Section 19.6.4.4. Microscopy

Once the vitrified specimen is inserted into the microscope and sufficient time is allowed (∼15 min) for the specimen stage to stabilize to minimize drift and vibration, microscopy is performed to generate a set of images that, with suitable processing procedures, can later be used to produce a reliable 3D reconstruction of the specimen at the highest possible resolution. To achieve this goal, imaging must be performed at an electron dose that minimizes beam-induced radiation damage to the specimen, with the objective lens of the microscope defocused to enhance phase contrast from the weakly scattering, unstained biological specimen, and under conditions that keep the specimen below the devitrification temperature and minimize its contamination.

The microscopist locates specimen areas suitable for photography by searching the EM grid at very low magnification () to keep the irradiation level very low (<0.05 e Å⁻² s⁻¹) while assessing sample quality: Is it vitrified and is the thickness optimal? Are the concentration and distribution of particles or is the size of the 2D crystal optimal? In microscopes operated at 200 keV or higher, where image contrast is very weak, it is helpful to perform the search procedure with the assistance of a CCD camera or a video-rate TV-intensified camera system. CCD cameras are gaining popularity because imaging conditions (defocus level, astigmatism, specimen drift or vibration etc.) can be accurately monitored and adjusted by computing the image Fourier transform online (Sherman et al., 1996) and also because in some cases the distribution of single particles can be seen at low or moderate magnifications (Olson et al., 1997). For some specimens, like thin 2D crystals, searching is conveniently performed by viewing the low-magnification high-contrast image produced by slightly defocusing the electron-diffraction pattern using the diffraction lens.

After a desired specimen area is identified, the microscope is switched to high-magnification mode for focusing and astigmatism correction. These adjustments are typically performed in a region ∼2–10 µm away from the chosen area at the same or higher magnification than that used for photography. The choice of magnification, defocus level, accelerating voltage, beam coherence, electron dose and other operating conditions is dictated by several factors. The most significant ones are the size of the particle or crystal unit cell being studied, the anticipated resolution of the images and the requirements of the image processing needed to compute a 3D reconstruction to the desired resolution. For most specimens at required resolutions from 3 to 30 Å, images are typically recorded at 25 000–50 000× magnification with an electron dose of between 5 and 20 e Å⁻² . Even lower magnification, down to 10 000×, can be used if high resolution is not required, and higher magnification, up to 75 000×, can be used if good specimen areas are easy to locate. These conditions yield micrographs of sufficient optical density (OD 0.2–1.5) and image resolution for subsequent image processing steps (Sections 19.6.4.5 and 19.6.4.6). Most modern EMs provide some mode of low-dose operation for imaging beam-sensitive, vitrified biological specimens. Dose levels may be measured directly (e.g. with a Faraday cup) or they may be estimated from a calibrated microscope exposure meter (e.g. Baker & Amos, 1978).

The intrinsic low contrast of unstained specimens makes it impossible to observe and focus on specimen details directly as is routine with stained or metal-shadowed specimens. Focusing, aimed to enhance phase contrast in the recorded images but minimize beam damage to the desired area, is achieved by judicious defocusing on a region that is adjacent to the region to be photographed and preferably situated on the microscope tilt axis. The appropriate focus level is set by adjusting the appearance of either the Fresnel fringes that occur at the edges of holes in the carbon film or the `phase granularity' from the carbon support film (e.g. Agar et al., 1974).

Unfortunately, electron images do not give a direct rendering of the specimen density distribution. The relationship between image and specimen is described by the contrast-transfer function (CTF) which is characteristic of the particular microscope used, the specimen and the conditions of imaging. The microscope CTF arises from the objective-lens focal setting and from the spherical aberration present in all electromagnetic lenses, and varies with the defocus and accelerating voltage according to equation (19.6.4.1), an expression which includes both phase and amplitude contrast components. First, however, it might be useful to describe briefly the essentials of amplitude contrast and phase contrast, two concepts carried over from optical microscopy. Amplitude contrast refers to the nature of the contrast in an image of an object which absorbs the incident illumination or scatters it in any other way so that a proportion of it is lost. As a result, the image appears darker where greater absorption occurs. Phase contrast is required if an object is transparent (i.e. it is a pure phase object) and does not absorb but only scatters the incident illumination. Biological specimens for cryo EM are almost pure phase objects and the scattering is relatively weak, so the simple theory of image formation by a weak phase object applies (Spence, 1988; Reimer, 1989). An exactly in-focus image of a phase object has no contrast variation since all the scattered illumination is focused back to equivalent points in the image of the object from which it was scattered. In optical microscopy, the use of a quarter wave plate can retard the phase of the direct unscattered beam, so that an in-focus image of a phase object has very high `Zernicke' phase contrast. However, there is no simple quarter wave plate for electrons, so instead phase contrast is created by introducing phase shifts into the diffracted beams by adjustment of the excitation of the objective lens so that the image is slightly defocused. In addition, since all matter is composed of atoms and the electric potential inside each atom is very high near the nucleus, even the electron-scattering behaviour of the light atoms found in biological molecules deviates from that of a weak phase object, but for a deeper discussion of this the reader should refer to Reimer (1989) or Spence (1988). In practice, the proportion of `amplitude' contrast is about 7% at 100 kV and 5% at 200 kV for low-dose images of protein molecules embedded in ice.

The overall dependence of the CTF on resolution, wavelength, defocus and spherical aberration is where , ν is the spatial frequency (in Å⁻¹), is the fraction of amplitude contrast, λ is the electron wavelength (in Å), where (= 0.037, 0.025 and 0.020 Å for 100, 200 and 300 keV electrons, respectively), V is the voltage (in volts), is the underfocus (in Å) and is the spherical aberration of the objective lens of the microscope (in Å).

In addition, this CTF is attenuated by an envelope or damping function which depends upon the spatial and temporal coherence of the beam, specimen drift and other factors (Erickson & Klug, 1971; Frank, 1973; Wade & Frank, 1977; Wade, 1992). Fig. 19.6.4.4 shows a few representative CTFs for different amounts of defocus on a normal and a FEG microscope. Thus, for a particular defocus setting of the objective lens, phase contrast in the electron image is positive and maximal only at a few specific spatial frequencies. Contrast is either lower than maximal, completely absent, or it is opposite (inverted or reversed) from that at other frequencies. Hence, as the objective lens is focused, the electron microscopist selectively accentuates image details of a particular size. For this discussion, we ignore inelastic scattering, which makes some limited contribution at low resolution to images as a result of the effect of chromatic aberration on the energy-loss electrons in thick specimens or samples embedded in thick layers of vitrified water (Langmore & Smith, 1992). Inelastically scattered electrons can be largely removed by use of microscopes equipped with electron-energy filtering devices (e.g. Langmore & Smith, 1992; Koster et al., 1997; Zhu et al., 1997), but this also leaves fewer electrons to form the image.

Figure 19.6.4.4| top | pdf | Representative plots of the microscope contrast-transfer function (CTF) as a function of spatial frequency, for two different defocus settings (0.7 and 4.0 µm underfocus) and for a field-emission (light curve) or tungsten (dark curve) electron source. All plots correspond to electron images formed in an electron microscope operated at 200 kV with objective-lens aberration coefficients  mm and assuming amplitude contrast of 4.8% (Toyoshima et al., 1993). The spatial coherence, which is related to the electron source size and expressed as β, the half-angle of illumination, for tungsten and FEG electron sources was fixed at 0.3 and 0.015 mrad, respectively. Likewise, the temporal coherence (expressed as ΔE, the energy spread) was fixed at 1.6 and 0.5 eV for tungsten and FEG sources. The combined effects of the poorer spatial and temporal coherence of the tungsten source leads to a significant dampening, and hence loss of contrast, of the CTF at progressively higher resolutions compared to that observed in FEG-equipped microscopes. The greater number of contrast reversals with higher defocus arises because of the greater out-of-focus phase shifts as described in Section 19.6.4.4.

Images are typically recorded 0.8–3.0 µm underfocus to enhance specimen features in the 20–40 Å size range and thereby facilitate phase-origin and specimen-orientation search procedures carried out in the image-processing steps (Section 19.6.4.8), but this level of underfocus also enhances contrast in lower-resolution maps, which may help in interpretation. To obtain results at better than 10–15 Å resolution, it is essential to record, process and combine data from several micrographs that span a range of defocus levels (e.g. Unwin & Henderson, 1975; Böttcher, Wynne & Crowther, 1997). This strategy assures good information transfer at all spatial frequencies up to the limiting resolution but requires careful compensation for the effects of the microscope CTF during image processing. Also, the recording of image focal pairs or focal series from a given specimen area can be beneficial in determining origin and orientation parameters for processing of images of single particles (e.g. Cheng et al., 1992; Trus et al., 1997; Conway & Steven, 1999).

Many high-resolution cryo EM studies are now performed with microscopes operated at 200 keV or higher and with FEG electron sources (e.g. Zemlin, 1992; Zhou & Chiu, 1993; Zemlin, 1994; Mancini et al., 1997). The high coherence of a FEG source ensures that phase contrast in the images remains strong out to high spatial frequencies () even for highly defocused images. The use of higher voltages provides potentially higher resolution (greater depth of field – i.e. less curvature of the Ewald sphere – owing to the smaller electron-beam wavelength), better beam penetration (less multiple scattering), reduced problems with specimen charging of the kind that plague microscopy of unstained or uncoated vitrified specimens (Brink et al., 1998) and reduced phase shifts associated with beam tilt.

Images are recorded on photographic film or on a CCD camera with either flood-beam or spot-scan procedures. Film, with its advantages of low cost, large field of view and high resolution (∼10 µm), has remained the primary image-recording medium for most cryo EM applications, despite disadvantages of high background fog and need for chemical development and digitization. CCD cameras provide image data directly in digital form and with very low background noise, but suffer from higher cost, limited field of view, limited spatial resolution caused by poor point spread characteristics and a fixed pixel size (24 µm). They are useful, for example, for precise focusing and adjustment of astigmatism (e.g. Krivanek & Mooney, 1993; Sherman et al., 1996). With conventional flood-beam imaging, the electron beam (generally >2–5 µm diameter) illuminates an area of specimen that exceeds what is recorded in the micrograph. In spot-scan imaging, which decreases the beam-induced specimen drift often seen in flood illumination, a 2000 Å or smaller diameter beam is scanned across the specimen in a square or hexagonal pattern while the image is recorded (Downing, 1991). This method is beneficial in the examination of 2D crystalline specimens at near-atomic resolutions (Henderson et al., 1990; Nogales et al., 1998) and has also been used to study some icosahedral viruses (e.g. Zhou et al., 1994; Zhao et al., 1995).

For studies in which specimens must be tilted to collect 3D data, such as with 2D crystals, single particles that adopt preferred orientations on the EM grid, or specimens requiring tomography, microscopy is performed in essentially the same way as described above. However, the limited tilt range (60–70°) of most microscope goniometers can lead to non-isotropic resolution in the 3D reconstructions (the `missing cone' problem), and tilting generates a constantly varying defocus across the field of view in a direction normal to the tilt axis. The effects caused by this varying defocus level must be corrected in high-resolution applications (Henderson et al., 1990) or they can be partially corrected during spot-scan microscopy if the defocus of the objective lens is varied in proportion to the distance between the beam and tilt axis (Zemlin, 1989).