Enhanced contrast in electron microscopy of unstained biological material. II. Defocused contrast of large objects

A contrast minimum is observed when 88 nm diameter polystyrene latex spheres are underfocused, which is related to the wide-angle scattering peaks. Images due to scattered and non-scattered wave components are displaced due to objective-lens spherical aberration and defocus. Maximum overlap of these components produces a contrast minimum at underfocus, related to the spherical aberration of the particular lens used. Similarly, a high-contrast band at carbon-film edges arises from spherical aberration and defocus separation of non-scattered and wide-angle scattered waves. This band increases in contrast with film thickness and in width with lens defocus. These geometrical effects account for the well-known ‘blinking’ of contrast of large biological objects upon swinging through focus without an objective aperture, and for the general contrast increase of defocused large objects. Fresnel fringes account for only a narrow band of enhanced contrast at distinct edges and cannot account for contrast enhancement of large objects lacking distinct edges.     

Enhanced contrast in electron microscopy of unstained biological material. I. Strioscopy (dark-field microscopy)

Electron-optical methods of enhancing contrast are being investigated in order to eliminate the artifacts associated with heavy-metal contrasting procedures. This report gives a quantitative discussion of the application of the strioscopic dark-field method to model biological objects. Beam-stopping apertures were constructed by microwelding fine platinum wires on to platinum objective apertures. These were precisely located and centred in the back focal plane of the objective lens. Contrast was evaluated by a sensitive micro-Faraday cage system. Bright-field and strioscopic contrast of organic layers up to 557 nm thickness were investigated and related to the transition from single to plural scattering. The spherical-aberration phase-contrast contribution could be separated from the amplitude contrast in bright-field microscopy by extrapolation to zero object thickness. Strioscopic contrast of thin layers was found to be much greater than bright field aperture contrast and shown to be non-linear with respect to thickness in the region 0·7–10 nm. For diffraction patterns made up of a few discrete beams the resolution is high, but is considerably less for diffusely scattering and thick objects. Strioscopy may be the method of choice for obtaining contrast of unstained or weakly-stained objects at high acceleration voltage.     

In-focus electron microscopy of frozen-hydrated biological samples with a Boersch phase plate

We report the implementation of an electrostatic Einzel lens (Boersch) phase plate in a prototype transmission electron microscope dedicated to aberration-corrected cryo-EM. The combination of phase plate, C_s corrector and Diffraction Magnification Unit (DMU) as a new electron-optical element ensures minimal information loss due to obstruction by the phase plate and enables in-focus phase contrast imaging of large macromolecular assemblies. As no defocussing is necessary and the spherical aberration is corrected, maximal, non-oscillating phase contrast transfer can be achieved up to the information limit of the instrument. A microchip produced by a scalable micro-fabrication process has 10 phase plates, which are positioned in a conjugate, magnified diffraction plane generated by the DMU. Phase plates remained fully functional for weeks or months. The large distance between phase plate and the cryo sample permits the use of an effective anti-contaminator, resulting in ice contamination rates of <0.6 nm/h at the specimen. Maximal in-focus phase contrast was obtained by applying voltages between 80 and 700 mV to the phase plate electrode. The phase plate allows for in-focus imaging of biological objects with a signal-to-noise of 5-10 at a resolution of 2-3 nm, as demonstrated for frozen-hydrated virus particles and purple membrane at liquid-nitrogen temperature.     

Phase and amplitude contrast in electron microscopy of stained biological objects.

For biological objects negatively stained with heavy atom material, electron microscope images show best contrast for image detail on the scale of 10--20 A when a small objective aperture is used. In images taken under the optimum phase contrast imaging conditions of Scherzer, the required image detail is lost in unwanted noise. Both of these conditions may be described in terms of phase contrast imaging for a thin phase object. Calculations of image intensities and noise are reported for a model object consisting of heavy and light atoms randomly distributed to simulate a negatively stained protein molecule. The results are consistent with experimental observations.     

Defocusing phase contrast effects in electron microscopy

The theory of defocusing phase contrast is presented in terms of Scherzer's (1949) ‘integrated-ray’ and Heidenreich's (1964) ‘single-ray’ approach to spherical aberration; contrast transfer theory in terms of the Scherzer expression is included. Fine structure in transverse sections, negative stain and carbon film is shown to be the result of defocusing phase contrast. Examples of interpretative confusion are given for various macromolecular systems and quantitative support is found for a simplified ‘rule-of-thumb’ approach to phase-contrast enhancement. High-resolution studies of carbon-fibre lattices demonstrate contrast reversal, hence it is possible to show that a practical expression for the phase change due to spherical aberration falls between the Scherzer and the Heidenreich terms; the defocus term is also found to require some adjustment.     

Phase contrast electron microscopy of biological materials

The properties of two electron microscope phase contrast imaging methods are compared. The first is the conventional bright-field method in which dark phase contrast is created by defocusing; the second is a phase plate method in which bright phase contrast is created by means of a suitably shaped electric field. Using some negatively stained biological specimens which have a well-known repeating structure as the test object, it is shown that the phase plate method has some important advantages over the bright-field method. Its contrast transfer characteristics are such that it can provide a more faithful representation of the high resolution detail in the object. Moreover, by producing bright, rather than the normal dark, phase contrast it is able to simultaneously enhance the detail in the specimen and weaken the detail in the stain; this latter property enables the method to display information about the specimen that it would not be possible to detect with the bright-field method.     

Topographic and material contrast in low-voltage scanning electron microscopy

Two scintillation backscattered electron (BSE) detectors with a high voltage applied to scintillators were built and tested in a field emission scanning electron microscope (SEM) at low primary beam energies. One detector collects BSE emitted at low take-off angles, the second at high takeoff angles. The low take-off detector gives good topographic tilt contrast, stronger than in the case of the secondary electron (SE) detection and less sensitive to the presence of contamination layers on the surface. The high take-off detector is less sensitive to the topography and can be used for detection of material contrast, but the contrast becomes equivocal at the beam energy of 1 keV or lower.     

Dark-field electron microscopy of unstained and unshadowed DNA

The vastly increased contrast made possible by tilted-beam dark-field electron microscopy enables strands of DNA prepared by the Kleinschmidt technique to be clearly imaged without the heavy-metal staining or shadowing usually required. This technique is applicable over a wide range of magnifications and is easily accomplished with several commercial instruments.     

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.     

Improved Zernike‐type phase contrast for transmission electron microscopy

Zernike phase contrast has been recognized as a means of recording high‐resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image typical phase objects such as unstained biological molecules or cryosections of biological tissue. According to the original proposal discussed in Danev and Nagayama (2001) and references therein, the Zernike phase plate applies a phase shift of π/2 to all scattered electron beams outside a given scattering angle and an image is recorded at Gaussian focus or slight underfocus (below Scherzer defocus). Alternatively, a phase shift of ‐π/2 is applied to the central beam using the Boersch phase plate. The resulting image will have an almost perfect contrast transfer function (close to 1) from a given lowest spatial frequency up to a maximum resolution determined by the wave length, the amount of defocus and the spherical aberration of the microscope. In this paper, I present theory and simulations showing that this maximum spatial frequency can be increased considerably without loss of contrast by using a Zernike or Boersch phase plate that leads to a phase shift between scattered and unscattered electrons of only π /4, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.     

Optimizing phase contrast in transmission electron microscopy with an electrostatic (Boersch) phase plate

Imaging of weak amplitude and phase objects, such as unstained vitrified biological samples, by conventional transmission electron microscopy (TEM) suffers from poor object contrast since the amplitude and phase of the scattered electron wave change only very little. In phase contrast light microscopy the imaging of weak phase objects is greatly enhanced by the use of a quarter-wave phase plate, which produces high signal contrast by shifting the phase of the scattered light. An analogous quarter-wave plate for the electron microscope, designed as an electrostatic einzel lens, was proposed by Boersch in 1947 but the small dimensions of the device have impeded its realization up to now. We here present the first fabrication and application of a miniaturized electrostatic einzel lens driven as TEM quarter-wave phase plate. Phase modulation is generated by the electrostatic field confined to the inside of a microstructured ring electrode. This field affects the phase velocity of the unscattered part of the electron wave. By varying its strength the phase shift of the primary beam can be adjusted to pi/2, producing strong phase contrast independent of spatial frequency. The phase plate proves to be mechanically stable and does not impair image quality, in particular it does not reduce the high-resolution signal. The expected residual lens effect of the einzel lens is minimal. Our microlens is supported by conducting rods arranged in a threefold symmetry. This particular geometry provides optimized single-sideband signal transfer for spatial frequencies otherwise obstructed by the supporting rods.     

The direct determination of magnetic domain wall profiles by differential phase contrast electron microscopy.

A new technique for the quantitative investigation of magnetic structures in ferromagnetic thin films is proposed. Unlike previous techniques the detected signal is simply related to the magnetic induction in the film, and as such the direct determination of domain wall profiles is possible. The technique utilizes a differential phase contrast mode of scanning transmission electron microscopy in which the normal bright field detector is replaced by a split-detector lying symmetrically about the optic axis of the system. The difference signal from the two halves of the detector provides the required magnetic information. Analysis of the image formation mechanism shows that, using a commercially available scanning transmission electron microscope equipped with a field emission gun, wall profiles should be obtainable directly from most structures of interest in Lorentz microscopy. Furthermore, signal-to-noise considerations indicate that these results can be obtained in acceptably short recording times. Finally, experimental results using both polycrystalline and single crystal specimens are presented, which confirm the theoretical predictions.     

Imaging of three-dimensional objects in emission electron microscopy

Under investigation by emission electron microscopy, the shape and size of three-dimensional objects are distorted because of the appearance of a characteristic potential relief and a possible contact potential difference between the particles and the substrate. An estimation of these effects for spherical particles is made. It is shown that the apparent size of particles observed in an emission electron microscope (EEM) could be increased as well as decreased depending on the relation between the work functions of the particle and the substrate. The corresponding formulae are given and several possibilities are shown which permit us to determine from the EEM image the real size of particles and their work function relative to the substrate.     

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 × 10 × 5 mm³) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 °C, are then fixed at 4 °C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 °C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.     

Advantages of megavolt electron microscopy in biological research.

New electron microscopes, operating within the megavolt range have opened important prospects for scientific research. These microscopes were, at first, mainly used by physicists and metallurgists; but nowadays more and more biologists are interested in high voltage electron microscopy: they have obtained important and significant results. The present paper gives more information concerning experiments that have been achieved in Toulouse with our two big instruments working at 1 million volts (1 MV) and three million volts (3 MV).     

Combining accurate defocus with low-dose imaging in high resolution electron microscopy of biological material

High resolution (< 2 nm) electron microscopy of biological specimens requires three exacting conditions to be met simultaneously: (a) fine specimen detail must be protected from destruction by the electron beam (low dose), (b) the electron optics must be adjusted to be capable of imaging that detail interpretably (accurate defocus), and (c) a suitable field of interest must be identified. We describe a method encompassing all three with an 80% success rate using only minor modifications to a transmission electron microscope, and no expensive on-line computing.