LS-4-O-1658 Phase-plate Imaging for Cryo-TEM: Types, Benefits and Applications
Vitreously frozen specimens for cryo-TEM retain a near-native state, free of artifacts due to chemical fixation or stains that limit resolution, but their low-Z composition yields weak amplitude contrast. Imaging by phase contrast, at low electron dose, is required. Thus, SNR maximization is important; it is improved by zero-loss energy filtering [1] and noise-free “counting” imaging detectors [2], which, along with use of an FEG and 300 kV, bring us closer to “all that physics will allow”. However, the oscillating CTF of traditional defocus phase-contrast imaging remains a spatial-frequency-dependent hindrance to good SNR. Recording “in-focus” images with a phase plate can be the next step in optimizing imaging [3].
Phase plates increase the SNR--especially at lower spatial frequencies--for cryo-electron tomography as well as for particle-picking and alignment of sub-100 kDa molecules in single-particle cryo-EM. They have been used to reveal structures in 3D not seen before [4].
While there are many types are in use, under development or proposed [review: 5], a typical phase plate creates a phase shift between the unscattered beam and electrons scattered by the specimen. The most-common type uses a thin film to shift the phase [3,6]. While scattering in the film slightly reduces contrast and may degrade the envelope function at the highest spatial frequencies, this type is easy to make and operate, and is the only one in routine use.
In principle, phase plates without films should be preferred. Those using a voltage applied to an electrode in or near the path of the unscattered beam [e.g. 7,8] have adjustable phase shift (either positive or negative), which can be optimized for accelerating voltage and intrinsic phase shift. Phase shift can also be created by a magnetic field [9] or by a laser [10]. Also, blocking half of the back-focal-plane diffraction pattern over a range of low spatial frequencies boosts phase contrast over that range [11].
We will discuss applications and operating considerations of various types of phase plate, and present our recent work on construction and use of Zernike phase plates [12].
[1] R. Grimm et al., J. Microsc. 183(1996)60-68.
[2] X. Li et al, Nat. Meth. 10(2013)584-590.
[3] R. Danev et al., Ultramicroscopy 109(2009)312-325.
[4] R. Rochat et al., J. Virol. 85(2011)1871-1874.
[5] R. Glaeser, Rev. Sci. Instr. 84(2013)11101
[6] M. Malac et al., Ultramicroscopy 118(2012)77-89.
[7] R. Cambie et al., Ultramicroscopy 107(2007) 329-339.
[8] S. Hettler et al., Microsc. Microanal. 18(2012)1010-1015.
[9] C. Edgecomb et al., Ultramicroscopy 120(2012)78-85.
[10] H. Müller et al., New J. Phys. 12 (2010)073011.
[11] B. Buijisse et al., Ultramicroscopy 111(2011)1696-1705.
[12] M. Marko et al., J. Struct. Biol. 184(2013)237-244.
Supported by NIH grant 8R01GM103555.
Fig. 1: 1. Examples of film-type phase plates in the objective-lens back focal plane (not to scale). A. Zernike; unscattered electrons go through central hole, others shifted by π/2 [3,12]. B. Hilbert; half of diffraction plane shifted by π [3]. Hole-free; unscattered electrons phase-shifted by induced, charged-up spot [6]. |
Fig. 2: 2. Some film-free phase-plate types (in back focal plane; not to scale). A. Central electrode or ring-magnet shifts phase of unscattered beam [5,7,9]. B. Coaxial cable: inner-conductor potential forms electrostatic field at unscattered beam [8]. C. Single-sideband blocking of low frequencies gives uniform transfer at 0.5 over that range [11]. |