Towards higher resolution in electron beam sensitive specimens of biological origin

Abstract

SUMMARY The resolution of structurally significant detail in electron micrographs of organic polymers is limited by damage to the specimen during electron irradiation. The factors affecting the kinetics of the degradation of characteristic high angle reflections in the electron diffraction patterns of Cellulose I and II have been studied in an attempt to determine quantitatively the conditions under which meaningful high resolution micrographs may be recorded. In Cellulose I it was established that the decay of the 002 reflection during irradiation (in the range 0.6‐6.0 Am−2) is independent of dose‐rate. Moreover the meridional 040 and equatorial 002 reflections were found to decay at different rates. This latter finding indicates the importance of selecting the appropriate conditions for imaging a particular structural repeat in a specimen. Perhaps the most significant conclusion arising from this work is that specimen cooling in conjunction with low‐beam densities may be used to considerable advantage in obtaining meaningful higher resolution images than hitherto possible in beam‐sensitive materials.

Image analysis in the electron microscopy of cellulose protofibrils

“…No information is obtained about the internal structure of the protofibrils. In fact radiation damage by the electron beam will certainly have destroyed the crystal structure of the cellulose during the process of recording the image [22]. In future work it will be necessary to evaluate the effect of radiation damage on the morphology of cellulose protofibrils.…”

Transmission electron microscopy of cellulose. Part 2: technical and practical aspects

“…The significant damage created by the electrons crossing a low-density polymer specimen rapidly affects the resolution of smaller details and decreases the contrast of the objects. Molecular excitations and ionization phenomena are induced in the material by the inelastic scattering of the electrons, resulting in the breaking of covalent bonds, diffusion of created free radicals and emission of volatile species (Dobb and Murray 1974;Grubb 1974). The consequences can be mass loss, melting, vaporization and crystallinity decrease, which have a detrimental impact on the amplitude, diffraction and Fresnel contrasts.…”

“…In addition to increasing the accelerating voltage, the radiation damage can be significantly slowed down (but not suppressed) by keeping the specimen at low temperature during the observation, using a cryo-holder cooled down with liquid nitrogen (Dobb and Murray 1974).…”

Radiation damage of polyethylene single crystals in electron microscopy between 1 and 2.5 MV. II. The influence of temperature

“…This result has been theoretically demonstrated for general transformation systems [29]. No experimental proof of this influence has been given yet for polymers [10,32] but it can be noted that the range of variable intensity lies only over 1 order of magnitude and that, if our specimen is comparable to living cells [29], an effect would be detected only when the intensity is changed by a factor of 100 or 1 000. Thus, this prediction is very important for the use of STEM where the beam is very concentrated and for the use of pulsed beams.…”