PMC

SEM image of one example of a hybrid double-sideband/single-sideband (tulip) aperture that was made by milling a hole with the desired shape into a thin metal foil, using a focused gallium-ion beam (FIB). The perspective in this image is foreshortened because the aperture was tilted to give an impression of the thickness as well as the shape of the tulip-like feature. The opaque half-circle at the center has a small notch at the center, where the straight edge is off-set from the diameter in one direction on the left, and by the same amount in the opposite direction on the right.

Schematic diagram showing how the vector field of the magnetic potential, , encircles the magnetic field, , when the latter is confined to a circular ring. The full, three-dimensional vector field of is generated by rotating the two-dimensional vector field, shown schematically here, about the axis of the ring. The web site http://en.wikipedia.org/wiki/Toroidal_inductors_and_transformers is recommended for a more detailed description. The line integral (i.e., two-dimensional projection) of the vector potential thus has opposite sign for electron rays passing inside the ring and for rays passing outside the ring. As a result, such a structure will produce a relative phase shift for electrons passing inside vs outside the ring.

In-focus cryo-EM image (obtained with a tulip aperture) of an area containing a monolayer crystal of streptavidin. Reproduced with permission from R. M. Glaeser , Ultramicroscopy 135, 6 (2013). Copyright 2013 Elsevier. (a) The raw image shown here exhibits a “shadowed” character due to the single-sideband contrast transfer produced by the tulip aperture. (b) The image shown here is a processed version of the raw image shown in (a), in which a systematic correction has been applied to compensate for the single-sideband contrast transfer. The full amount of contrast provided by this aperture is retained when applying this correction. As a result, individual streptavidin tetramers, examples of which are indicated by the arrows, can be identified quite easily in the non-crystalline area of the specimen, even though their molecular weight is only ∼55 kDa.

Images that illustrate the design of the anamorphic phase plate and the (somewhat) related Zach phase plate. (a) Conceptual drawing of the anamorphic phase plate. The electrons propagate parallel to the z axis, and the unscattered beam in the electron diffraction pattern is focused at the intersection of the x and y axes. Furthermore, the diffraction pattern is greatly magnified in the x-direction while at the same time it is greatly demagnified in the y-direction. The biased electrodes produce a localized electrostatic potential that is different at the center of the diffraction pattern than what it is elsewhere. Reproduced with permission from H. Rose, Ultramicroscopy 110, 488 (2010). Copyright 2010 Elsevier. (b) SEM image of a Zach phase plate, which is a layered beam that extends from the rim to the center of the aperture. The tip of the beam is thus placed close to the center of the electron diffraction pattern. The close-up view on the right shows the biased electrode is exposed at the center of the tip, with a layer of insulation separating it from the surrounding, grounded electrode. This panel is a modified version of a figure published in Ref. .

Schematic diagram of the structure of the thin-carbon-film phase plate, and some representative examples of images that have been obtained with such a device. (a) Cartoon showing one implementation of this type of phase plate. A carbon film (typically <20 nm thick) is supported on a molybdenum aperture, and a focused ion-beam tool is used to drill a hole though the carbon film, at the center of the aperture. This “core” structure is then coated conformally with a second carbon film, which is about 5 nm thick. The purpose of the coating is to cover up any contaminants that may have been deposited in the earlier steps of fabrication. Reproduced by permission from R. Danev, R. M. Glaeser, and K. Nagayama, Ultramicroscopy 109, 312 (2009). Copyright 2009 Elsevier. (b) Example of cryo-EM images of unstained GroEL, showing the extraordinary amount of contrast that is produced in in-focus images. Reproduced with permission from R. Danev and K. Nagayama, J. Struct. Biol. 161, 211 (2008). Copyright 2008 Elsevier. (c) A second example of the remarkable amount of contrast that is produced in in-focus images of cryo-EM samples, this time of ice-embedded influenza virus particles. Reproduced with permission from M. Yamaguchi, R. Danev, K. Nishiyama, K. Sugawara, and K. Nagayama, J. Struct. Biol. 162, 271 (2008). Copyright 2008 Elsevier.

Two examples of designs that have been proposed for electrostatic phase plates. (a) Scanning electron microscope (SEM) image showing one design for an einzel lens phase plate, which in this case is supported at the center of the aperture by three beams extending from the rim of the aperture. The inset shows a close-up view of the central hole of the einzel lens, revealing the two layers of insulating material that separate the grounded layers of gold, top and bottom, from the biased layer of gold at the center of the device. Reproduced with permission from E. Majorovits, B. Barton, K. Schultheiss, F. Perez-Willard, D. Gerthsen, and R. R. Schroder, Ultramicroscopy 107, 213 (2007). Copyright 2007 Elsevier. (b) SEM image of the drift tube design of an electrostatic phase plate. The top panel shows an overall view of the aperture, and the lower panel shows a close-up view of the central, biased electrode surrounded by a grounded electrode. The focused, unscattered electron beam passes through the center of the biased tube, and thus experiences a phase shift relative to the scattered electrons, which pass outside the grounded electrode. Reproduced with permission from R. Cambie, K. H. Downing, D. Typke, R. M. Glaeser, and J. Jin, Ultramicroscopy 107, 329 (2007). Copyright 2007 Elsevier.