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Female Niger essay education
In-Focus Electrostatic Zach Phase Plate Imaging For Transmission Electron Microscopy With Tunable Phase Contrast Of Frozen Hydrated Biological Samples
Nicole Frindt, Marco Oster, Simon Hettler, Björn Gamm, Levin Dieterle, Wolfgang Kowalsky, Dagmar Gerthsen, And Rasmus R. Schröder
1 CellNetworks, BioQuant, Universitatsklinikum Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany
2Laboratorium fur Elektronenmikroskopie, Karlsruhe Institut fur Technologie (KIT), Engesserstr 7, 76128 Karlsruhe, Germany
3Institut fUr Hochfrequenztechnik, Technische Universitat Braunschweig, Schleinitzstr 22, 38106 Braunschweig, Germany
4Innovation Lab GmbH, Heidelberg, Speyerer Str. 4, 69115 Heidelberg, Germany
5CAM Centre of Advanced Materials, Universitat Heidelberg, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
Abstract: Transmission electron microscopy (TEM) images of beam sensitive weak-phase objects such as biological cryo samples usually show a very low signal-to-noise ratio. These samples have almost no amplitude contrast and instead structural information is mainly encoded in the phase contrast. To increase the sample contrast in the image, especially for low spatial frequencies, the use of phase plates for close to focus phase contrast enhancement in TEM has long been discussed. Electrostatic phase plates are favorable in particular, as their tunable potential will allow an optimal phase shift adjustment and higher resolution than film phase plates as they avoid additional scattering events in matter. Here we show the first realization of close to focus phase contrast images of actin filament cryo samples acquired using an electrostatic Zach phase plate. Both positive and negative phase contrast is shown, which is obtained by applying appropriate potentials to the phase plate. The dependence of phase contrast improvement on sample orientation with respect to the phase plate is demonstrated and single-sideband artifacts are discussed. Additionally, possibilities to reduce contamination and charging effects of the phase plate are shown.
Key words: electrostatic phase plate, cryo transmission electron microscopy, weak-phase object, phase contrast, frozen hydrated biological samples, cryo samples, single-sideband effect, charging, contamination
The improvement of close to focus phase contrast imaging for transmission electron microscopy (TEM) of weak-phase objects (WPOs) by using phase plates (PPs) has been a topic of great interest in the TEM community in the last years (Danev & Nagayama, 2001; Nagayama, 2008). In the field of biology, frozen hydrated samples in cryo-TEM are typical WPOs with images showing very low signal-to-noise ratio (SNR) when acquired at low dose imaging conditions. As a consequence of the low atomic number of the protein and its embedding ice, the resulting interaction between the incident electron wave and the sample is very weak. This results in an insignificantly small modification of the amplitude and a small modulation of the phase of the exit electron wave with respect to the plane incident electron wave. To enhance the phase contrast of such WPO images in conventional cryo-TEM, a phase shift between scattered and unscattered electrons is induced by defocusing the objective lens and exploiting the spherical aberration of the imaging lens system. Defocus and spherical aberration lead to transfer of the object information, which is modulated in frequency space by the sine curve like phase contrast Transfer function (pCTF). However, the use of strong defocus values leads to contrast and information delocalization, which often results in a loss of image resolution and information. To overcome these limitations the use of Pps in TEM has been intensively investigated (Danev & Nagayama, 2008; Murata et al., 2010). The task of a PP is to improve the phase contrast, which results from the interference of the scattered and unscattered electron wave. By applying an additional phase shift of ± p / 2 between these two electron wave parts, the sine curve like pCTF is transformed to a cosine-like function. This results in an improved transfer of low spatial frequencies. An additional use of very small defocus values, as described by Danev and Nagayama (2001), leads to an optimized transfer band for the pCTF. As described so far, most Pps induce this additional phase shift between the scattered and unscattered part of the electron wave by using the inner potential of a thin carbon film (Danev & Nagayama, 2001; Danev et al., 2002) or a homogeneous electrostatic field (Majorovits et al., 2007). Pps are mounted in the back focal plane (BFP) of the objective lens or any other conjugated plane, since in those planes scattered and unscattered electrons are spatially separated.
The group of Nagayama demonstrated the application of Zernike and Hilbert film Pps on biological samples (Nagayama, 2008) and also for single particle analysis (Danev & Nagayama, 2008). Murata et al. (2010) presented the application of the Zernike film PP for single particle reconstruction and also for tomography. The drawback of the film PP is the production of positive phase contrast only with a fixed amount of phase shift, which depends—for a given acceleration voltage of the electron microscope—on material and film thickness. Furthermore, the image resolution is decreased due to scattering events inside the film. To overcome these drawbacks, other groups focused on electrostatic Pps, which allow tunable phase shifts and higher image resolution (Schultheiss et al., 2006; Cambie et al., 2007) . The first electrostatic PP, based on an original idea of Boersch (1947), was proposed by Matsumoto and Tonomura (1996) and first realized and studied in detail by Schultheiss et al. (2006) and Majorovits et al. (2007). These electrostatic Pps consist of a five-layered annular lens, which is positioned in the BFP by three supporting rods. Unscattered electrons passing through the electrostatic field inside the annular lens gain an additional relative phase shift with regard to the scattered electrons passing through the outer regions.
It should be noted that several other approaches have been investigated to overcome the drawbacks of the film Pps. For example, Buijsse et al. (2011) reported about a hybrid double-sideband/single-sideband (Schlieren) objective aperture suitable for electron microscopy, and Glaeser et al. (2013) discussed the use of evaporated carbon to minimize the electrostatic charging of such an aperture that is used to produce in-focus phase contrast in the TEM.
The most serious drawback of the Boersch PP design is the information loss resulting from electrons being obstructed by the annular lens electrode and the supporting rods. To overcome this drawback and at the same time use the relative benefits of positive and negative phase contrast, possible only with an electrostatic PP (Danev & Nagayama, 2011) , Zach (2008) proposed an obstruction-minimized PP design, consisting of only one remaining bar with an open electrode at the tip. The first fabricated electrostatic Zach PP and its application for phase contrast imaging of material science samples were shown by Schultheiss et al. (2010). In that work another essential advantage of the electrostatic PP over the film Pps was discussed, namely the option to apply a variable potential and thus to deterministically tune the phase shift. This property can be used for an exit wave reconstruction as proposed by Gamm et al. (2010).
In this article we present the first application of an electrostatic Zach PP for close to focus phase contrast imaging with tunable phase contrast of frozen hydrated samples. For this purpose we use the Zach PP with a conventional TEM to image actin filaments embedded in vitreous ice. We show that the phase contrast images of actin filaments depend on the relative orientation of the PP rod and the filament axis. This results from the mixing of phase contrast and PP-produced single-sideband contrast. Furthermore, very promising solutions to keep the PP tip free from unwanted charged contaminations are presented.
MATERIALS AND METHODS
The electrostatic Zach PP consists of a five-layered structure with a geometry that is comparable to a micro-scaled coaxial cable. Figure 1a shows an overview scanning electron microscopy (SEM) image of a Zach PP in a top perspective. The dark region corresponds to the aperture-like opening of the PP. The electrode-supporting bar has an arrowheadlike shape to improve its stability. The PP itself is implemented in a silicon chip with a freestanding low-stress Si3+XN4_X membrane. Details of the complex fabrication procedure are discussed in Hettler et al. (2012) and Schultheiss et al. (2010). Figure 1b shows a SEM image of the electrode tip with its five-layered structure. A Zach PP consists of a gold electrode in the center, which is surrounded by insulating Al2O3 and Si3+XN4_X layers and shielding gold layers. To reduce the obstruction of electrons in the BFP by the PP bar, the tip has a small width of 0.8-1.0 mm. When applying a voltage to the PP, an anisotropic electrostatic potential leaks out of the open PP tip. Mounted in a diffraction plane of the microscope, the unscattered electrons are aligned such that they pass through the steep gradient of the potential distribution of the field close to the electrode tip. Thereby they gain an additional phase shift with regard to the scattered electrons (Schultheiss et al., 2010). The additional phase shift can be positive or negative, depending on the sign of the applied voltage. The magnitude of the resulting phase shift pPP depends on the distance of the zero-order beam of unscattered electrons from the PP tip and is calculated from the integral of the potential distribution along the electron path:
Where A is the electron wave length, e the elementary electric charge, E0 the electron rest energy, E the kinetic energy of the electron, and F ( z ) the electrostatic potential along the z direction.
Contaminations on the surface and the PP tip lead to strong charging effects and distorted phase shifts. This is indicated by the distorted Thon rings in the power spectra of the acquired PP TEM images in Figures 1c, 1e, and 1f. To reduce these contaminations and charging effects two strategies are used: First, a heating device is implemented on the silicon chip in the vicinity of the PP structure. Details on the micro-structured heating device are described in Hettler et al. (2012). From experiments with this device it was deduced that heating for 4 h at 60°C after a day of cryo experiments was sufficient to remove hydrocarbons and water molecules adhering to the PP surface. Heating of the PP structure is always performed several hours before PP imaging, to avoid PP drift due to thermal material expansion. Second, the PP is plasma cleaned with argon gas and then an about 2.5 nm thin amorphous carbon layer is deposited around the five-layered PP structure by carbon rod evaporation. Following this the PP tip is polished by milling with a focused Ga+ ion beam. This second proce Dure is carried out outside the microscope before the PP is again mounted into the vacuum system. For plasma cleaning a systematic time series has been done to determine the optimal conditions (Frindt, 2013). It was found that 30 min of plasma cleaning was sufficient to remove contamination. Carbon coating was carried out following standard electron microscopy procedures.
The TEM experiments were performed using a Zeiss 923 Omega and a Zeiss Libra 200 DMU (diffraction magnification unit), both equipped with a field emission gun operated at electron energy of 200 keV, a cooling system for cryo-TEM application and a TVIPS TemCam F416 CMOS camera. For the Zeiss 923 Omega, the Zach PP is inserted into the BFP of the objective lens with focal length 2.7 mm. Here no dedicated objective aperture is used for image Acquisition. For the Zeiss Libra 200 DMU the PP is mounted into a DMU. In the DMU the objective lens focal length of 4. 6 m m is enlarged to an effective focal length of 15.1 mm, resulting in a 3.3X magnified conjugated plane of the objective lens BFP. Additionally, the BFP is further magnified by a factor of 1.7 compared to the BFP of the Zeiss 923 Omega due to a 1.7X larger focal length. This leads to an overall 5.6X magnification of the conjugated plane inside the DMU compared to the BFP of the objective lens of the Zeiss 923 Omega. The dimensions and layer thicknesses of the Pps used in the two microscopes are given in Table 1.
The samples used for the experiments were cryo samples of rabbit filamentous actin embedded in vitreous ice on Quantifoil carbon grids (Zeiss 923 Omega) and negative stained bovine liver catalase crystals on Quantifoil carbon grids (Zeiss Libra 200) for the power spectra shown in Figures 1e-1h. For the cryo sample preparation original Quantifoil grids without additional carbon film over the holes is used. Three ml of filamentous actin suspension is pipetted directly onto the grid and quick frozen by plunging it into liquid ethane. Thereby a cooling rate of 107 K/s is reached, which ensured the transformation from liquid water into vitreous ice (Dubochet et al., 1982). The preparation was done using a FEI Vitrobot Mark IV. The sample preparation took place at a temperature of 4°C and air humidity of close to100% inside the preparation chamber. For the negative stained bovine liver catalase crystal sample, 1 ml of catalase buffer solution (SERVA, Germany) on a sample grid is stained two times with 5 ml unbuffered 2% aqueous uranyl. Excessive staining solution is dried with filter paper.
RESULTS AND DISCUSSION
Reduction of Charging Effects of Electrostatic Pps Heating the PP with an integrated heating system reduces the charging of the PP tip drastically. Figure 1c shows a power spectrum of a filamentous actin cryo sample with a charged PP structure, indicated by the red dashed lines. This PP was mounted in the BFP of the Zeiss 923 Omega. The distorted Thon rings and the bright area next to the PP tip are typical indications of charging. Figure 1d shows the power spectrum with the same PP structure after heating the device for about 4 h at 60°C. The distortions of the Thon rings have vanished and the PP structure no longer shows any effects of charging. A cooling period after heating and before PP TEM of ~ 3 h is always applied to stabilize thermal PP movements. The heating procedure needs to be repeated after ~ 4 h of operation.
The additional beneficial influence of plasma cleaning and carbon coating on PP charging is demonstrated in Figures 1e-1h. Here the PP was mounted into the DMU of the Zeiss Libra 200 DMU. Due to the largely reduced magnetic field in the DMU compared to the field strength in the conventional BFP of an objective lens, additional electrical fields, arising from PP charging, have a stronger effect on the electron trajectories. Thus, remnant contamination after heating and resulting charging effects have to be further reduced. This is visualized in the power spectrum in Figures 1e and 1f, which was acquired with a PP with heating device but without carbon coating. Figure 1e is acquired close to focus and Figure 1f at 2 mm under focus. Apart from reflections from the catalase crystals it shows strong charging and thus resulting in strong deformation of the Thon rings in the power spectrum. The situation changes completely after argon plasma cleaning and subsequent coating with amorphous carbon, as demonstrated in Figure 1g acquired close to focus and Figure 1h acquired at 2 mm under focus. Figure 1g shows that even subtle CTF artifacts, arising, for example, from charging, which are Most visible in close to focus power spectra, are removed after carbon coating. In addition, the Thon rings in the power spectra of images acquired at 2 mm under focus in Figure 1h have the required circular shape without charging artifacts. Contamination after extended operation of the carbon coated PP can then again be removed by using the heating system as described above.
Argon plasma cleaning helps to remove organic and oxide contamination. However, hydrocarbons and water vapor layers on the gold surface of the PP are very difficult to remove completely, even after argon plasma cleaning under high vacuum conditions. This conclusion was drawn by comparing charging of a plasma cleaned PP after mounting it into the Zeiss 923 Omega and the Zeiss Libra 200 DMU. We found that residual hydrocarbons on the surface and thus arising charging induced smaller phase distortions in the Zeiss 923 Omega, whereas remnant contamination showed a much stronger effect in the Zeiss Libra 200 DMU. This result is in good agreement with similar observations of gold surface contamination reported by Walker et al. (2010) and Weber et al. (1996). However, the combination of argon plasma cleaning and additional coating of the PP with amorphous carbon has led to significantly reduced PP charging.
The binding of hydrocarbons or water molecules is based on physisorption and its strength is strongly dependent on the absorbing substrate. In the case of gold or other highly conductive surfaces, higher binding energies reaching those of chemisorbed molecules can occur due to the interaction with image charges. This might be the reason why argon plasma cleaning and PP heating is not sufficient to remove all hydrocarbons from the gold surface of the PP. The very inhomogeneous charging effects that are observed on Pps without carbon coating are most probably attributed to cracked hydrocarbons, which are formed on the PP during electron irradiation. This leads to polymerization by cross-linking, formation of charges on the gold surface and hence to induced charges in the metal surface. The electron irradiation of the PP surface might also lead to the production of secondary electrons, which then form a charge cloud around the PP, thus producing an additional fluctuation of the electrical field around the PP. The coating of the PP with amorphous carbon reduces the binding energies for physisorption of the hydrocarbon and water molecules. Even areas with insulating character could be present, due to disordered regions of C-atoms with mainly sp3-diamondlike hybridization, which also is a factor for reduced binding energy. Another explanation for reduced adhesion of hydrocarbons when using a carbon layer is the smaller surface energy of carbon compared to that of gold according to Vitos et al. (1998).
Tunable PP cryo-TEM of Filamentous Actin Filaments
Rabbit actin filaments embedded in vitreous ice was used as a sample to demonstrate tunable and invertible phase contrast of a true WPO with an electrostatic Zach PP. The actin Monomers are arranged as a helical double-strand filament, with a diameter of 5-9 nm and a helical repeat of 35.7 nm (Holmes et al., 1990; Alberts et al., 2008). The PP equipped with a heating device but without an additional carbon layer was mounted into the Zeiss 923 Omega operated at 200 kV. Heating the PP was sufficient to remove visible charging effects because the PP was mounted in the BFP of the objective lens. Four images of the same sample area with their corresponding power spectra are shown in Figure 2. Each image is recorded with an electron dose of 1,500 e—/nm2. To give an overview of all present filaments in the sample area, Figure 2a shows an image acquired at 6.5 mm underfocus, without inserted PP. Figures 2b-2d were acquired close to focus and with a PP next to the zero-order beam. No potential is applied to the electrode in Figure 2b. As expected for a close to focus image acquisition, most filaments do not show any contrast compared to Figure 2a, except those filaments with an orientation from the left lower to the right upper image corner, labeled with white arrows. This contrast results from the single-sideband effect, which can be understood in the following way: spatial frequencies corresponding to the diameter structure of the filament are oriented perpendicular to the filament and appear in the same direction as the obstructing PP bar in diffraction space. This applies to the filaments marked by the white arrows in Figure 2b. In the corresponding power spectrum (Fig. 2f) the spatial frequencies of these filaments are aligned along the green double arrow. These spatial frequencies are partially obstructed by the PP, which is indicated by the red dashed lines. We note that, due to Friedel symmetry in the power spectra, the PP structure is visible twice at mirrored positions with respect to the zeroorder beam. Nevertheless, frequency obstruction in the BFP occurs only in one half plane, leading to the single-sideband contrast, which is responsible for the high contrast of the filaments marked with white arrows. In Figures 2c and 2d potentials of +0.9 and —0.9 V were applied to the Zach PP. A change in phase contrast, even with contrast inversion, is now visible. Actin filaments indicated with black arrows display positive phase contrast in Figure 2c and negative phase contrast in Figure 2d. The phase contrast of perpendicularly oriented filaments also shows a slight change in contrast, but no contrast inversion is visible, since contrast is dominated by the effect. Furthermore, the Bragg contrast of a crystalline ice region is marked with a blue ellipse. The contrast of the crystalline ice particle is strongly delocalized due to the high defocus value as can be inferred from the separated bright spots in Figure 2a. Delocalization is hardly Recognizable in Figures 2b-2d, which confirms that images are acquired close to Gaussian focus. In addition, in this crystalline ice region, a contrast inversion is visible by comparing Figure 2c and 2d.
The tunable phase contrast for different PP potentials is further investigated on two filament sections with perpendicular orientation to each other. For this, the mean image intensities are equalized. Two filament areas are selected and aligned to each other. Since images were recorded at low SNR, application of a noise reducing anisotropic diffusion filter was necessary. The magnified filament regions are marked with a red and a blue box in Figure 3a. The change in phase contrast, depending on applied PP potential and filament orientation, are shown in Figures 3b-3e. In the first image row (Filament No. 1) the change in phase contrast of the filament is visible for different applied voltages to the PP. In contrast, the filament imaged in the second row (Filament No. 2) shows no change in phase contrast for different applied PP voltages. Based on these images, line scans are taken across the filament structure and averaged over a line width of 150 pixels. The orientation of these line scans is marked by a red and a blue arrow in Figure 3b, respectively. The corresponding graphs are shown in Figure 4. The line scans for Filament No. 1 in Figure 4a show an intensity and corresponding phase contrast inversion at + 0.9 and —0.9 V, which indicate a phase shift of p / 2 and —p/2. For Filament No. 2, all three line scans for +0.9, 0.0, and —0.9 V show a comparable intensity profile with a Minimum at 14 nm (Fig. 4c), which indicates negative phase contrast. Furthermore, the amplitude is slightly more pronounced for —0.9 V than for the application of 6.5 mm under focus, without a PP (Fig. 4d). However, both line scans show comparable object profile widths. This effect might be caused by the steep phase gradient of the Zach PP, which produces an unwanted delocalization of the imaged object.
Spatial Frequency Obstruction by PP Structures and Single-Sideband Correction
The size of the PP tip and its distance to the zero-order beam in the BFP plays a major role in image formation and influence the visibility of WPOs. The cryo-TEM phase contrast images of the actin filaments show that singlesideband artifacts affect filaments with an orientation perpendicular to the PP tip. This artifact dominates and thus suppresses the effect of phase contrast improvement. These image artifacts appear as spatial frequencies are obstructed by the PP structure from one side. The image artifacts are most pronounced for large structural features and corresponding small spatial frequencies. This is due to the larger fraction of obstructed Friedel symmetric spatial frequencies by the PP structure, compared with higher spatial frequencies with larger radial distance to the zero-order beam. Nevertheless, it also influences small sample structures such as the diameter of the actin filaments.
In our experiments shown in Figures 2 and 3, the PP tip had a distance of 240 nm from the zero-order electron beam. This corresponds to a beginning obstruction frequency for single-sideband contrast kssb = 0.035 nm—1, by using the equation kssb = r/( fA) (Reimer & Kohl, 2008), where r = 240 nm is the distance of the PP tip to the zero-order beam, f = 2.7 mm is the focal length of the objective lens, and A = 2.51 pm is the relativistic wavelength of the electron wave for an acceleration voltage of 200 kV. This means that sample details with a size smaller than xssb = 1/kssb, resulting in xssb = 27.72 nm, are influenced by the single-sideband contrast. Hence, the actin filaments with a diameter of 5-9 nm are affected as observed.
Actin filaments with an orientation parallel to the PP tip show phase contrast inversion, which is not based on single-sideband artifacts. Furthermore, the change in phase contrast by tuning the PP voltage shows that the steep gradient of the spatial electrostatic potential distribution produces a "soft" cut-on frequency, that is, a gradual change between phase shifted and not phase shifted spatial frequencies. This effect is beneficial with respect to contrast oscillation artifacts, which occur if a step-like change of phase shift occurs at blocking structures in the BFP like, for example, for the micro-scaled electrostatic lens of a Boersch PP as described in Schultheiss et al. (2010).
The remaining single-sideband artifact can be corrected numerically for WPOs. For an ideal WPO the information from spatial frequencies that are obstructed by the PP tip from one side in the BFP is still contained in the Friedel symmetric frequencies. Therefore, information is Not lost completely for the obstructed region, but the intensity values in these regions are reduced by a factor of two in the according power spectra. The numerical correction is analogous to the correction for the use of a Boersch PP as described in Majorovits et al. (2007), but the correction has to be performed for one instead of three supporting bars. For correction of the single-sideband artifact the exact position of the PP and its Friedel symmetric image have to be determined from the power spectrum of the PP TEM image. If one wants the images to be corrected in a way that the single-sideband affected filament are eliminated from the image, then the frequency coordinates (kx, ky) of the PP rod position and its Friedel symmetric image in the Fourier transformed image have to be multiplied by the value 2sign(kx). Otherwise the single-sideband affected filaments can show positive or negative phase contrast after the correction, if the frequency coordinates (kx,ky) are multiplied by the value 2(kx) or —2(kx). All other frequency coordinates of the Fourier transformed image have been multiplied by the value 1. Afterwards a back Fourier transformation has to be done to receive the corrected image. In Figure 5 we show the result of singlesideband correction for the images of actin filaments performed such that only the non single-sideband phase contrast Is visible, therefore increasing visibility of the true Ppmediated phase contrast. Uncorrected and corrected images (with eliminated single-sideband affected filaments) are shown in Figures 5a and 5b for U = 0 V, Figures 5c and 5d for U = +0.9 V, and Figures 5e and 5f for U = - 0 . 9 V. Filaments showing single-sideband contrast (marked by white arrows in Fig. 2) are now almost invisible, as expected for close to focus images. Only filaments with pure improved phase contrast induced by the PP potential are now visible. In Figure 6 we show the result of single-sideband correction performed such that the single-sideband affected filaments show either positive (Fig. 6a) or negative (Fig. 6b) phase contrast. These corrections were calculated for the positive phase contrast images shown in Figure 5c. Figure 6c shows the corresponding power spectrum after singlesideband correction. This correction procedure does not compromise the contrast improvement, as is expected for the applied correction algorithm. Line scans across filaments in these single-sideband corrected images show the same profile as in Figure 4.
We show the first application of an electrostatic Zach PP for close to focus phase contrast imaging and tunable positive/ negative phase contrast of a typical frozen hydrated biological sample. In our experiments we demonstrate the advantage of variable phase shifts enabled by the tunable phase shifting potential of the electrostatic Zach PP. This enables for the first time close to focus image acquisition of WPOs with increased and tunable phase contrast. Compared to film Pps the electrostatic Zach PP produces not only tunable positive and negative phase contrast, but also provides potentially higher resolution images, by preventing coherence loss in otherwise unavoidable additional scattering events in film Pps. Compared to the electrostatic Boersch PP, the Zach PP offers considerably less spatial frequencies obstruction. Spatial frequency information obstructed from one side by the Zach PP leads to single-sideband contrast. For WPOs the object information could be reconstructed exploiting Friedel symmetric information in frequency space in the BFP.
To illustrate tunable image contrast an image series of close to focus PP TEM images of frozen hydrated actin filaments Are shown. An inversion of the phase contrast is visible for different applied PP potentials. This is compared to the image contrast by simply defocusing the objective lens. Images and line scans across the actin filament show that phase contrast visibility depends on the sample orientation with regard to the obstructive PP structure. Single-sideband artifacts occur only for actin filament with an orientation perpendicular to the PP tip. This results from the one-sided obstruction of appropriate spatial frequencies by the PP tip in the BFP, which dominates the general image appearance. For filaments with an orientation parallel to the PP tip, no obstruction of corresponding spatial frequencies in the BFP occurs and hence phase contrast inversion is visible. The shown filaments illustrate this particular imaging behavior of the Zach PP, but, of course, this anisotropic contrast will be present for any imaged object. Single sideband effects can be corrected by image processing following a method described, for example, in Majorovits et al. (2007). The anisotropic but steep gradient electrostatic potential distribution of the electrostatic Zach PP, providing a soft cut-on frequency did not disturb the phase contrast image formation as reported in Schultheiss et al. (2010). An interesting finding of our experiments was the reduction of charging effects by cleaning the PP with argon plasma and subsequent coating with amorphous carbon. Power spectra of images with inserted PP before and after carbon coating show the strong improvement of Thon ring appearance, which is normally compromised by contamination on the PP.
As an outlook for further experiments and application possibilities we conclude that electrostatic Pps are now promising new tools for high-resolution applications. Multiple elastic scattering as well as incoherent inelastic scattering in the diffraction plane is completely absent in contrast to the usual thin-film Pps. Moreover, with tunable contrast object-wave reconstruction and hence compensation of aberrations can now be performed that ultimately limits the imaging system inherent resolution to the information limit of the microscope.
We thank Katrin Schultheiss for her support and advice concerning electrostatic phase plates and Setsuko Fujita Becker for the actin suspension preparation. This project was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft) Sch 424/11 and Ge 841/16. Lewin Dieterle, Wolfgang Kowalsky, and Rasmus R. Schroder acknowledge the funding by the German Federal Ministry of Education and Research (FZ 13N 10794).
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