Peter Rez, Lothar Houben, Shahar Seifer, Michael Elbaum
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Contrast by electron microscopy in thick biological specimens.
The contributions of coherent bright-field phase and incoherent dark-field amplitude contrast are investigated for thick biological specimens. A model for a T4 phage is constructed and images simulated for both TEM and STEM phase contrast using a multislice code. For TEM, the fraction of the illumination intensity available for phase contrast imaging is limited by the fraction of electrons in the zero loss peak, the plasmon peak, or the Landau distribution peak for very thick specimens. These were measured from electron energy loss spectra recorded from various thicknesses of vitreous ice. The incoherent amplitude contrast is simulated using the Penelope Monte Carlo code. Noise limits the features that can be distinguished under the low-dose conditions required for cryo-EM, even for high electron exposures of 100 electrons/Å2. Since in STEM post specimen optics are not used to form the image inelastically scattered electrons contribute to the recorded intensity. In principle STEM should have an advantage over TEM not just for incoherent amplitude contrast but also for coherent phase contrast beyond the limit of weak phase. The simulations suggest that it should be possible to image features in the phage embedded in 1 µm of vitreous ice when collection angles are optimised for bright or dark-field signals, with best contrast achieved for accelerating voltages of about 700 keV.
期刊介绍:
The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit.
The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens.
Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.