Practical factors affecting the performance of a thin-film phase plate for transmission electron microscopy.

2.1.

Preparation of thin carbon-film phase plates

The phase plates were prepared in several steps. First, a core carbon film with thickness ~15 nm was evaporated on a freshly cleaved mica surface by a JEE-420T (JEOL, Tokyo) carbon arc vacuum evaporator at a vacuum level of ~1.5 × 10 −4 Pa or by a BAF-060 (BAL-TEC AG, Balzers) electron beam evaporator at a vacuum level of ~1.0 × 10 −6 Pa. Both evaporation systems are equipped with turbo-molecular pumps. In the case of arc evaporation the carbon rods had a diameter of 5mm, specific resistance of 4 mΩ/cm, purity of 99.9999% (Nisshin EM Co. Ltd., Tokyo), and the thickness of the film was judged by observing the darkening of a partially shadowed filter paper. In the case of electron beam evaporation the carbon rods had a diameter of 3mm (LZ 02217 KN, BAL-TEC AG, Balzers) and the thickness of the film was monitored by a quartz crystal thickness monitor. The carbon-coated mica was cut by scissors into ~5 × 5mm square pieces. A mica piece was gradually lowered into a thoroughly cleaned glass Petri dish filled with commercially purchased distilled water (049–16787, Wako Pure Chemical Industries Ltd., Osaka), thus floating the carbon film on the water surface. The film was then picked up by a platinum wire loop and lowered onto specially ordered molybdenum grids resting on top of two to three layers of Whatman Grade No. 1 filter paper. The film was left to dry on the grids for at least 12 h. These grids, which we refer to below as “aperture discs”, are 2mm in diameter and contain a 7 × 7 array of 100 µm diameter holes with a 100 µm spacing between the edges of adjacent holes (Daiwa Techno Systems, Tokyo). The hole in the center of the phase plates was drilled by a JFIB-2300 (JEOL, Tokyo) focused ion beam machine. In addition, four fiducial markers, in the form of holes similar in size to the central hole, were drilled close to the edge of the apertures. Each marker points to a specific direction of the aperture grid (+ X , − X , + Y , − Y ) looking from the central hole. The fiducial markers simplify finding the central hole during the initial alignment of a phase plate. First, a marker is found by following the aperture edge. Then, by using the aperture drive that moves in a direction perpendicular to the edge, the central hole is brought into the TV-rate camera’s field of view. The final step in the phase-plate manufacturing process is to evaporate ~5–7 nm of amorphous carbon onto both sides of the “core” of the phase plate, a process that we describe as “wrapping” the core with an additional layer of evaporated carbon. Fig. 1 shows a schematic of a finished phase plate. The role of the final wrapping is to cover any contaminants on the surface of the core film that may have been accumulated during the manufacturing process. In particular, the water surface transfer process can deposit inorganic salts or other impurities present in the water or dissolved from the filter paper supporting the grid. Such contaminants, unlike vacuum system fluids, cannot be desorbed by heating. Wrapping by a fresh layer of carbon traps the impurities and prevents the formation of surface charges by the electron beam. 2.2.

Electron microscopy

All cryo-EM images of biological samples were recorded on a JEOL JEM-3100FFC electron microscope, fitted with a top-entry, liquid-helium cold stage [ 12 ]. The cold stage was normally used at a temperature of ~55 K. Our recent tests (unpublished) showed that at that temperature specimen stability and contrast of cryo-specimens were both improved. Maintaining the stage temperature at ~55 K is achieved by initially cooling the inner pot of the Dewar with helium, waiting for it to evaporate, and then topping up the level of liquid nitrogen in the outer shield tank at ~5 h intervals to ensure long-term stability of the temperature. This instrument was fitted with a modified objective lens, which has a larger than usual gap, and modified specimen cartridges were used to lower the position of the specimen within the lens. The design of this lens allows highly parallel incident illumination to be focused at the plane of the phase-contrast aperture with a focal length (for the equivalent thin lens) of 5 mm. The larger gap also allowed the standard, 60 µm diameter objective aperture to be inserted as usual, but through a port different from that used for the phase-plate aperture rod. The standard objective aperture was thus used to provide a room-temperature, thermal shield between the cryo-specimen and the heated phase-contrast aperture. A custom-built holder was designed by JEOL to allow heating of the phase-contrast apertures. Fig. 2 shows a photograph of the tip of the aperture rod. A total of four aperture discs, described above, are mounted at one time. The principal features of this aperture rod are that it is fitted with vacuum feed-throughs for the current that is supplied to the micro-ceramic heater (Sakaguchi E.H VOC Corp., Tokyo), and a ceramic thermal insulator is inserted between the heated tip and the main section of the rod. The temperature of the tip is estimated from the known temperature dependence of the resistivity of the heating element. The heated tip is electrically grounded to the main part of the aperture rod with a thin wire, and a metal plate encloses the cavity housing the ceramic heater. A metal enclosure also surrounds the end of the ceramic thermal insulator, as can be seen in Fig. 2 . Current for the heater was provided by a model PAR36-3H regulated power supply (Kenwood, Melrose, MA). Movements from one phase-plate position to another within a given aperture disc, and approximate centering of the hole of the phase-contrast aperture, are performed with the mechanical drives provided for the custom-built JEOL aperture rod. These alignment steps, and in particular keeping track of which particular phase plate is being used, are facilitated by initially aligning rows of individual phase plates parallel to the long axis of the aperture rod when the aperture disc is placed into the tip of the rod. Centering of a selected phase plate with a precision of ~0.5 µm is monitored in the defocused diffraction (“Search”) mode, and the hole in the phase plate is placed close to a predetermined fiducial mark that is spotted onto the viewing monitor for the Gatan TV camera. The final, precise centering of the unscattered beam into the hole of the phase plate is carried out by making small adjustments to the beam-tilt angle. In this case the condenser-lens values are set to those used in the “Focus” mode, which produces both a more intense and a more convergent beam than is used in the Photo mode. As a result, the unscattered beam then has a radius larger than that of the hole in the phase plate, and tilting the illumination to make the unscattered beam concentric with the hole is monitored by eye, looking directly at the viewing screen. The Minimum Dose System (MDS) software provided with the microscope was used to switch between pre-set values for the Search, Focus, and Photo modes mentioned above. A 50 µm diameter second-condenser-lens aperture was used for all modes. The available in-column energy filter was normally operated with an energy slit of 20 eV, placed symmetrically about the zero-loss beam. Images were recorded with a retractable Gatan (Pleasanton, CA) MegaScan 795 2K × 2K CCD camera, which had previously been fitted with a scintillator that was optimized by Gatan for use with 400 keV electrons. The pixel size of this camera is 30 µm. The microscope magnification was set to 60,000, unless stated otherwise, which resulted in an actual magnification of about 100,000 at the plane of the CCD camera, which was mounted below the film camera. Cryo-EM images were recorded with an electron exposure of 2500 electrons/nm 2 , using an exposure time of 2 s. The microscope was also fitted with a stationary Gatan 622 SC TV camera, mounted below the housing for the CCD camera. The TV camera was used for video-rate observation of images produced in Search mode. 2.3. Sample preparation A Vitribot (FEI, Hillsboro, Oregon) was used to prepare cryo-EM samples of all specimens on Quantifoil grids, using a fairly standardized protocol. The sample chamber was equilibrated at 4 °C and the relative humidity was generally set to 95% or 100%. Two-microliter volumes of sample were applied to glow-discharge treated Quantifoil ® R 1.2/1.3 (Quantifoil Micro Tools GmbH, Jena) EM grids, and the grids were immediately blotted and plunged into liquid ethane to freeze the sample. The blotting time was typically set to 5–15 s. There was no “drain-time” allowed between blotting and plunging. It should be noted that the applied sample was not initially at a temperature of 4 °C. Although some evaporation of sample buffer is expected to occur under these conditions, it was found empirically that this standard protocol produced a good density of particles for sample concentrations in the range of 1–10 mg/ml, depending upon the sample material. 2.4. Simulation of image contrast for “strong” phase objects Simulations of phase-contrast TEM imaging were performed using custom scripts for the DigitalMicrograph ® software platform provided by Gatan. The simulations consisted of a few steps. A complex object wave was prepared that corresponded to a pure phase object, for example a uniform sphere with π/256 phase shift per nm of thickness. The diffracted wave was calculated as the Fourier transform of the object wave. A complex form of the wave aberration (due to defocus and spherical aberration) and phase-plate functions were applied (by multiplication) to the diffracted wave. The modified diffracted wave was then inverse-Fourier transformed, producing the image wave. The image intensity was calculated as the square of the image wave.