Chapter 3.4. Mounting and setting of specimens for X-ray crystallographic studies

Informative accounts of the powder method of recording diffraction patterns have been given by Klug & Alexander (1954), D'Eye & Wait (1960) and Dent Glasser (1977). There are three principal methods of preparing polycrystalline specimens for mounting in powder cameras:

The most common method of preparing samples of polycrystalline materials is to encase them in thin-walled capillary tubes, for Debye–Scherrer camera work, or into sample holders, for Guinier camera and diffractometer measurements. This technique has the advantage that the sample can be readily protected from attack by oxygen, carbon dioxide and water vapour, and, if necessary, the sample preparation can be undertaken in an inert atmosphere (Lange & Haendler, 1972; D'Eye & Wait, 1960). The precise details of sample preparation and mounting will be dependent on the type of camera or diffractometer used, but the particle size should be generally less than 10 µm for stationary samples and diffractometer work. A slightly larger particle size, 45 µm, can be used for Debye–Scherrer camera work if the specimen is rotated. Foit (1982) has described a simple method of filling thin-walled capillaries using an ultrasonic vibrator. A frequent problem affecting intensity measurements from powder specimens is caused by preferred orientation when powder samples are packed or pressed. McMurdie, Morris, Evans, Paretzkin & Wong-Ng (1986) have described a method of sample preparation suitable for a diffractometer that minimizes this problem.

Capillaries made from lithium beryllium borate (Lindemann glass), borosilicate (e.g. Pyrex glass), or fused silica are commercially available in a variety of internal diameters. For very high temperatures, thin-walled ceramic or metal capillaries can be used. The diffraction pattern of the metal can be used as an internal standard. Capillaries that are suitable for materials that react with glass can be made from various organic polymers. Table 3.4.1.1 lists details of capillaries and other containers suitable for encasing powder specimens.

In the bonded method, the polycrystalline material is mixed with an adhesive such as gum tragacanth or ethyl cellulose, and the mixture is wetted with water or aqueous alcohol to form a viscous paste. The paste is then rolled between two glass slides or extruded through a glass capillary, using a glass or metal piston, to form a cylindrical sample. This can be cut to length and either glued, fixed with plasticine, or cemented (for high-temperature work) to the camera mounting pin. Alternatively, the sample can be compressed and compacted in a die to form a solid rod, or, for diffractometers, into a disc. In the case of very small quantities of material, the powder can be smeared with silicone vacuum grease over the surface of a disc-shaped silica crystal. The silica can then be used as an internal standard.

In the fibre-supported method, a silica, Lindemann, or borosilicate glass fibre moistened with adhesive (Canada balsam diluted with xylene, collodion, gum tragacanth and water, dilute fish glue) is dipped into the powder. Experience has shown that powder adhesion to the fibre is often improved if non-drying glues or viscous oils are employed. Hairs of fine organic filaments have been used in place of glass fibres, and for high temperature above 1270° C metal wires are useful. Once again, the metal diffraction patterns can act as internal standards. For extruded metal wires, the wire itself acts as the specimen, and the diameter can be reduced by etching if it is too large, or a glancing-angle diffraction technique can be employed. Various specialized holders for diffraction studies of polycrystalline samples can be found in annual conference proceedings such as EPDIC (European Powder Diffraction Conference, Switzerland: Trans Tech Publications) and Advances in X-ray Analysis (Proceedings of the Annual Conference on the Applications of X-ray Diffraction, New York/London: Plenum). The journals Reviews of Scientific Instruments (American Institute of Physics) and Nuclear Instruments and Methods (Elsevier, North-Holland) also provide useful sources of information.

A number of devices have recently been described to study polycrystalline specimens under non-ambient conditions. Rink, Mathias & Schlenoff (1994) have designed a portable sample housing for work at room temperature with samples that are air or moisture sensitive. A review of designs and desirable features for high-temperature furnaces suitable for X-ray diffractometers has been given by McKinstry (1970). More recently, Puxley, Squire & Bates (1994) have described an in situ cell fitted to a Siemens D-500 powder diffractometer that allows samples in flowing or static reactive gas environments at atmospheric pressure and at temperatures up to 1273 K. These authors also review other developments in the field of high-temperature furnaces for polycrystalline X-ray diffraction published since the McKinstry article in 1970. Brown, Swapp, Bennett & Navrotsky (1993) have devised methods to minimize the uncertainties in temperature at the sample and in the position of the sample itself. Tarling, Barnes & Mackay (1984) have adapted a Guinier–Lenné high-temperature powder camera to include a gas rinsing system and a specially designed mini-environment cell in which conditions of industrial furnacing can be simulated. In the neutron area, Lorenz, Neder, Marxreiter, Frey & Schneider (1993) have developed a mirror furnace working at up to 2300 K and suitable for polycrystalline or single-crystal samples.

A comprehensive account of cryogenic studies pertinent to both polycrystalline and single-crystal samples is given by Rudman (1976). Nieman, Evans, Heal & Powell (1984) have described a device for the preparation of low-temperature samples of noxious materials. The device is enclosed in a vanadium can and is therefore only suitable for neutron diffraction studies. Ihringer & Küster (1993) have described a cryostat for powder diffraction, temperature range 8–300 K, for use on a synchrotron-radiation beam line at HASYLAB, Germany (Arnold et al., 1989).)

Small single crystals of inorganic and organic materials, suitable for intensity data collection, are normally glued to the end of a glass or vitreous silica fibre, or capillary (Denne, 1971b; Stout & Jensen, 1968). A simple device that fits onto a conventional microscope stage to facilitate the procedure of cementing a single crystal to a glass fibre has been constructed by Bretherton & Kennard (1976). The support is in turn fixed to a metal pin that fits onto a goniometer head. For preliminary studies, plasticine or wax are useful fixatives, since it is then relatively easy to alter the orientation of the support, and hence the crystal, as required. For data-collection purposes, the support should be firmly fixed or glued to the goniometer head pin. The fibre should be sufficiently thin to minimize absorption effects but thick enough to form a rigid support. The length of the fibre is usually about 10 mm. Kennard (1994) has described a macroscope that allows specimens to be observed remotely during data collection and can also be used for measurement of crystal faces for absorption correction. Large specimens can be directly mounted onto a camera or onto a specially designed goniometer (Denne, 1971a; Shaham, 1982). A method using high-temperature diffusion to bond ductile single crystals to a metal backing, for strain-free mounting, has been described by Black, Burdette & Early (1986).

Prior to crystal mounting, it is always prudent to determine the nature of any spatial constraints that are applicable for the proposed experiment. Some diffractometers have relatively little translational flexibility, and the length of the fibre mount or capillary is critical. For some low-temperature devices where the cooling gas stream is coaxial with the specimen mount, the orientation of the fibre (and crystal) on the goniometer head may also need careful alignment.

Many proprietary adhesives can be used (see Table 3.4.1.2), but it should be remembered that adhesives such as epoxy resins are often permanent, and attempts to dismount specimens lead to crystal damage. Some adhesives contain organic solvents that may react with the sample, and others may be X-ray sensitive and deteriorate with exposure. In low-temperature work, some adhesives shrink or become brittle. Ideally, the adhesive should have the same thermal characteristics as the crystal and its mount. An account of how strong stresses on adhesives, typically used to mount single crystals, induced by low and high temperatures is given by Argoud & Muller (1989a). The stresses appear to cause anisotropic modifications to secondary extinction, leading to discrepancies in the intensities of symmetry-related reflections. Beeswax and paraffin wax were found to be free from such stresses. Crystals that are sensitive to air can be mounted inside capillary tubes or other containers, as listed in Table 3.4.1.1. A useful summary of the methods available has been provided by Rao (1989). All adhesives and containers will give diffraction patterns, typically comprising diffuse bands, that contribute to the general background, and that may change with ageing. Minimal amounts of adhesive and thin-walled capillaries should be used. If the background diffraction is critical, it is highly recommended that diffraction patterns of the container and/or adhesive are recorded separately as controls.

The morphology of a given crystal will normally dictate the way that it is mounted, particularly for data-collection purposes. Thus, prismatic crystals and needle-shaped crystals are usually mounted with the longest dimension parallel to the fibre, in order to minimize systematic errors due to absorption. Jeffery (1971) and Wood, Tode & Welberry (1985) have described devices for shaping crystals into spheres and cylinders, respectively. A solvent lathe whereby a string moistened with solvent is used to shape the crystal is described by Stout & Jensen (1968).

As in the case of polycrystalline samples, a number of devices have been described to study single crystals at elevated pressures and at a range of temperatures. The mounting of the sample is very dependent on the device and radiation used. In recent years, the field of high-pressure crystallography has expanded significantly, and several sample holders based on the diamond-anvil cell have been reported for pressures up to 10 GPa. Recent papers include those by Alkire, Larson, Vergamini, Schirber & Morosin (1985) for neutron diffraction, and Malinowski (1987) and Leszczynski, Podlasin & Suski (1993) for X-ray diffraction.

Various types of furnace have been designed for high-temperature studies of single crystals. These are based either on radiative heat transfer mechanisms [e.g. Swanson & Prewitt (1986); temperatures up to 1400 K], electrically heated gas streams [e.g. Tsukimura, Sato-Sorensen & Ghose (1989); temperatures up to 1600 K], or flame heaters [e.g. Miyata, Ishizawa, Minato & Iwai (1979); temperatures up to 2600 K]. Furnaces specific to Weissenberg geometry (Adlhart, Tzafaras, Sueno, Jagodzinski & Huber, 1982) and Laue diffraction (Bhat, Clark, El Korashy & Roberts, 1990) have also been reported. There are many techniques available for mounting single crystals for high-temperature diffraction (Hazen & Finger, 1982), and a detailed account using an MgO-based ceramic cement is given by Swanson & Prewitt (1986). A recent paper by Peterson (1992) summarizes previous work in the design of high-temperature furnaces and describes a flame-heated gas-flow furnace operating in the range 373 to 1573 K. For this system, the crystals can be mounted either directly onto the thermocouple bead with a paste of fine platinum particles and oil, particularly useful if the crystal is to be exposed to a gas mixture that controls oxygen fugacity, or sealed under high vacuum in an ampoule made from 0.2 or 0.3 mm diameter silica capillary. In the latter case, the main support for the crystal is a 0.2 mm Pt wire threaded through a 0.3 mm diameter silica glass capillary. A 0.05 mm Pt/13Rh lead is welded to the end of this support to form the thermocouple bead. The wire is then wound around the outside of the capillary. A 0.05 mm Pt lead is welded to the other end of the 0.2 mm Pt wire and is threaded through a hole in the capillary near the base. The whole assembly can be mounted on a goniometer head that has only translational adjustments. For neutron diffraction, Lorenz, Neder, Marxreiter, Frey & Schneider (1993) have described a mirror furnace operating upto 2300 K. The sample support is normally a thin ceramic tube or rod of Al₂O₃ or ZrO₂ to which the sample may be glued with a ceramic cement. Neder, Frey & Schulz (1990) have described a versatile holder for high-temperature neutron studies. One part of the crystal is ground away to leave a stem, which is then fixed to an alumina rod with a ceramic glue based on zirconia. The ceramic glue is surrounded by a cylinder of BN to minimize spurious scattering.

A comprehensive account of low-temperature diffraction is given by Rudman (1976). Procedures for the selection and transfer of crystals to diffractometers have been described by Boese & Bläser (1989) and Kottke & Stalke (1993). These procedures are applicable down to temperatures of 213 and 193 K, respectively. The latter authors do not recommend the use of capillaries, but describe a device employing the oil-drop mounting technique pioneered by Hope (1987, 1988). Lippman & Rudman (1976) have used a mechanically refrigerated gas stream to achieve temperatures down to approximately 150 K, and the use of liquid nitrogen extends the range to 77 K. Devices such as the Oxford Cryostream can be readily fitted to diffractometers and other types of camera. Closed-cycle refrigerators, liquid-helium-based devices (e.g. Henriksen, Larsen & Rasmussen, 1986; Argoud & Muller, 1989b; Zobel & Luger, 1990; Graafsma, Sagerman & Coppens, 1991; Toyoshima, Hoya & Ohshima, 1991) further extend the low-temperature limit to 5 K, but often involve substantial blind regions and collision zones. For sample mounting in these devices, it is essential to have good heat conduction to the crystal. Zobel & Luger (1990) describe a taper-formed sample holder made of special copper screwed to the cold head. A steel injection needle with a Be wire inside (0.3 mm diameter and exactly 2 mm in length) is fitted into a 0.5 mm bore hole. The crystal is glued with Araldite to the Be needle, which has little X-ray absorption but good heat conduction. In addition to diffractometer-based devices, Moret & Dallé (1994) have described an adaptation of the closed-cycle refrigerator for a precession goniometer, and various authors have reported systems utilizing Weissenberg geometry for both X-rays and neutrons (e.g. Hohlwein & Wright, 1981; Adlhart & Huber, 1982; Allen et al., 1982). Reference should be made to the individual papers for methods of mounting, including spatial and any other constraints.

Crystals of biological macromolecules are very susceptible to radiation damage, and this can severely limit the amount and quality of diffraction data that can be collected per crystal. There have been relatively few systematic studies of this phenomenon (Young, Dewan, Nave & Tilton, 1993; Gonzalez & Nave, 1994; Nave, 1995), but one of the first effects of radiation damage is the deterioration of the high-resolution regions of the pattern, followed by increasing loss of crystallinity. Improvement of crystal lifetime in X-ray beams has been obtained by the addition of free-radical scavengers (Zaloga & Sarma, 1974) and the replacement of the mother liquor with solutions containing 10–20% polyethylene glycol 4000 or 20000 (Cascio, Williams & McPherson, 1984). The use of synchrotron radiation has also led to improved data-per-crystal ratios (Lindley, 1988). The high intensity allows fast collection of data, and the high collimation permits different sections of the same crystal to be used for data collection. This is particularly useful for prismatic crystals, which can be mounted along their largest morphological axis. An alternative method of surmounting this problem, however, is to freeze the protein crystal. As the temperature is decreased, the rate of diffusion of free radicals is reduced, with a corresponding reduction in radiation damage. Appreciable reduction in diffusion rate is achieved even at 250 K, and at 100 K diffusion essentially ceases. Cryogenic measurements not only minimize radiation damage but often lead to improved resolution owing to decrease in thermal motion in the crystal. Increasing the crystal lifetime may be particularly important with respect to multiwavelength anomalous-dispersion measurements in order to derive phase information. Since crystals of biological macromolecules contain substantial amounts of solvent, typically between 35 and 80% by volume, the technical problem is to force the solvent to cool in an amorphous glass-like state, rather than as crystalline ice. The latter normally degrades the crystallinity by expansion and gives rise to powder rings, which complicate data measurement.

Cryoprotectants are normally required to avoid ice formation, and the choice of cryoprotectant will depend on the nature of the mother liquor from which the crystals have been grown. Crystals grown from high salt will usually require high salt concentration in the cryobuffer to avoid dissolution, although the addition of organic solvents may be a useful alternative. Table 3.4.1.3 lists commonly used cryoprotectants and their typical concentrations (Gamblin & Rogers, 1993).

The introduction of the cryoprotectant can be achieved through: (a) crystal growth in the cryoprotectant; (b) direct transfer of crystal from mother liquor into cryoprotectant buffer either in a single step or in steps of increasing cryoprotectant concentration; (c) dialysis, either direct or stepwise; or (d) exchange of liquor using a flow cell and a gradient maker.

Experience indicates that small crystals are better for cryogenic purposes, presumably because the rate of diffusion of small molecules and the rate of heat loss during rapid freezing is significantly faster than for large crystals. In most cases, there is an increase in the mosaicity (typically by a factor of 2–3), and in large specimens the increase may render the crystals useless for data collection. Successful freezing is often indicated by the crystal remaining transparent. Opacity usually indicates considerable breakdown in the crystallinity. Three commonly used methods for mounting crystals of biological macromolecules for cryogenic measurements are detailed below.

Several airstream devices have been described to cool protein crystals to around 250 K [Marsh & Petsko (1973), temperature range 253 to 303 K; Rossi (1989), temperature range 242 to 335 K; Machin, Begg & Isaacs (1984), 258 to 293 K; Fischer, Moras & Thierry (1985), temperature range 263 to 293 K; Fraase Storm & Tuinstra (1986), 250 to 350 K; Arndt & Stubbings (1987), 248 to 353 K]. The devices of Machin, Begg & Isaacs, Fraase Storm & Tuinstra and Arndt & Stubbings involve thermoelectric modules utilizing the Peltier effect. The space available to accommodate the sample is usually very limited and care has to be taken with the length of the capillary and other aspects of crystal mounting. Hovmöller (1981) has designed an extension to the cooling delivery tube that minimizes air turbulence at the sample. Various devices have been described that operate down to near liquid-nitrogen temperature and that can be fitted to a variety of data-collection systems. These include the rotation camera (Bartunik & Schubert, 1982), and a universal cooling device for precession cameras, rotation cameras and diffractometers (Hajdu, McLaughlin, Helliwell, Sheldon & Thompson, 1985). One of the more versatile devices is the cryostream described by Cosier & Glazer (1986), which uses a pump to effectively separate the liquid-nitrogen supply from the gas outflow; this arrangement eliminates instabilities in the cooling-gas stream; the device works in the range 77.4 to 323.0 K and is commercially available (Oxford Cryosystems, England).

Cryocrystallography not only minimizes the effects of radiation damage but also often allows the collection of high-quality, high-resolution data from a single specimen. In the case of very labile systems such as ribosomal particles, it is sometimes the only means of obtaining useful diffraction data. Further, cryocrystallography permits the study of temperature effects on the structure and dynamics of biological macromolecules. In this latter regard, examples include multiple-temperature crystallographic studies on sperm whale myoglobin (Frauenfelder, Petsko & Tsernoglou, 1979; Hartmann et al., 1982; Frauenfelder et al., 1987) and, more recently, ribonuclease-A (Tilton, Dewan & Petsko, 1992; Rasmussen, Stock, Ringe & Petsko, 1992). The future will no doubt see the routine emergence of cryogenic techniques for data collection, using both conventional and synchrotron X-ray sources, from biological macromolecules, with consequent improvement in structure quality and detail.

The azimuthal and back-reflection Laue methods for setting crystals with relatively small unit cells have been described by Jeffery (1971). The former is capable of achieving an accuracy of setting of ±0.05°, whereas the latter is important in metallurgy, where the Laue method is often the only possibility because of the large size of the specimens. Schiller (1985) has emphasized the importance of the back-reflection Laue technique for setting specimens with a precision of 0.1° needed in semiconductor surface preparation.

In recent years, there has been a resurgence of the Laue technique, in conjunction with synchrotron radiation, to record intensity data from biological macromolecules in very short time scales. The overall experimental strategies involved are described by Helliwell et al. (1989) and Clifton, Elder & Hajdu (1991). Crystals are not usually set in a precise orientation for these types of experiment prior to data acquisition because of radiation damage. The post-determination of the precise crystal orientation with respect to the instrument axes from the recorded Laue pattern therefore forms an essential part of the data processing. Most methods are based on the indexing procedure of Riquet & Bonnet (1979), and an interactive computer program for the interpretation and simulation of Laue patterns has been written by Laugier & Filhol (1983). An orientation-matrix approach has been reported by Jacobson (1986), and the work of Helliwell et al. (1989) has led to a comprehensive set of Laue processing programs. In addition to enabling trial-and-error visual matching of images, this program suite includes an autoindexing procedure based on a known unit cell, and refinement of the orientational parameters. More recently, Carr, Cruickshank & Harding (1992) have developed a method whereby a gnomonic projection of the Laue diffraction pattern can be used to determine the cell dimensions and orientation of a crystal. The axial ratios and interaxial angles can be determined precisely, but the absolute scaling of the cell is dependent on the accuracy with which the minimum wavelength used in the experiment is known.

More recently, the azimuthal method has proved of great value in the rapid alignment of crystals with large unit cells prior to data collection on devices using rotation geometry. After optical alignment, a `still' photograph taken with monochromatic radiation (or a very small angle rotation photograph, typically 0.05–0.20°), is used to locate a zero-layer reciprocal-lattice plane (Fig. 3.4.2.1 ). Such a plane will record on a flat detector placed at a distance D mm from the crystal, C, as an ellipsoidal trace of maximum dimension S mm from the direct-beam position, O′. In order to make the plane perpendicular to the X-ray beam (i.e. the real axis parallel to the X-ray beam), it must be rotated through an angle such that .

Figure 3.4.2.1| top | pdf | A zero-layer reciprocal-lattice plane will record on a flat-plate detector placed at a distance D from the crystal C as an ellipsoid of maximum dimension S from the direct-beam position O′.

If the vector O′P makes an angle α with the rotation axis, the angle can be resolved into a vertical component, , corresponding to a rotation of the spindle axis, and a horizontal component, , corresponding to a rotation of the goniometer arc whose axis is perpendicular to the X-ray beam (assuming a perpendicular and parallel setting of the goniometer head). Rotation of the reciprocal-lattice plane within its own plane can then be achieved with the goniometer arc whose axis is parallel to the beam. This technique is also applicable to preliminary setting on a precession camera.

However, with very radiation sensitive crystals, it is inadvisable to waste time accurately setting the crystal prior to data collection, since the crystal is subject to continuous radiation damage from the beginning of the first exposure (Rossmann & Erickson, 1983). In this case, two `still' images are collected, preferably separated by a 90° rotation, after data collection but before the crystal is irretrievably damaged. In principle, the orientation can be determined from a single still, but the precise crystal orientation is better determined by identifying and measuring the orientations of two real axes relative to the camera axes, from the sets of ellipses on two stills. The orientation of the reciprocal axis, perpendicular to these two real axes, can then be calculated, and, provided that the unit-cell dimensions are known, the orientation of the third real axis readily determined. Given the directions of the three real axes, the direction cosines of the reciprocal axes can be computed and a matrix determined that specifies the crystal orientation with respect to the camera axes. This method obviates the need to index the `partial' reflections on still images (Jones, Bartels & Schwager, 1977).