In this theoretical study we analyze contrast transfer of weak-phase objects in a transmission electron microscope, which is equipped with an aberration corrector (C(s)-corrector) in the imaging lens system and a physical phase plate in the back focal plane of the objective lens. For a phase shift of pi/2 between scattered and unscattered electrons induced by a physical phase plate, the sine-type phase contrast transfer function is converted into a cosine-type function. Optimal imaging conditions could theoretically be achieved if the phase shifts caused by the objective lens defocus and lens aberrations would be equal to zero. In reality this situation is difficult to realize because of residual aberrations and varying, non-zero local defocus values, which in general result from an uneven sample surface topography. We explore the conditions--i.e. range of C(s)-values and defocus--for most favourable contrast transfer as a function of the information limit, which is only limited by the effect of partial coherence of the electron wave in C(s)-corrected transmission electron microscopes. Under high-resolution operation conditions we find that a physical phase plate improves strongly low- and medium-resolution object contrast, while improving tolerance to defocus and C(s)-variations, compared to a microscope without a phase plate.
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007.
In the last few years the use of carbon film for Zernike-type phase contrast in the TEM has been explored extensively [1]. Several problems such as contamination and charging of the film let some groups consider an alternative approach, which is the use of an electric potential to affect the phase of the electron wave. This technical solution for obtaining phase contrast in TEM was first proposed by Boersch [2]. Matsumoto and Tonomura suggested a feasible realization of the device [3]. Recently two groups have succeeded to build such electrostatic phase plates in the form of an einzel lens [4] and a drift tube [5], respectively, and have shown the first experimental proof of phase contrast mediated by an electric potential. One remaining problem with both published realizations of the electrostatic phase plate [4,5] is the obstruction of electrons in the diffraction plane by structures that are necessary to form the correct, spatially confined electric fields. In case of the electrostatic einzel lens (Fig.1.A) such structures are the ring electrode of the lens itself and its supporting connectors. To avoid the obstruction, we propose a novel electrostatic phase plate shown in Fig.1.B. Instead of using special structures for confining the electric fields, we employ a highly anisotropic field distribution, which is placed at an anamorphotic image of the diffraction plane, i.e. in a plane where the diffraction image is compressed in one direction. The field shifts the phase of a thin stripe of the electron wave. We incorporate two phase plates, each of which is positioned at one of two crossed anamorphotic diffraction planes as shown in Fig.2. Each phase plate shifts the phase by 45˚ for optimal phase contrast of a weak phase object. Simulated phase contrast images are shown in Fig.3 for this mode of operation. We compare our einzel lens results [4] for diffraction planes without additional magnification (Fig.3.B) and a 7-fold magnification ( Fig.3.C) with results for anamorphotic designs of different aspect ratios of the diffraction plane. The new anamorphotic phase plates can be incorporated into future aberration correctors that provide strongly anamorphotic images of the diffraction plane.
Frozen-hydrated biological specimen yield low object contrast in conventional TEM images recorded typically at electron energies of 200 300 keV. This property is inherent to weak phase objects such as ice-embedded biological or soft matter material. In addition, the contrast and thereby the attainable resolution are limited by the characteristic electron dose that the specimen can tolerate before its structure is irrevocably destroyed. However, improved image contrast is crucial when visualizing cryospecimen e.g. for alignment procedures used in single particle analysis to obtain 3D structural information of biological macromolecules.The conventional method to improve image contrast is defocusing. However, defocusing leads to information gaps in the contrast transfer function and in general to attenuated high resolution.. Another emerging technique is the use of phase plates [1], which should yield optimal contrast transfer but lacks practicability so far [2]. Here we investigate the contrast enhancement obtained by zero-loss filtering [3] combined with imaging at low electron energies.We used Cs-corrected Energy Filtering TEMs (Zeiss LIBRA200 Cs) equipped with TVIPS TemCam F416 cameras to measure the improvement of image contrast with decreasing electron energy. Figure 1 shows a pair of zero-loss filtered images of TMV embedded in vitrified ice recorded at 40kV (A) and at 80kV (B) in focus. The applied electron dose was 2,5 e -/Å 2 and 3,8 e -/Å 2 , respectively, and the ice thickness was determined to be about 40nm in both cases. The image acquired at 40kV clearly shows better image contrast despite the lower applied electron dose. This is confirmed quantitatively by equals 9,5% (figure 1C) while at 80kV the contrast decreases to 4,9% ( figure 1D). The analysis of a whole set of images of TMV recorded at electron energies of 20, 40, 60 and 80 kV is shown in figure 2. The graph shows averaged contrast measurements in dependence of electron energy for zero-loss filtered (blue markers) and unfiltered (red markers) images. The energy dependence can be nicely fitted with a function proportional to the scattering cross section : C ~ ~ 1/v 2 (v = electron speed). Furthermore, zero-loss filtering yields a contrast improvement be a factor of two for all examined electron energies, as expected from previous work [3].The gain in contrast has to be seen in context with the applicable electron dose D e . Our data confirm that beam damage is inversely proportional to the scattering cross section, D e ~ 1/ ~ v 2 (data not shown). Since the dose limited resolution is proportional to ~ C -1 D e -1/2 ~ v, the attainable resolution for typical cryo-specimen should increase with decreasing electron energy. First results show a trend in this direction (data not shown). However, a competing effect is the increasing contribution of inelastic scattering in a sample of finite thickness at lower electron energy (e.g. 20kV), which leads to a reduced zero-loss signal. In this case the electron dose limited resolution is further constrain...
Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007
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