic membrane structure in amoeba (Chaos carolinensis) mitochondria determined by electron microscopic tomography. J. Struct. Biol. 127:231–239.PubMedCrossRefGoogle Scholar
  11. Dorset, D. L. and Parsons, D. F. (1975). The thickness determination of wet protein microcrystals: use of Laue zones in cross-grating diffraction patterns. J. Appl. Phys. 46:938–940.CrossRefGoogle Scholar
  12. Egerton, R. F., Li, P. and Malac, M. (2004). Radiation damage in the TEM and SEM. Micron 35:399–409.PubMedCrossRefGoogle Scholar
  13. Giddings, T. H., Jr, O’Toole, E.T., Morphew, M., Mastronarde, D.N., McIntosh, J. R. and Winey, M. (2001). Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol. 67:27–42.PubMedCrossRefGoogle Scholar
  14. Gillis, J.M. and Wibo, M. (1971). Accurate measurement of the thickness of ultrathin sections by interference microscopy. J. Cell Biol. 49:947–949.PubMedCrossRefGoogle Scholar
  15. Glaeser, R. M. and Taylor, K. A. (1978). Radiation damage relative to transmission electron microscopy of biological specimens at low temperature: a review. J. Microsc. 112:127–138.PubMedGoogle Scholar
  16. Glauert, A. M. (1998). Biological Specimen Preparation for Transmission Electron Microscopy. Portland Press.Google Scholar
  17. Grubb, D.T. (1974). Radiation damage and electron microscopy of organic polymers. J. Mater. Sci. 9:1715–1736.CrossRefGoogle Scholar
  18. Gunning, B. E. S. and Hardham, A. R. (1977). Estimation of the average section thickness in ribbons of ultra-thin sections. J. Microsc. 109:337–340.Google Scholar
  19. Harlow, M., Ress, D., Koster, A., Marshall, R. M., Schwarz, M. and McMahan, U. J. (1998). Dissection of active zones at the neuromuscular junction by EM tomography. J. Physiol. Paris 92:75–78.PubMedCrossRefGoogle Scholar
  20. Harlow, M. L., Ress, D., Stoschek, A., Marshall, R. M. and McMahan, U. J. (2001). The architecture of active zone material at the frog’s neuromuscular junction. Nature 409:479–484.PubMedCrossRefGoogle Scholar
  21. Hayat, M.A. (2000). Principles and Techniques of Electron Microscopy. Cambridge University Press.Google Scholar
  22. Hayward, S. B. and Glaeser, R.M. (1979). Radiation damage of purple membrane at low temperature. Ultramicroscopy 4:201–210.CrossRefGoogle Scholar
  23. He, W., Cowin, P. and Stokes, D. L. (2003). Untangling desmosomal knots with electron tomography. Science 302:109–113.PubMedCrossRefGoogle Scholar
  24. Heuser, J. E. (1989). Development of the quick-freeze, deep-etch, rotary-replication technique of sample preparation for 3-D electron microscopy. Prog. Clin. Biol. Res. 295:71–83.PubMedGoogle Scholar
  25. Heuser, J. E., Keen, J. H., Amende, L. M., Lippoldt, R. E. and Prasad, K. (1987). Deep-etch visualization of 27S clathrin: a tetrahedral tetramer. J. Cell Biol. 105:1999–2009.PubMedCrossRefGoogle Scholar
  26. Hirose, K., Lenart, T. D., Murray, J. M., Franzini-Armstrong, C. and Goldman, Y. E. (1993). Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP. Biophys. J. 65:397–408.PubMedGoogle Scholar
  27. Horowitz, R. A., Agard, D. A., Sedat, J.W. and Woodcock, C. L. (1994). The three-dimensional architecture of chromatin in situ: electron tomography reveals fibers composed of a continuously variable zig-zag nucleosomal ribbon. J. Cell Biol. 125:1–10.PubMedCrossRefGoogle Scholar
  28. Jesior, J. C. (1982). A new approach for the visualization of molecular arrangement in biological micro-crystals. Ultramicroscopy 8:379–384.PubMedCrossRefGoogle Scholar
  29. Jesior, J. C. and Wade, R. H. (1987). Electron-irradiation-induced flattening of negatively stained 2D protein crystals. Ultramicroscopy 21:313–319.PubMedCrossRefGoogle Scholar
  30. Koster, A. J., Grimm, R., Typke, D., Hegerl, R., Stoschek, A., Walz, J. and Baumeister, W. (1997). Perspectives of molecular and cellular electron tomography. J. Struct. Biol. 120:276–308.PubMedCrossRefGoogle Scholar
  31. Kremer, J. R., O’Toole, E.T., Wray, G. P., Mastronarde, D. M., Mitchell, S. J. and McIntosh, J. R. (1990). Characterization of beam-induced thinning and shrinkage of semi-thick section in H.V.E.M. In Proceedings of the XIIth International Congress for Electron Microscopy (Peachey, L.D. and Williams, D.B., eds). San Francisco Press Inc., San Francisco, pp. 752–753.Google Scholar
  32. Ladinsky, M. S., Mastronarde, D. N., McIntosh, J. R., Howell, K. E. and Staehelin, L. A. (1999). Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J. Cell Biol. 144:1135–1149.PubMedCrossRefGoogle Scholar
  33. Lamvik, M. K. (1991). Radiation damage in dry and frozen hydrated organic material. J. Microsc. 161:171–181.Google Scholar
  34. Landis, W. J., Hodgens, K. J., Song, M. J., Arena, J., Kiyonaga, S., Marko, M., Owen, C. and McEwen, B. F. (1996). Mineralization of collagen may occur on fibril surfaces: evidence from conventional and high-voltage electron microscopy and three-dimensional imaging. J. Struct. Biol. 117:24–35.PubMedCrossRefGoogle Scholar
  35. Lefman, J., Zhang, P., Hirai, T., Weis, R. M., Juliani, J., Bliss, D., Kessel, M., Bos, E., Peters, P. J. and Subramaniam, S. (2004). Three-dimensional electron microscopic imaging of membrane invaginations in Escherichia coli overproducing the chemotaxis receptor Tsr. J. Bacteriol. 186:5052–5061.PubMedCrossRefGoogle Scholar
  36. Lenzi, D., Crum, J., Ellisman, M. H. and Roberts, W. M. (2002). Depolarization redistributes synaptic membrane and creates a gradient of vesicles on the synaptic body at a ribbon synapse. Neuron 36:649–659.PubMedCrossRefGoogle Scholar
  37. Lenzi, D., Runyeon, J.W., Crum, J., Ellisman, M. H. and Roberts, W.M. (1999). Synaptic vesicle populations in saccular hair cells reconstructed by electron tomography. J. Neurosci. 19:119–132.PubMedGoogle Scholar
  38. Liu, J., Reedy, M. C., Goldman, Y. E., Franzini-Armstrong, C., Sasaki, H., Tregear, R. T., Lucaveche, C., Winkler, H., Baumann, B. A., Squire, J. M., Irving, T. C., Reedy, M. K. and Taylor, K. A. (2004a). Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges. J. Struct. Biol. 147:268–282.PubMedCrossRefGoogle Scholar
  39. Liu, J., Taylor, D.W. and Taylor, K.A. (2004b). A 3-D reconstruction of smooth muscle alpha-actinin by CryoEm reveals two different conformations at the actin-binding region. J. Mol. Biol. 338:115–125.PubMedCrossRefGoogle Scholar
  40. Lucic, V., Forster, F. and Baumeister, W. (2005). Structural studies by electron tomography: from cells to molecules. Annu. Rev. Biochem. 74:833–865.PubMedCrossRefGoogle Scholar
  41. Luther, P. K., Lawrence, M. C. and Crowther, R.A. (1988). A method for monitoring the collapse of plastic sections as a function of electron dose. Ultramicroscopy 24:7–18.PubMedCrossRefGoogle Scholar
  42. Marsh, B. J. (2005). Lessons from tomographic studies of the mammalian Golgi. Biochim. Biophys. Acta 1744:273–292.PubMedCrossRefGoogle Scholar
  43. Marsh, B. J., Volkmann, N., McIntosh, J. R. and 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 101:5565–5570.PubMedCrossRefGoogle Scholar
  44. McEwen, B. F. and Frank, J. (2001). Electron tomographic and other approaches for imaging molecular machines. Curr. Opin. Neurobiol. 11:594–600.PubMedCrossRefGoogle Scholar
  45. McIntosh, R., Nicastro, D. and Mastronarde, D. (2005). New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol. 15:43–51.PubMedCrossRefGoogle Scholar
  46. Muller, W. H., Koster, A. J., Humbel, B. M., Ziese, U., Verkleij, A. J., van Aelst, A. C., van der Krift, T. P., Montijn, R. C. and Boekhout, T. (2000). Automated electron tomography of the septal pore cap in Rhizoctonia solani. J. Struct. Biol. 131:10–18.PubMedCrossRefGoogle Scholar
  47. Ohno, S. (1980). Morphometry for determination of size distribution of peroxisomes in thick sections by high-voltage electron microscopy: I. Studies on section thickness. J. Electron Microsc. (Tokyo) 29:230–235.Google Scholar
  48. Padron, R., Alamo, L., Craig, R. and Caputo, C. (1988). A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension. J. Microsc. 151:81–102.PubMedGoogle Scholar
  49. Peachey, L.D. (1958). Thin sections. I. A study of section thickness and physical distortion produced during microtomy. J. Biophys. Biochem. Cytol. 4:233–242.PubMedCrossRefGoogle Scholar
  50. Perkins, G. A., Renken, C. W., Song, J. Y., Frey, T. G., Young, S. J., Lamont, Martone, S. M., Lindsey, E. S. and Ellisman, M. H. (1997). Electron tomography of large, multicomponent biological structures. J. Struct. Biol. 120:219–227.PubMedCrossRefGoogle Scholar
  51. Perkins, G. A., Renken, C.W., van der Klei, I. J., Ellisman, M. H., Neupert, W. and Frey, T. G. (2001). Electron tomography of mitochondria after the arrest of protein import associated with Tom19 depletion. Eur. J. Cell Biol. 80:139–150.PubMedCrossRefGoogle Scholar
  52. Rader, R. S. and Lamvik, M. L. (1992). High conductivity amorphous Ti88Si22 substrates for low temperature electron microscopy. J. Microsc. 168:71–77.Google Scholar
  53. Rath, B. K., Marko, M., Radermacher, M. and Frank, J. (1997). Low-dose automated electron tomography: a recent implementation. J. Struct. Biol. 120:210–218.PubMedCrossRefGoogle Scholar
  54. Reimer, L. (1989). Transmission Electron Microscopy. Springer-Verlag, Berlin.Google Scholar
  55. Saxton, W. O., Baumeister, W. and Hahn, M. (1984). Three-dimensional reconstruction of imperfect two-dimensional crystals. Ultramicroscopy 13:57–70.PubMedCrossRefGoogle Scholar
  56. Shimoni, E. and Muller, M. (1998). On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device. J. Microsc. 192:236–247.PubMedCrossRefGoogle Scholar
  57. Sjostrom, M., Squire, J. M., Luther, P., Morris, E. and Edman, A. C. (1991). Cryoultramicrotomy of muscle: improved preservation and resolution of muscle ultrastructure using negatively stained ultrathin cryosections. J. Microsc. 163:29–42.PubMedGoogle Scholar
  58. Slot, J. W. and Geuze, H. J. (1985). A new method of preparing gold probes for multiple-labeling cytochemistry. Eur. J. Cell Biol. 38:87–93.PubMedGoogle Scholar
  59. Small, J.V. (1968). Measurements of section thickness. In Proceedings of the 4th European Congress on Electron Microscopy (S. Bocciareli, ed.). Vol. 1, pp. 609–610.Google Scholar
  60. Sosa, H., Popp, D., Ouyang, G. and Huxley, H.E. (1994). Ultrastructure of skeletal muscle fibers studied by a plunge quick freezing method: myofilament lengths. Biophys. J. 67:283–292.PubMedGoogle Scholar
  61. Spencer, M. (1982). Fundamentals of Light Microscopy. IUPAB Biophysics Series.Google Scholar
  62. Stenn, K. and Bahr, G. F. (1970). Specimen damage caused by the beam of the transmission electron microscope, a correlative reconsideration. J. Ultrastruct. Res. 31:526–550.PubMedCrossRefGoogle Scholar
  63. Studer, D., Graber, W., Al-Amoudi, A. and Eggli, P. (2001). A new approach for cryofixation by high-pressure freezing. J. Microsc. 203:285–294.PubMedCrossRefGoogle Scholar
  64. Trachtenberg, S., Pinnick, B. and Kessel, M. (2000). The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryoelectron tomography of freeze-substituted cells and projection studies of negatively stained envelopes. J. Struct. Biol. 130:10–26.PubMedCrossRefGoogle Scholar
  65. Unwin, P.N. (1974). Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. II. The influence of electron irradiation of the stain distribution. J. Mol. Biol. 87:657–670.PubMedCrossRefGoogle Scholar
  66. Uzawa, S., Li, F., Jin, Y., McDonald, K. L., Braunfeld, M. B., Agard, D. A. and Cande, W. Z. (2004). Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+. Mol. Biol. Cell. 15:5219–5230.PubMedCrossRefGoogle Scholar
  67. van Marle, J., Dietrich, A., Jonges, K., Jonges, R. de Moor E., Vink, A., Boon, P. and van Veen, H. (1995). EM-tomography of section collapse, a non-linear phenomenon. Microsc. Res. Tech. 31:311–316.PubMedCrossRefGoogle Scholar
  68. Williams, M.A. and Meek, G. A. (1966). Studies on thickness variation in ultrathin sections for electron microscopy. J. R. Microsc. Soc. 85:337–352.Google Scholar
  69. Winkler, H. and Taylor, K.A.(2006). Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography. Ultramicroscopy 106:240–254.PubMedCrossRefGoogle Scholar
  70. Yang, G. C. H. and Shea, S. M. (1975). The precise measurement of the thickness of ultrathin sections by a ‘re-sectioned’ section technique. J. Microsc. 1103:385–392.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Pradeep K. Luther
    • 1
  1. 1.Biomedical Sciences DivisionImperial College LondonLondonUK

Personalised recommendations