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The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules

  • Richard Henderson (a1)


Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associatedenergy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of ˜ 105 molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10000 particles are required. Below 105 molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.



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Abramowitz, M. & Stegun, I. A. (1965). Handbook of Mathematical Functions with Formulas, Graphs and Mathematical Tables. New York: Dover Publications.
Adrian, M., Heggeler-Bordier, B. T., Wahli, W., Stasiak, A. Z., Stasiak, A. & Dubochet, J. (1990). Direct visualisation of supercoiled DNA molecules in solution. EMBO J. 9, 45514554.
Arndt, U. (1984). Optimum X-ray wavelength for protein crystallography. J. Appl. Cryst. 17, 118119.
Baker, T. S., Newcomb, W. W., Olson, N. H., Cowsert, L. M., Olson, C. & Brown, J. D. C. (1991). Structures of bovine and human papillomaviruses – analysis by cryoelectron microscopy and 3-dimensional image-reconstruction. Biophys. J. 60, 14451456.
Breedlove, J. R. & Trammell, G. T. (1970). Molecular microscopy: fundamental limitations. Science, 170, 13101313.
Cowley, J. M. (1975). Diffraction Physics Amsterdam: North-Holland.
Darwin, C. G. (1914). The theory of X-ray reflexion. Phil. Mag. 27, 315333.
De Rosier, D. J. & Klug, A. (1968). Reconstruction of three-dimensional structures from electron micrographs. Nature 217, 130134.
Erickson, H. & Klug, A. (1971). Measurement and compensation of defocusing and aberrations by Fourier processing of electron micrographs. Phil. Trans. Roy. Soc. Lond., B 261, 105118.
Gabor, D. (1948). A new microscopic principle. Nature, 161, 777778.
Glaeser, R. M. & Ceska, T. A. (1989). High-voltage electron diffraction from bacteriorhodopsin (purple membrane) is measurably dynamical. Acta Cryst. A45, 620628.
Gonzalez, A., Thompson, A. W. & NAVE, C. (1992). Cryo-protection of protein crystals in intense X-ray beams. Rev. Sci. Instrum. 63, 11771180.
Henderson, R. (1990). Cryo-protection of protein crystals against radiation damage in electron and X-ray diffraction. Proc. Roy. Soc. B 241, 68.
Henderson, R. (1992). Image contrast in high resolution electron microscopy of biological macromolecules: TMC in ice. Ultramicroscopy 46, 118.
Henderson, R. & Glaeser, R. M. (1985). Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals. Ultramicroscopy 16, 139150.
Henderson, R., Baldwin, J. M., Ceska, T. A., Beckmann, E., Zemlin, F. & Downing, K. (1990). A model for the structure of bacteriorhodopsin based on high resolution electron cryomicroscopy. J. Mol. Biol. 213, 899929.
Hoppe, W. (1983). Electron diffraction with the transmission electron microscope as a phase-determining diffractometer - from spatial frequency filtering to the three-dimensional structure analysis of ribosomes. Angew. Chem. Int. Ed. Engl. 22, 456485.
Hubbell, J. H., Gimm, H. A. & Øverbø, I. (1980). Pair, triplet and total atomic cross sections (and mass attenuation coefficients) for 1Mev-100Gev photons in elements Z = 1 to 100. J. Phys. Chem. Ref. Data 9, 10231147.
Jacobsen, C., Williams, S., Anderson, E., Browne, M. T., Buckley, C. J., Kern, D., Kirz, J., Rivers, M. & Zhang, X. (1991). Diffraction-limited imaging in a scanning transmission X-ray microscope. Optics Commun. 86, 351364.
Jap, B. K., Walian, P. J. & Gehring, K. (1991). Structural architecture of an outermembrane channel as determined by electron crystallography. Nature 350, 167170.
Jeng, T.-W., Crowther, R. A., Stubbs, G. & Chiu, W. (1989). Visualisation of alphahelices in tobacco mosaic virus by cryo-electron microscopy. J. Mol. Biol. 205, 251257.
Kirz, J., Ade, H., Jacobsen, C., Ko, C.-H., Lindaas, S., McNulty, I., Sayre, D., Williams, S. & Zhang, X. (1992). Soft X-ray microscopy with coherent X-rays. Rev. Set. Instrum. 63, 557563.
Kühlbrandt, W. & Wang, D. N. (1991). Three-dimensional structure of plant lightharvesting complex determined by electron crystallography. Nature 350, 130134.
Kühlbrandt, W., Wang, D. N. & Fujiyoshi, Y. (1994). Atomic model of plant lightharvesting complex by electron crystallography. Nature 367, 614621.
Langmore, J. P. & Smith, M. F. (1992). Quantitative energy-filtered electron microscopy of biological molecules in ice. Ultra-microscopy 46, 349373.
Licht, H. (1991). Optimum focus for taking electron holograms. Ultramicroscopy 38, 1322.
Penczek, P., Radermacher, M. & Frank, J. (1992). Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy 40, 3353.
Reimer, L. (1989). Transmission Electron Microscopy, 2nd ed. Berlin: Springer-Verlag.
Sayre, D. & Chapman, H. N. (1994). X-ray microscopy. Acta Cryst. A51 (In press).
Sayre, D., Kirz, J., Feder, R., Kim, D. M. & Spiller, E. (1977). Transmission microscopy of unmodified biological materials: comparative radiation dosages with electrons and ultrasoft X-ray photons. Ultramicroscopy 2, 337349.
Sears, V. F. (1992). Neutron scattering lengths and cross-sections. Neutron News 3(3), 2637.
Smith, M. F. & Langmore, J. P. (1992). Quantitation of molecular densities in cryoelectron microscopy: determination of the radial density distribution of tobacco mosaic virus. J. Mol. Biol. 226, 763774.
Spence, J. C. H. (1988). Experimental High-Resolution Electron Microscopy, 2nd ed., Oxford University Press.
Stewart, P. L., Burnett, R. M., Cyrklaff, M. & Fuller, S. D. (1991). Image reconstruction reveals the complex molecular organisation of Adenovirus. Cell 67, 145154.
Steyerl, A. (1989). Ultracold neutrons: production and experiments. Physica B, 156, 528533.
Steyerl, A., Drexel, W., Ebisawa, T., Gutsmiedle, E., Steinhauser, K.-A., Gahler, R., Mampe, W. & Ageron, P. (1988). Neutron microscopy. Rev. Phys. Appl. 23, 171180.
Thon, F. (1966). Zur Defokussierungsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung. Z. Naturforsh. 219, 476478.
Tonomura, A. (1992). Electron-holographic interference microscopy. Adv. Physics 41, 50103.
Toyoshima, C., Sasabe, H. & Stokes, D. L. (1993). Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane. Nature 362, 469471.
Toyoshima, C. & Unwin, N. (1988a). Contrast transfer for frozen-hydrated specimens: determination from pairs of defocused images. Ultramicroscopy 25, 279292.
Toyoshima, C. & Unwin, N. (1988b). Ion channel of acetyl-choline receptor reconstructed from images of postsynaptic membrane. Nature 336, 247250.
Zernike, F. (1955). How I discovered phase contrast. Science 121, 345349.


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