OPTIMAL STRATEGIES FOR IMAGING THICK BIOLOGICAL SPECIMENS - EXIT WAVE-FRONT RECONSTRUCTION AND ENERGY-FILTERED IMAGING

In transmission electron microscopy (TEM) of thick biological specimen s, the relationship between the recorded image intensities and the pro jected specimen mass density is distorted by incoherent electron-speci men interactions and aberrations of the objective lens. It is highly d esirable to develop a strategy for maximizing and extracting the coher ent image component, thereby allowing the projected specimen mass dens ity to be directly related to image intensities, For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM . Because electron energy-loss filtered imaging allows the contributio ns of inelastically scattered electrons to be removed, it is potential ly advantageous for imaging thick, biological samples, In this paper, exit wavefront reconstruction is used to quantitatively analyse the im aging properties of an energy-filtered microscope and to assess its ut ility for thick-section microscopy. We found that for imaging thick bi ological specimens (> 0.5 mu m) at 200 keV, only elastically scattered electrons contribute to the coherent image component, Surprisingly li ttle coherent transfer was seen when using energy-filtering at the mos t probable energy loss (in this case at the first plasmon energy-loss peak), Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at remov ing the effects of multiple elastic and inelastic scattering and micro scope objective lens aberrations than either technique by itself, Opti mization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV), These results have important impli cations for the accurate recording of images of thick biological speci mens as, for instance, in electron microscope tomography.