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FIGURE 10.2 Laser scanning confocal microscopy. A laser beam is scanned across a particular x-y plane within a sample. By combining successive x-y planes, it is possible to create a three-dimensional image.

fat spreads (Blonk and van Aalst 1993); to monitor creaming of droplets (Brakenoff et al. 1988); and to follow the desorption of proteins from the surface of emulsion droplets.

10.3.1.3. Electron Microscopy

Electron microscopy is widely used to examine the microstructure of food emulsions, particularly those that contain structural components which are smaller than the lower limit of resolution of optical microscopes (ca. <1 |im) (e.g., protein aggregates, small emulsion droplets, fat or ice crystals, micelles, and interfacial membranes) (Chang et al. 1972, Tung and Jones 1981, Hunter 1986, Aguilera and Stanley 1990, Heertje and Paques 1995). It can be used to provide information about the concentration, dimensions, and spatial distribution of structural entities within a specimen, provided the microstructure of the specimen is not significantly altered by the sample preparation (Heertje and Paques 1995). With suitable sample preparation, electron microscopy can be used to analyze both oil-in-water and water-in-oil emulsions that are either liquid or solid. Electron microscopes are fairly large pieces of equipment which are relatively expensive to purchase and maintain (Smart et al. 1995). For this reason, they tend to be available only at fairly large research laboratories or food companies.

Electron microscopes use electron beams, rather than light beams, to provide information about the structure of materials (Aguilera and Stanley 1990, Heertje and Paques 1995). These beams are directed through the microscope using a series of magnetic fields rather than optical lenses. Electron beams have much smaller wavelengths than light and so can be used to examine much smaller objects. In principle, the smallest size that can be resolved is about 0.2 nm, but in practice, it is usually about 1 nm due to limitations in the stability and performance of the magnetic lenses. Two types of electron microscope are commonly used to examine the structure of food systems: transmission electron microscopy (TEM) and scanning electron microscopy (SEM). In both of these techniques, it is necessary to keep the microscope under high vacuum because electrons are easily scattered by atoms or molecules in a gas, and this would cause a deterioration in the image quality. This also means that specimens must be prepared so that they are free of all volatile components that could evaporate (e.g., water and organic molecules).

Transmission Electron Microscopy. A cloud of electrons, produced by a tungsten cathode, is accelerated through a small aperture in a positively charged plate to form an electron beam (Figure 10.3). This beam is focused and directed through the specimen by a series of magnetic lenses. Part of the electron beam is either adsorbed or scattered by the specimen, while the rest is transmitted. The beam of transmitted electrons is magnified by a magnetic

FIGURE 10.3 Schematic diagram of TEM. An electron beam is directed through the sample, and the intensity of the transmitted beam is measured.

lens and then projected onto a fluorescent screen to create an image of the specimen. The fraction of electrons transmitted by a substance depends on its electron density: the lower the electron density, the greater the fraction of electrons transmitted and the more intense the image. Components with different electron densities therefore appear as regions of different intensity on the image. Images are typically 100 to 500,000 times larger than the portion of specimen being examined, which means that structures as small as 0.4 nm can be observed (Hunter 1993).

Electrons are highly attenuated by most materials, and therefore specimens must be extremely thin in order to allow enough of the electron beam through to be detected (Aguilera and Stanley 1990, Heertje and Paques 1995). Specimens used in TEM are therefore much thinner (~0.05 to 0.1 |im) than those used in light microscopy (~1 |im to a few millimeters). The difference in electron densities of food components is quite small, and therefore it is difficult to distinguish between them. For this reason, the density contrast between components is usually enhanced by selectively staining the sample with various heavy metal salts (which have high electron densities), such as lead, tungsten, or uranium. These salts bind selectively to specific components, which enables them to be distinguished from the rest of the sample. The metal salts may bind to the component itself (positive staining) or to the surrounding material (negative staining). In positive staining, a dark specimen is seen against a light background, whereas in negative staining, an illuminated specimen is seen against a dark background. The need to have very thin and dehydrated specimens, that often require staining, means that sample preparation is considerably more time consuming, destructive, and cumbersome than for other forms of microscopy.

The information produced by TEM is usually in the form of a two-dimensional image that represents a thin slice of the specimen (Figure 10.4). Nevertheless, it is possible to obtain some insight about the three-dimensional structure of a sample using a technique called metal shadowing (Figure 10.5). A vapor of a heavy metal, such as platinum, is sprayed onto the surface of a sample at an angle (Hunter 1993). The sample is then dissolved away using a strong acid, which leaves a metal replica of the sample. When a beam of electrons is

FIGURE 10.4 Comparison of electron micrographs of emulsions produced by (a) SEM (of butter) and (b) TEM (of margarine). TEM normally produces a two-dimensional image of a sample, whereas SEM produces a more three-dimensional image. The SEM of butter: g = fat droplet, f= fat crystals. The TEM of margarine: W = water droplets, F = fat continuous matrix, c = fat crystals. (Photographs kindly provided by I. Heertje.)

FIGURE 10.4 Comparison of electron micrographs of emulsions produced by (a) SEM (of butter) and (b) TEM (of margarine). TEM normally produces a two-dimensional image of a sample, whereas SEM produces a more three-dimensional image. The SEM of butter: g = fat droplet, f= fat crystals. The TEM of margarine: W = water droplets, F = fat continuous matrix, c = fat crystals. (Photographs kindly provided by I. Heertje.)

FIGURE 10.5 TEM can be used to obtain images of the surface topography of a sample using a technique known as metal shadowing.

transmitted through the metal replica, the "shadows" formed by the specimen are observed as illuminated regions which have characteristic patterns from which the topography of the specimen can be deduced.

Scanning Electron Microscopy. SEM is used to provide images of the surface topography of specimens (Heertje and Paques 1995). It relies on the measurement of secondary electrons generated by a specimen when it is bombarded by an electron beam, rather than the electrons which have traveled through the specimen. A focused electron beam is directed at a particular point on the surface of a specimen. Some of the energy associated with the electron beam is absorbed by the material and causes it to generate secondary electrons, which leave the surface of the sample and are recorded by a detector. An image of the specimen is obtained by scanning the electron beam in an x-y direction over its surface and recording the number of electrons generated at each location. Because the intensity at each position depends on the angle between the electron beam and the surface, the electron micrograph has a three-dimensional appearance (Figure 10.4).

Sample preparation for SEM is considerably easier and tends to produce fewer artifacts than TEM. Because an image is produced by secondary electrons generated at the surface of a specimen, rather than by an electron beam that travels through a specimen, it is not necessary to use ultrathin samples. Even so, specimens often have to be cut, fractured, fixed, and dehydrated, which may alter their structures. The resolving power of SEM is about 3 to 4 nm, which is an order of magnitude worse than TEM but about three orders of magnitude better than optical microscopy. Another major advantage of SEM over optical microscopy is the large depth of field, which means that images of relatively large structures are all in focus (Hunter 1993).

Electron microscopy is widely used by food scientists to determine the size of emulsion droplets; the dimensions and structure of flocs; the surface morphology of droplets and air bubbles; the size, shape, and location of fat crystals; and the microstructure of three-dimensional networks of aggregated biopolymers (Chang et al. 1972, Kalab 1981, Tung and Jones

1981, Hermansson 1988, Aguilera and Stanley 1990, Bucheim and Dejmek 1990, Lee and Morr 1992, Heertje and Paques 1995). TEM and SEM electron micrographs of two water-in-oil emulsions are shown in Figure 10.4. TEM gives a two-dimensional cross-section of the sample, whereas SEM gives a more three-dimensional image. As mentioned earlier, the major limitation of the technique is the difficulty in preparing samples without altering their structure. In addition, the use of high-energy electron beams can change the structure of delicate specimens. Many of these problems are being overcome as the result of recent advances in the design of electron microscopes and sample preparation techniques (Smart et al. 1995).

10.3.1.4. Atomic Force Microscopy

Atomic force microscopy (AFM) has the ability to provide information about structures at the atomic and molecular levels and is therefore complementary to the other forms of microscopy mentioned above (Miles and McMaster 1995). The technique has only recently been developed as a commercial instrument, although these instruments are still fairly expensive to purchase. For this reason, the application of AFM to foods is still largely in its infancy (Kirby et al. 1995) and is only used by a small number of research laboratories. Nevertheless, the technique has the potential to provide a wealth of valuable information about the structure and organization of molecules in food emulsions and will increase our fundamental understanding of these complex systems.

The AFM creates an image by scanning a tiny probe (similar to the stylus of a record player, but only a few micrometers in size) across the surface of the specimen being analyzed (Figure 10.6). When the probe is held extremely close to the surface of a material, it experiences a repulsive force, which causes the cantilever to which it is attached to be bent away from the surface. The extent of the bending is measured using an extremely sensitive optical system. By measuring the deflection of the stylus as it is moved over the surface of the material, it is possible to obtain an image of its structure. In practice, it is more common to measure the force required to keep the deflection of the stylus constant, as this reduces the possible damage caused by a stylus as it moves across the surface of a sample. The resolution of AFM depends principally on the size and shape of the probe and the accuracy to which it can be positioned relative to the sample. Samples to be analyzed are usually dissolved in a suitable solvent and then dried onto the surface of an extremely flat plate such as mica.

Atomic force measurements have been used to observe the structure of individual and aggregated polysaccharide and protein molecules (e.g., xanthan, pectin, acetan, starch, col

FIGURE 10.6 Principles of AFM. A probe is scanned over the surface of a material, and its displacement is measured using an accurate laser detection system. The force required to keep the probe in the same location, or the deflection of the probe, is measured.

Cantilever

FIGURE 10.6 Principles of AFM. A probe is scanned over the surface of a material, and its displacement is measured using an accurate laser detection system. The force required to keep the probe in the same location, or the deflection of the probe, is measured.

Cantilever

Cantilever Displacement Detector lagen, myosin, and bovine serum albumin) (Miles and McMaster 1995, Kirby et al. 1995). This type of study is useful for examining the relationship between the structure and interactions of biopolymer molecules and the type of gels they form. AFM has also been used to study the molecular organization of emulsifiers at planar oil-water and air-water interfaces (Kirby et al. 1995).

10.3.2. Static Light Scattering 10.3.2.1. Principles

Static light scattering is used to determine particle sizes between about 0.1 and 1000 | m and is therefore suitable for characterizing the droplets in most food emulsions. When a beam of light is directed through an emulsion, it is scattered by the droplets (Dickinson and Stainsby 1982, Farinato and Rowell 1983, Hiemenz 1986, Hunter 1986, Everett 1988). A measurement of the degree of scattering can be used to provide information about the droplet size distribution and concentration. Analytical instruments based on this principle have been commercially available for many years (Mikula 1992) and are widely used in the food industry for research, development, and quality control purposes. Most of these instruments are fully automated, simple to use, and provide an analysis of an emulsion within a few minutes. Even so, they tend to be fairly expensive to purchase, which has limited their application somewhat.

The interaction of an electromagnetic wave with an emulsion is characterized by a scattering pattern, which is the angular dependence of the intensity of the light emerging from the emulsion, /(O) (Figure 10.7). The size and concentration of droplets in an emulsion are ascertained from the scattering pattern using a suitable theory (Farinato and Rowell 1983). Theories that relate light-scattering data to droplet size distributions are based on a mathematical analysis of the propagation of an electromagnetic wave through an ensemble of particles (van de Hulst 1957, Bohren and Huffman 1983). A number of theories are available, which vary according to their mathematical complexity and the type of systems to which they can be applied. The interaction between light waves and emulsion droplets can be conveniently divided into three regimes, according to the relationship between the droplet radius (r) and the wavelength (X): (1) long-wavelength regime (r < X/20), (2) intermediate-wavelength regime (X/20 < r < 20X), and (3) short-wavelength regime (r > 20X). A characteristic scattering pattern is associated with each of these regimes (Figure 10.8).

FIGURE 10.7 The scattering pattern from an emulsion is characterized by measuring the intensity of the scattered light, / (O), as a function of angle (O) between the incident and scattered beams. The scattering pattern is particularly sensitive to particle size relative to the wavelength of light.

LWR IWR SWR

FIGURE 10.8 Typical scattering patterns for droplets in the long-, intermediate-, and short-wavelength regimes.

LWR IWR SWR

FIGURE 10.8 Typical scattering patterns for droplets in the long-, intermediate-, and short-wavelength regimes.

Long-Wavelength Regime. The simplest equation for relating the characteristics of the particles in a suspension to the scattering pattern produced when a monochromatic light beam passes through it was derived by Lord Rayleigh over a century ago and is applicable in the long-wavelength regime (Sherman 1968b):

where Ii is the initial intensity of the light beam in the surrounding medium, ^ is the dispersed-phase volume fraction, R is the distance between the detector and the scattering droplet, and n0 and n are the refractive indices of the continuous phase and droplets, respectively. This equation cannot be used to interpret the scattering patterns of most food emulsions because the size of the droplets (typically between 0.1 and 100 |im) is of the same order or larger than the wavelength of light used (typically between 0.2 and 1 |im). Nevertheless, it can be applied to suspensions which contain smaller particles, such as surfactant micelles or protein molecules (Hiemenz 1986). Equation 10.1 also provides some useful insights into the factors which influence the scattering profile of emulsions. It indicates that the degree of scattering from an emulsion is linearly related to the droplet concentration and increases as the refractive indices of the materials become more dissimilar.

Intermediate-Wavelength Regime. As mentioned earlier, most food emulsions contain droplets which are in the intermediate-wavelength regime. The scattering profile in this regime is extremely complex because light waves scattered from different parts of the same droplet are out of phase and therefore constructively and destructively interfere with one another (Shaw 1980, Hiemenz 1986). For the same reason, the mathematical relationship between the scattering pattern and the particle size is much more complex. A mathematician called Mie developed a theory which can be used to interpret the scattering patterns of dilute emulsions that contain spherical droplets of any size (van de Hulst 1957, Kerker 1969). The Mie theory is fairly complicated, but it can be solved rapidly using modern computers. This theory gives excellent agreement with experimental measurements and is used by most commercial particle-sizing instruments. It should be pointed out that the Mie theory assumes that the light waves are only scattered by a single particle, and so it is only strictly applicable to dilute emulsions. In more concentrated emulsions, a light beam scattered by one droplet may subsequently interact with another droplet, and this alters the scattering pattern (Ma et al. 1990). For this reason, emulsions must be diluted prior to analysis to a concentration where multiple scattering effects are negligible (i.e., ^ < 0.05%).

Short-Wavelength Regime. When the wavelength of light is much smaller than the particle diameter, the droplet size distribution can be determined directly by optical microscopy (Section 10.3.1).

Dilute Detector

Emulsion

FIGURE 10.9 Design for a particle-sizing instrument that utilizes angular scattering.

Dilute Detector

Emulsion

FIGURE 10.9 Design for a particle-sizing instrument that utilizes angular scattering.

10.3.2.2. Measurement Techniques

Angular Scattering Methods. Most modern particle-sizing instruments, based on static light scattering, measure the angular dependence of the scattered light (Farinato and Rowell 1983, Mikula 1992). The sample to be analyzed is diluted to an appropriate concentration and then placed in a glass measurement cell (Figure 10.9). A laser beam is generated by a helium-neon laser (X = 632.8 nm) and directed through the measurement cell, where it is scattered by the emulsion droplets. The intensity of the scattered light is measured as a function of scattering angle using an array of detectors located around the sample. The scattering pattern recorded by the detectors is sent to a computer, where it is stored and analyzed. The droplet size distribution that gives the best fit between the experimental measurements of /(O) versus O and those predicted by the Mie theory is calculated. The data are then presented as a table or graph of droplet concentration versus droplet size (Section 1.3.2). Once the emulsion sample has been placed in the instrument, the measurement procedure is fully automated and only takes a few minutes to complete. Before analyzing a sample, the instrument is usually blanked by measuring the scattering profile from the continuous phase in the absence of emulsion droplets. This scattering pattern is then subtracted from that of the emulsion to eliminate extraneous scattering from background sources other than the droplets (e.g., dust or optical imperfections). The range of droplet radii that can be detected using this type of experimental arrangement is about 0.1 to 1000 |im. A number of instrument manufacturers have developed methods of determining droplet sizes down to 0.01 |im, but there is still much skepticism about the validity of data at these low sizes. This is highlighted by the fact that measurements on exactly the same emulsion using a number of different commercial instruments often give very different particle size distributions (Coupland and McClements 1998).

Spectroturbidimetric Methods. These techniques measure the turbidity of a dilute emulsion as a function of wavelength (Walstra 1968, Pearce and Kinsella 1978, Reddy and Fogler 1981, Pandolfe and Masucci 1984). The turbidity (t) is determined by comparing the intensity of light which has traveled directly through an emulsion (/) with that which has traveled directly through the pure continuous phase (/): t = -ln(///)/d, where d is the sample path length. The greater the scattering of light by an emulsion, the lower the intensity of the transmitted wave, and therefore the larger the turbidity. The turbidity of a dilute emulsion is linearly related to the dispersed-phase volume fraction, and so turbidity measurements can be

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