Ultramicroscopy 116 (2012) 62–72

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Ultramicroscopy

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Practical aspects of Boersch phase contrast electron microscopyof biological specimens

Andreas Walter a, Heiko Muzik b, Henning Vieker b, Andrey Turchanin b, Andre Beyer b, Armin Golzhauser b,Manfred Lacher c, Siegfried Steltenkamp c, Sam Schmitz c, Peter Holik c, Werner Kuhlbrandt a, Daniel Rhinow a,n

a Max-Planck-Institute of Biophysics, Department of Structural Biology, Max-von-Laue-Str. 3, D-60439 Frankfurt, Germanyb University of Bielefeld, Physics of Supramolecular Systems and Surfaces, Universitatsstr. 25, D-33615 Bielefeld, Germanyc Caesar Research Center, Ludwig-Erhard-Allee 2, D-53175 Bonn, Germany

a r t i c l e i n f o

Article history:

Received 5 December 2011

Received in revised form

7 March 2012

Accepted 10 March 2012Available online 17 March 2012

Keywords:

Phase plate

Transmission electron microscopy

Aberration correction

X-ray photoelectron spectroscopy

Auger electron spectroscopy

Helium ion microscopy

91/$ - see front matter & 2012 Elsevier B.V. A

x.doi.org/10.1016/j.ultramic.2012.03.009

esponding author. Tel.: þ49 69 6303 3050.

ail address: daniel.rhinow@biophys.mpg.de (D

a b s t r a c t

Implementation of physical phase plates into transmission electron microscopes to achieve in-focus contrast

for ice-embedded biological specimens poses several technological challenges. During the last decade several

phase plates designs have been introduced and tested for electron cryo-microscopy (cryoEM), including thin

film (Zernike) phase plates and electrostatic devices. Boersch phase plates (BPPs) are electrostatic einzel lenses

shifting the phase of the unscattered beam by an arbitrary angle. Adjusting the phase shift to 901 achieves the

maximum contrast transfer for phase objects such as biomolecules. Recently, we reported the implementation

of a BPP into a dedicated phase contrast aberration-corrected electron microscope (PACEM) and demonstrated

its use to generate in-focus contrast of frozen–hydrated specimens. However, a number of obstacles need to be

overcome before BPPs can be used routinely, mostly related to the phase plate devices themselves. CryoEM

with a physical phase plate is affected by electrostatic charging, obliteration of low spatial frequencies, and

mechanical drift. Furthermore, BPPs introduce single sideband contrast (SSB), due to the obstruction of Friedel

mates in the diffraction pattern. In this study we address the technical obstacles in detail and show how they

may be overcome. We use X-ray photoelectron spectroscopy (XPS) and Auger electron spectroscopy (AES) to

identify contaminants responsible for electrostatic charging, which occurs with most phase plates. We

demonstrate that obstruction of low-resolution features is significantly reduced by lowering the acceleration

voltage of the microscope. Finally, we present computational approaches to correct BPP images for SSB

contrast and to compensate for mechanical drift of the BPP.

& 2012 Elsevier B.V. All rights reserved.

1. Introduction

Physical phase plates promise to increase the inherently lowsignal-to-noise ratio in electron micrographs of unstained biologicalsamples, and to enable in-focus transmission electron microscopy(TEM) of weak phase objects [1–7]. Although proposed by Boersch asearly as 1947 [8], it has not been possible to devise and test phaseplates (PPs) suitable for electron cryo-microscopy (cryoEM) untilrecently [9]. The simplest PP is a thin amorphous carbon film with asmall hole for the central beam to pass through, which is placed inthe back-focal plane of the objective lens [9]. Thin carbon film(Zernike) PPs are analogous to quarter-wave zone plates in lightmicroscopy. Depending on the film thickness, the phase of thescattered electrons is shifted by approximately 901 by the innerpotential of the carbon film to maximise phase contrast. Zernike-typePPs have been applied in a variety of cryoEM studies [10–13]. As analternative to thin film PPs, electrostatic PPs have been developed,including Boersch-type einzel-lens phase plates (BPPs) [2,5,7,14,15],a drift tube [3], and other devices [6]. The principle of phase contrast

ll rights reserved.

. Rhinow).

TEM with a BPP is shown in Fig. 1a. Fig. 1b and c shows BPP devicesimaged in a helium ion microscope, indicating alternating layers of

conductive (Au) and isolating materials (Si3N4) of the electrode ring.

The locally confined electrostatic field of the electrode ring shifts the

phase of the unscattered electrons by an angle that depends on the

applied voltage, and can be adjusted freely in the range from þ901 to

�901, thus enabling in-line holography [16]. Fig. 1d demonstrates

the generation of in-focus contrast for ice-embedded specimens

using a BPP (see also Supplementary Fig. S1). Recent simulations

predict that PPs will be particularly useful for single-particle cryoEM

of unsymmetrical molecules with masses as low as 100 kDa [17,18].A number of technical problems remain to be solved until BPPs can

be used routinely in cryoEM. First, surface contamination and electro-static charging are proving to be serious problems for all PPs [19]. Inthis work we investigate possible sources of BPP contamination byhelium ion microscopy (HIM), Auger electron spectroscopy (AES) andX-ray photoelectron spectroscopy (XPS), and suggest possible solu-tions. Another problem that has to be addressed is mechanical drift ofthe PP device. This is of particular concern when images of the samespecimen area are to be recorded in a time frame of minutes tohours, as in the acquisition of tomographic tilt series. To overcomethis obstacle, we have developed a routine to correct for BPP drift

Fig. 1. Phase contrast TEM with a BPP. (a) The BPP is an electrostatic einzel lens that shifts the phase of the unscattered beam by 901 to maximise in-focus phase contrast of weak-

phase objects. (b), (c) HIM images of the central electrode show alternating layers of conductive and insulating materials in the BPP. (d) Comparison of defocus contrast and BPP in-

focus contrast for ice-embedded tobacco mosaic virus (TMV) in Fourier transforms of CCD images. Each column contains Fourier transforms of a series of images recorded from the

same area using an electron dose of 6 e�/A2 per image. Red arrows indicate the 3rd layer line of TMV at 1/2.3 nm�1. Images were recorded at 200 kV acceleration voltage.

A. Walter et al. / Ultramicroscopy 116 (2012) 62–72 63

automatically. A third problem is the cut-on frequency of the BPP, sincethe central electrode ring of the device blocks contrast transfer at lowresolution. The phase contrast aberration-corrected electron micro-scope (PACEM) includes a diffraction magnification unit (DMU) toreduce obliteration of low spatial frequencies [7]. However, dependingon the particular BPP used, a lower cut-on frequency is often desired.We demonstrate that this demand can be largely met by reducing theacceleration voltage. Finally, we address the effects of single-sidebandcontrast caused by the rods supporting the central BPP electrode, andpresent a computational method to correct for this effect.

2. Materials and methods

2.1. Boersch phase plates

BPPs were manufactured by the micro-systems technology groupat Caesar Research Center, as described recently [7]. BPPs were usedin a phase contrast aberration-corrected microscope (PACEM) [7].The PACEM includes a diffraction magnification unit (DMU) and anincreased pole piece gap to increase the cut-on frequency, and thusto reduce obliteration of low spatial frequencies.

Argon sputtering of BPPs was conducted with an argon fluxof �10 mA/cm2 and an acceleration voltage of 1 kV. The base pressureof the UHV system was 5�10�10 mbar.

2.2. Helium ion microscopy (HIM)

Helium ion microscopy (HIM) was performed with a Carl ZeissOrion Pluss instrument. The Heþ beam was operated at 32–34 kV

acceleration voltage, and secondary electrons were collected byan Everhart–Thornley detector. Rutherford backscattered ions(RBI) were detected by a microchannel plate (MCP) detector.

2.3. X-ray photoelectron spectroscopy (XPS)

XPS data were recorded in a multi-technique UHV instrument(Multiprobe, Omicron Nanotechnology) using a monochromated AlX-ray source (1486.7 eV, 225 W) and a hemispherical electronenergy analyzer (Sphera) in constant analyzer energy mode. Thechamber pressure was 5�10�10 mbar. For the analysis of the XPspectra a Shirley background subtraction procedure was employed.

2.4. Auger electron spectroscopy (AES)

SEM imaging and AES were performed with a Zeiss Geminiscanning electron microscope at a beam energy of 3 kV and anelectron current of 2 nA. The Auger electrons were detected by ahemispherical electron energy analyser (NanoSAM Energy AnalyserPackage, Omicron Nanotechnology) in constant retard ratio mode.The experiments were performed at a pressure of 3�10�10 mbar.

3. Results and discussion

3.1. Initial surface contamination of BPPs

Images recorded with fresh BPPs often show characteristicflower-like distortions of the Fourier transform (Fig. 2a). These

Fig. 2. Initial surface contamination of BPPs. (a) Phase effects due to charging are

observed in Fourier transforms of BPP images of amorphous carbon, recorded

immediately after loading a BPP chip into the microscope. (b) The phase effects

are reduced by heating at 120 1C for 1 day in the microscope. Both images were

recorded at similar defocus.

A. Walter et al. / Ultramicroscopy 116 (2012) 62–7264

distortions are indicative of electrostatic charging, most likelycaused by non-conductive contaminants on the BPP surface. Werefer to this initial charging effect as type-I charging. We observedthat heating the BPP chip in the PACEM to T¼120 1C is sufficientto reduce type-I charging considerably. Fig. 2b shows Thon ringpatterns obtained from a defocused BPP image of carbon filmrecorded after 1 day of heating the BPP chip at T¼120 1C in themicroscope. The flower-like Thon ring pattern is no longerpresent. Nonetheless, non-linear contributions to the contrasttransfer function (CTF) originating from electrostatic chargingare still manifest in the equally-spaced CTF minima [20].

We analysed the chemical nature of contaminants responsible fortype-I charging by X-ray photoelectron spectroscopy (XPS). Theoutline of the experiment is sketched in Fig. 3a. First, the BPP chipwas transferred from atmosphere into the ultra-high vacuum of theXPS system, heated in situ to 200 1C for 2 h, and analysed by XPS.Afterwards, an area of �2 cm2 on the chip was sputter-cleaned withAr for 5 min in the same vacuum line, and analysed again by XPS.Then the BPP chip was transferred back to atmosphere, re-transferredinto the XPS system after 5 min, and the experiment was repeatedwithout heating. Immediately after the BPP chip had been transferredinto the spectrometer vacuum, XPS analysis revealed only carbon andoxygen as surface contaminants (Fig. 3d and e top). Much loweramounts of contamination (95% less carbon, 70% less oxygen) weredetected on BPP chips heated to 200 1C in the XPS (Fig. 3b and c top).This was analogous to heating the BPP in the microscope, and thusprovided a link between the level of surface contamination and theobserved distortions of the Thon ring pattern (Fig. 2). In both cases,in situ sputter cleaning of the chip with Ar for 5 min was sufficient toreduce carbon and oxygen contaminations below the XPS detectionlimit (Fig. 3b–e, below). We chose pure Ar sputtering for cleaning BPPchips, whereas Zernike carbon film PPs have been cleaned in O2/Arplasma [20] instead. However, plasma cleaning with molecularoxygen results in the formation of Au2O3 on the gold surface [21],which is non-conductive and might cause charging.

The carbon signal detected by XPS is most probably due to air-borne hydrocarbons, which coat the gold surface when the BPP chipis kept under normal atmospheric conditions. Carbon contaminationreappears within minutes on sputter-cleaned chips when the chip istransferred back to ambient air (Fig. 3d top). The oxygen signalobserved by XPS has several possible origins. First, most air-borneorganic compounds also contain oxygen. Second, it is known thatgold oxide can form under certain conditions, even though chemi-sorption of O2 to gold surfaces has a high enthalpy of activation.However, gold oxide forms only under harsh conditions that prevailfor example when gold surfaces are cleaned with an oxygen-containing plasma [21] or, at lower activation energies, on goldnanoparticles [22,23]. Finally, the oxygen signal may be due to wateradsorbed on the gold surface. This is likely since the oxygen peak inthe X-ray photoelectron spectrum reappears as soon as the sputter-cleaned chips are exposed to air, and the spectrum does not indicatethe presence of gold oxide (data not shown).

We conclude that carbon and oxygen contaminations areresponsible for the type-I charging that is observed immediatelywhen a new BPP chip is placed into the microscope. Heating the BPPreduces the type-I contamination significantly, but sputter cleaningwith Ar is even more effective. To completely avoid type-I contam-ination the phase plate should be sputter cleaned first, and thentransferred into the TEM under vacuum. For this purpose it wouldbe necessary to devise a turbo-pumped pre-chamber, with a sputtercleaner and vacuum transfer system in front of the phase plate port.

3.2. Performance of BPPs

Before use, all BPPs are checked for charging effects by theprocedure outlined in Fig. 4a–d. First, the microscope is aligned

and the BPP is mechanically centred with a piezo-controlledmanipulator [7]. Next, the unscattered beam is moved to one ofthe phase plate sectors (Fig. 4a) using the beam tilt alignmentcoils. The unscattered beam is then moved towards the centralelectrode in several discrete steps (Fig. 4b) with the same coils.Charging effects are particularly noticeable close to the centralelectrode by image distortions (see Fig. 4j). Finally, the beam iscentred on the central BPP electrode with the beam-tilt coils, andan electrode voltage applied, which shifts the phase of theunscattered beam (Fig. 4c and d).

An example is shown in Fig. 4e–h. Fig. 4e1–h1 shows zero-lossfiltered BPP images of negatively stained ferritin on ultrathincarbon nanomembranes (CNM) [24], with the corresponding

Fig. 3. Analysis of surface contamination by X-ray photoelectron spectroscopy (XPS). (a) Outline of the experiment. (b), (c) XPS analysis of carbon and oxygen

contaminations after transferring the BPP chip from atmosphere into vacuum and heating to 200 1C for 2 h (black) and after additionally sputter cleaning the BPP chip for

5 min with Ar (red). (d), (e) Carbon and oxygen contaminations after transferring the chip from atmosphere into the vacuum without heating (black). After sputter cleaning

with Ar, carbon and oxygen are not detected by XPS (red).

A. Walter et al. / Ultramicroscopy 116 (2012) 62–72 65

Fourier transforms in Fig. 4e2–h2. Moving the unscattered beamtowards the central electrode causes only minor changes in theimage (Fig. 4f), indicating the absence of strong charging. Tocalibrate the phase shift, highly defocused images were recordedat increasing BPP voltages. The phase shift was determined from afit of the rotationally averaged Thon ring patterns using defocusand phase shift as fitting parameters. For this particular phaseplate, applying a voltage of 100 mV to the centred BPP led to a 901phase shift, resulting in contrast inversion of the ferritin particles(Fig. 4g and h). The BPP images have been recorded with anadditional defocus. Obviously, the low-resolution information ofthe ferritin particles is initially located within a maximum of theCTF, which changes its sign upon applying a 901 phase shift.

In some cases BPPs give rise to uncontrolled phase effects, evenafter heating in the microscope. An example with unstained ferritinparticles on CNM is shown in Fig. 4i–l. This particular BPP producesmajor image distortions as soon as the unscattered beam approachesthe central electrode (Fig. 4j). Though the diameter of the innerelectrode was the same in both cases (Fig. 4e–h and Fig. 4i–l), thesecond BPP blurs the outlines of the ferritin iron cores completely(Fig. 4k and l), indicative of strong charging, which did not disappearupon heating. We refer to this kind of charging as type-II charging.Type-II charging effectively increases the cut-on frequency of theBPP, so that object features in a resolution range which should notbe obstructed by the BPP geometry are nevertheless invisible.Fig. 4k demonstrates that ferritin cores, which measure 6–8 nmacross, are obstructed by this phase plate, even though its theore-tical cut-on frequency is larger than 1/8 nm�1. It is worth notingthat image distortions due to type-II charging are observed early onin the life of a BPP and thus cannot be attributed to aging.

3.3. Analyzing further sources of charging

Surface contaminants cannot be buried by coating, for examplewith extra layers of gold, without short-circuiting the ringelectrode of the BPP. Apart from carbon and oxygen discussedabove, other possible surface contaminants include gallium oxidedeposited and subsequently oxidised during focused ion-beam(FIB) milling of the central hole, or silicon nitride. To identify the

contaminants responsible for the observed artefacts (Fig. 4i–l) weanalyzed single BPPs by helium-ion microscopy (HIM) and Augerelectron spectroscopy (AES).

Compared to classical scanning electron microscopy (SEM),HIM provides better chemical contrast [25] and is thereforeparticularly suitable for analyzing composite materials such asBPPs. Fig. 5a shows a HIM image of a BPP, which produced strongphase effects due to type-II charging. HIM images of the centralelectrode were acquired with secondary electrons (SEs; Fig. 5band d) and Rutherford backscattered (RBS) helium ions (Fig. 5cand e). As the yield of RBS helium ions is particularly large forheavy elements, gold can be recognised in RBS images by itsbright grey level (Fig. 5c and e). Therefore, the bright area on theupper left of the central hole in Fig. 5e (arrow) is interpreted as athin film of gold re-deposited during FIB milling. Deposition ofgold in the central hole is most likely responsible for leak currentsor even short-circuits, which we observe with many BPPs.Furthermore, thin films consisting of gold islands might besusceptible to electrostatic charge build-up. Apart from someblack dots in the RBS image (Fig. 5c), which we attribute totopographical contrast due to pits in the gold surface, HIM imagesdo not indicate any gross contamination, such as large siliconnitride particles, in the vicinity of the central hole.

For quantitative information on the local chemical composi-tion we recorded Auger electron spectra (AES) from differentpositions on the BPP surface. AES analysis of fresh, unused BPPsrevealed gold as the only chemical species, apart from a smallcarbon signal, which most probably originates from the SEMvacuum chamber (see Supplementary Fig. S2). Fig. 6a shows anoverview SEM image of the same used BPP, which has beenanalysed by HIM. AE spectra were acquired at points located nearthe central electrode (position 1 in Fig. 6a), or on the gold surfaceof the chip (position 2 in Fig. 6a). Prior to AES the BPP was heatedto 200 1C for 2 h to remove volatile contaminants. AE spectra areshown in Fig. 6b and c. A strong carbon signal was detected in thevicinity of the central electrode (Fig. 6b), which obliteratedthe signal from the underlying gold substrate. By comparison,the gold signal is clearly visible in the AE spectra obtainedfrom the chip surface (Fig. 6c). To exclude carbon contamination

Fig. 4. Performance of BPPs in the PACEM. (a)–(d) Procedure for initial testing a BPP. (a) The unscattered beam is moved to a sector between two support rods. (b) The

unscattered beam is moved towards the central electrode in several distinct steps. Charging effects become evident as the beam approaches the central electrode. (c), (d)

Analysis of phase shift. (e1)–(h1) BPP images of negatively stained ferritin on ultrathin carbon nanomembranes and their Fourier transforms (e2–h2). Images were recorded

by the procedure outlined in (a)–(d). (e1) The unscattered beam passes between two support rods. (f1) No image distortions are visible when the unscattered beam is

positioned near the central electrode, indicating the absence of strong charging effects. (g1), (h1) Contrast inversion upon applying a voltage of 100 mV to the BPP central

electrode, resulting in a 901 phase shift. (i)–(l) Image distortions due to electrostatic charging. (i1)–(l1) BPP images of ferritin on carbon nanomembranes and (i2)–(l2) their

Fourier transforms. (j1) Strong image distortions are observed when the unscattered beam is placed near the ring electrode. (k1, l1) Object outlines are obscured by strong

charging near the central electrode. Images were recorded at 200 kV acceleration voltage.

A. Walter et al. / Ultramicroscopy 116 (2012) 62–7266

in the SEM as a source, we transferred the chip under highvacuum to a sputter cleaner. The chip was sputter-cleaned withAr for 5 min and transferred back into the SEM chamber underhigh vacuum. Fig. 6d shows an SEM image of the BPP aftersputtering. Note that the contamination, deposited by the elec-tron beam of the SEM prior to sputtering (Fig. 6a, white arrows),has disappeared. Again, we recorded AE spectra from a point inthe vicinity of the ring electrode (Fig. 6d, position 1) as well as AEspectra from a point on the chip surface (Fig. 6d, position 2). Aftersputtering, the strong carbon signal, visible in AE spectra recorded

at position 1 (Fig. 6b), remained virtually unchanged (Fig. 6e). Thecarbon contamination in the vicinity of the central electrode wasvisible on SEM images and persisted even after a second round ofsputter cleaning (Fig. 6g), whereas the AES signal from the goldsurface increases after sputtering (Fig. 6f), due to removal ofsurface contaminants. A small carbon signal is still visible, whichoriginates most probably from the SEM vacuum.

Strong carbon contamination near the ring electrode isobserved after extensive use of a BPP in the microscope. Weconclude that BPPs suffer from carbon contamination in the

Fig. 5. Helium ion microscopy (HIM) of BPPs. (a) HIM image of a BPP in secondary electron (SE) mode. (b) HIM image of the central electrode in SE mode and (c) the same

area in Rutherford backscattering (RBS) mode. (d) HIM image of the central hole and (e) the corresponding RBS image. In the RBS mode, gold appears particularly bright. An

area of gold re-deposited during the FIB process is highlighted (arrow).

Fig. 6. Auger electron spectroscopy (AES) of BPP surface contamination. The BPP has been used for several weeks in the electron microscope. (a)–(c) Analysis of the BPP

after heating to 200 1C for 2 h. (a) SEM image. AE spectra were recorded at positions 1 and 2 (red arrows). Contaminations on a BPP rod originating from the electron beam

of the SEM are highlighted (white arrows). (b) AE spectrum recorded at position 1 (near central ring electrode). A strong carbon peak (275 eV) is visible, which obscures the

AES signal from the gold substrate. (c) AE spectrum recorded at position 2 (chip surface). The AES signal from the gold substrate (74 eV, 154 eV) is clearly visible, in

addition to a small carbon peak, originating from the SEM vacuum. (d)–(f) BPP after subsequent sputter cleaning for 5 min in Ar plasma. (d) SEM image. Note that the

contamination (white arrows in (a)) has disappeared. (e) AE spectrum recorded at position 1 (near ring electrode) after sputter cleaning. The strong carbon peak is still

present. (f) AE spectrum recorded at position 2. The AES intensity of the gold substrate (74 eV) has increased considerably. (g) SEM image of the same BPP after a second

round of sputter cleaning. (h) SEM image of the BPP after incubation in a piranha solution (H2SO4/H2O2). (i) SEM image of the BPP after 60 min sputter cleaning with Ar.

Most of the carbon contamination has been removed. (j) AE spectrum obtained from the BPP after 60 min sputter cleaning (recorded from position 3).

A. Walter et al. / Ultramicroscopy 116 (2012) 62–72 67

A. Walter et al. / Ultramicroscopy 116 (2012) 62–7268

intense TEM beam. Because carbon is a conductor, this contam-ination may cause leak currents, which gradually increase overtime, as we observed with most BPPs. We investigated possiblemethods to remove the strong carbon contamination, thus recon-ditioning used BPPs. Incubation in a piranha solution (H2SO4/H2O2) was not sufficient to remove the strong carbon contamina-tion (Fig. 6h). In contrast, sputter cleaning the BPP with Ar for60 min removed most of the carbon contamination (Fig. 6i),which is also reflected in the AE spectrum (Fig. 6j).

However, carbon deposition in the electron beam is anunlikely reason for image distortions ascribed to type-II charging,since this type of charging occurs already early in the lifetime of aBPP. In conclusion, AES analysis showed that carbon is the onlydetectable contaminant that accumulates during use in the TEM,although it is possible that the strong carbon signal camouflagesother contaminants. Further causes for type-II charging include(1) charging of the insulating layers of the BPPs in the unscatteredbeam and (2) patch fields on the gold surface due to differentwork functions at different positions on the surface [26]. Furtherstudies are needed to analyse the origin of this type of charging.

Fig. 7. Low-voltage TEM reduces the cut-on frequency of the BPPs. (a) Relationship

between electrode diameter and cut-on frequency at acceleration voltages of 200 kV,

80 kV, and 20 kV, with the instrument parameters of the PACEM.

Fig. 8. (a)–(d) Simulated BPP images of ribosomes at 200 kV acceleration voltage (Cs¼

ribosomes at 80 kV acceleration voltage (Cs¼10 mm, Cc¼7.9 mm, Dz¼18 nm). (a), (e) W

(b), (f) Electrode diameter 3 mm. (c), (g) Electrode diameter 4.5 mm. (d), (h) Electrode dia

Fourier patterns by the BPP. Ribosomes were treated as pure phase objects.

3.4. Aberration-corrected low-voltage BPP TEM

Obstruction of low spatial frequencies by the electrode ring isan inherent drawback of the BPP for cryoEM, as object featureslarger than 1/kcut-on are not visible. The PACEM is equipped with adiffraction magnification unit (DMU), which magnifies the diffrac-tion pattern and thus minimises the obstruction of low spatialfrequencies [7]. Further reduction of the electrode dimensions isone possibility to enable additional low-resolution contrast trans-fer. Currently, the dimensions of the central ring and hole arelimited by the production process. Even smaller dimensions wouldcompromise both reproducibility and throughput of the productionprocess. Another possibility to reduce the cut-on frequency is tooperate the microscope at lower acceleration voltages (80 kV oreven 20 kV), which increases the electron wavelength and scatter-ing angles. The cut-on frequency is given by kcut-on¼r/lf, where r isthe distance from the centre to the edge of the electrode ring, l isthe electron wavelength, and f is the effective focal length. Asshown in Fig. 7, reducing the acceleration voltage from 200 kVdecreases the cut-on frequency by roughly 40% at 80 kV, or 70% at20 kV. Accordingly, for a given electrode diameter, the size ofbiological objects that can be imaged faithfully with a BPPincreases by a factor of �1.6 at 80 kV or �3.4 at 20 kV.

Fig. 8 shows simulated phase contrast images of ice-embeddedribosomes at acceleration voltages of 200 kV or 80 kV with 2 mmdefocus (Fig. 8a and e). In-focus BPP images with increasing dimen-sions of the central electrode are shown in Fig.8b–d andf–h. For all BPP dimensions, object outlines are clearer at 80 kV thanat 200 kV, because of limited obstruction of low spatial frequencies.Fig. 9a and b shows the low-resolution range of calculated CTFs forthe PACEM at 80 kV and 200 kV. For a BPP with a 6 mm diameter ofthe central electrode, the cut-on frequency is �1/13 nm�1 at 200 kVor �1/21 nm�1 at 80 kV. For an experimental validation, werecorded BPP images of amorphous carbon films in the PACEM at200 kV or 80 kV with the same phase plate (Fig. 9c and d). Line plotsalong the central electrode in Fourier space (Fig. 9e and f) demon-strate that reducing the acceleration voltage from 200 kV to 80 kVindeed increases transfer of low-resolution information. The observedcut-on frequency is reduced from �1/13.9 nm�1 at 200 kV to �1/20.8 nm�1 at 80 kV, in good agreement with the calculated values.

10 mm, Cc¼7.3 mm, defocus spread Dz¼7.7 nm). (e)–(h) Simulated BPP images of

ithout phase plate, 2 mm defocus. (b)–(d), (f)–(h) Simulated in-focus BPP images.

meter 6 mm. The insets show the Fourier masks used to simulate the obstruction of

Fig. 9. Cut-on frequency of BPPs in the PACEM, (a),(b) Calculated CTFs for in-focus BPP TEM compared to conventional defocus TEM. The cut-on frequency at 80 kV (b) is reduced

compared to that at 200 kV (a). (c)–(f) Experimental proof of reduced cut-on frequency at 80 kV in the PACEM. (c) Fourier transform of a defocused BPP image of an amorphous

carbon film at 200 kV. (e) A line section through the Fourier transform indicates a cut-on frequency of 1/13.9 nm�1. (d) Fourier transform of a defocused image of amorphous

carbon using the same BPP at 80 kV. (f) A line section through the Fourier transform reveals that the cut-on frequency has been reduced to 1/20.8 nm�1.

A. Walter et al. / Ultramicroscopy 116 (2012) 62–72 69

Low acceleration voltages may be useful for TEM imaging withelectrostatic phase plates other than BPPs, which are also affectedby cut-on frequency. For microscopes not equipped with a DMU,lowering the acceleration voltage is the only practical way toreduce the cut-on frequency, if phase plate dimensions cannot bereduced further. Acceleration voltages of 80 kV [27] or even 20 kV[28] have other advantages, such as increased contrast of lightelements and lower knock-on damage [29]. On the other hand,due to the increased elastic and inelastic cross-section at lowelectron voltages, radiation damage for biological specimens ismuch more severe [30], and the weak-phase approximation forbiomolecules might not hold. Furthermore, the contribution ofamplitude contrast to image formation can no longer be

neglected, particularly if an energy-filter is used. Finally, effectsof electrostatic charging will be more pronounced at lowerelectron energies. Indeed, Fig. 9c and d reveals slightly distortedThon rings, indicating electrostatic charging. Certainly, contrasttransfer in low-voltage TEM has to be analysed in more detail, andthe theory of image contrast using physical phase plates needs tobe adapted to low-voltage TEM.

The most important limiting factor in low-voltage phase contrastTEM is chromatic aberration of the objective lens. CTF calculationsindicate that temporal coherence reduces the information limit of thePACEM from �2.2 A (200 kV, BPP in-focus, Cs¼10 mm, Cc¼7.9 mm)to �4.4 A (80 kV, BPP in-focus, Cs¼10 mm, Cc¼7.3 mm), using the1/e2-criterion (Fig. 10a and b). Implementation of a Cc corrector [31]

Fig. 10. Effect of Cc correction on low-voltage phase contrast TEM with the instrument parameters of the PACEM. (a) At 80 kV (Cs¼10 mm, Cc¼7.9 mm) the information

limit of the PACEM is 0.44 nm compared to (b) 0.22 nm at 200 kV (Cs¼10 mm, Cc¼7.3 mm). (c)–(f) A Cc corrector (Cs¼10 mm, Cc¼10 mm) would result in optimal contrast

transfer for all spatial frequencies relevant for biological TEM.

A. Walter et al. / Ultramicroscopy 116 (2012) 62–7270

would produce maximal information transfer for in-focus BPP TEM at80 kV, covering all spatial frequencies of interest for biologicalsamples (Fig. 10c and d).

3.5. Single-sideband contrast

Partial obstruction of Friedel mates by the rods supportingthe central electrode results in single-sideband (SSB) contrasttransfer in small regions of Fourier space. Although the char-acteristic SSB effects are not evident in our BPP images, correc-tion for SSB contrast is nevertheless required to restore the exactimage phases. We developed an automatic computational rou-tine to correct BPP images for SSB contrast. Fig. 11a shows theclose-up of a BPP image of negatively stained ferritin. TheFourier transform (Fig. 11b) reveals the threefold symmetry ofthe BPP. Our image restoration software first detects the orientation ofBPP rods by radial averaging in Fourier space. Second, the electrode

diameter and width of the support rods are determined interactively.Third, the program automatically aligns and applies a mask, whichcorrects the image transform for SSB contrast, by applying the inverseoperation X¼1/CTFSSB along the rods. This ensures optimal contrasttransfer without loss of information (Fig. 11d). The signal-to-noiseratio in the regions covered by the support is reduced by 50%, since50% of the coherently scattered electrons are lost. This is accounted forby a factor of 0.5 in the CTF of the partially obstructed regions:CTFSSBðkÞ ¼�ð1=2ÞisignðkxÞ� e�isignðkxÞwðkÞ [2]. The wave aberration termw(k) contains the defocus and spherical aberration as experimentalparameters. Defocus is determined by a CTF fit. The sphericalaberration term is negligible because the PACEM is Cs corrected.Spatial frequencies obstructed by the central electrode are set tozero. The corrected image (Fig. 11c) reveals artefacts, which are dueto the sharp edges of the mask. It is known that such edges in Fourierspace cause oscillations and contrast reversals in real space [32] dueto the so-called Gibbs phenomenon. Edge effects are avoided by

Fig. 11. Correction of BPP images for single-sideband contrast. (a) BPP image of negatively stained ferritin and (b) Fourier transform of (a). (c) BPP image after correction

for single-sideband contrast using the mask in (d). Position and orientation of support rods are determined by radial averaging in Fourier space. Fourier transforms are

corrected for single-sideband contrast using the inverse operation X¼1/CTFSSB or �X¼�1/CTFSSB. Masking artefacts are visible. (e) BPP image after correction for single-

sideband contrast using the smoothed mask in (f). The image is free of artefacts. Images were recorded at 200 kV acceleration voltage.

Fig. 12. BPP drift. (a) Time series of BPP images of negatively stained TMV and gold nanoparticles, revealing phase plate drift during a 9-min time course. Fourier

transforms are shown in (b). (c) Feedback drift compensation in BPP images of amorphous carbon film. Initially the BPP is centred (00:00 min). Drift is determined after

8 min from the centre-of-intensity displacement (08:00 min, red dot), which is used as a feedback parameter. The centre-of-intensity is compared with its initial position

(00:00 min, green dot), the deviation is calculated and the computer-controlled goniometer re-centres the BPP. The procedure is repeated every 2–4 min.

A. Walter et al. / Ultramicroscopy 116 (2012) 62–72 71

smoothing the mask (Fig. 11f) with a Gaussian filter. This procedureresulted in artefact-free images corrected for single sideband contrastwith optimal contrast transfer (Fig. 10e).

3.6. Automatic correction of BPP drift

Because of the small dimensions of the central electrode, theBPP is extremely sensitive to mechanical drift. A time series

recorded with a BPP shows that it remains centred for �3 min(Fig. 12a and b). Drift is noticeable after �6 min (Fig. 12a and b),as indicated by a displacement of the centre of intensity (Fig. 12a,red dots) relative to its initial position (Fig. 12a, green dots). Thecentre of intensity is the mean location of image intensity. Itspixel location corresponds to the balance point of all intensitiesand is calculated by summing up the products of pixel intensitiesand their location. After �10 min the BPP obscures most of the

A. Walter et al. / Ultramicroscopy 116 (2012) 62–7272

unscattered beam, and needs to be re-centred before more imagescan be recorded. This would make it impractical to record tomo-graphic tilt series or cryo-images for single-particle processing. It istherefore necessary to compensate for BPP drift over time. Wetherefore developed a computer-guided feedback control to com-pensate for BPP drift, using the centre of intensity as a feedbackparameter. Fig. 12c shows the feedback control in action. First, theBPP is centred manually, a BPP image is recorded as a reference, andthe centre of intensity is calculated (Fig. 12c, 00:00 min, green dot).Then a new image is recorded every 2–4 min to determine the driftof the BPP. The centre of intensity for the new image is automaticallycalculated (Fig. 12c, 08:00 min, red dot), the displacement relative tothe reference image is determined, and the BPP is automaticallycentred by the computer-controlled piezo-driven BPP goniometer(Fig. 12c, 08:00 min corrected). Communication between the com-puter and the goniometer is established by a Matlab interface and aPCI card. Using the feedback control, we were able to keep the BPPcentred for more than 30 min (see Supplementary Fig. S3).

4. Conclusions

In this work we discuss a variety of technical obstacles, whichhave to be overcome for routine application of BPPs in cryoEM. Wehave developed routines that handle SSB artefacts and phase platedrift by automated image processing. Of the remaining problems, elec-trostatic charging of BPPs is the most complex and difficult to address.In our analysis of surface contamination of BPPs we have identifiedtwo types of BPP charging. XPS analysis revealed that one type ofcharging (type-I charging) of BPPs is mainly caused by insulatingsurface contaminants from ambient air, particularly hydrocarbons andoxygen-containing compounds, which can be effectively removed byin-situ sputter cleaning. Furthermore, we demonstrate by AES thatstrong carbon contamination, deposited in the electron beam of theTEM, can be removed by sputter cleaning as well. However, the originof other more serious charging effects (type-II charging) remainsunclear and requires further analysis. HIM analysis revealed that FIBmilling of BPPs is accompanied by undesired re-deposition of gold inthe central hole. This problem is to be addressed in further studies. Wehave shown that lower acceleration voltages reduce the obstruction oflow spatial frequencies by the central electrode, but further work isneeded to extend the theory of image contrast to lower accelerationvoltages of 80 kV or less.

Acknowledgements

The PACEM project is funded by the Deutsche Forschungsge-meinschaft (DFG) within the Cluster of Excellence Frankfurt,‘‘Macromolecular Complexes’’. Angelika Sehrbrock (Caesar Insti-tute, Bonn, Germany) assisted in the phase plate fabrication. Wethank Gerd Benner and Marko Matijevic from Carl Zeiss NTS forassistance with the 80 kV alignment of the PACEM.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.ultramic.2012.03.009.

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