SCANNING ELECTRON MICROSCOPY

Scanning electron microscopy (SEM) has been comprehensively applied to the analysis of food structure since the 1960s and earlier applications are well summarized in a number of books, book chapters (Aguilera and Stanley 1999; Holcomb and Kalab 1981), and review articles (Heertje 1993; Holcomb 1990; Sargant 1988). Unlike transmission electron micros-copy (TEM), the sample for SEM does not have to be electron transparent, removing many of the sample preparation difficulties inherent in TEM. However, until relatively recently, SEM (like TEM) has been a technique that required the sample be exposed to high vacuum conditions.

The basic components of a conventional SEM are shown in Figure 3.5 with the enlarged area highlighting the aspects of the technique that provide both its main advantages (high resolution, high depth of field) and disadvantages (high vacuum required, conducting specimen best). SEM is a high-resolution imaging technique, the ultimate resolution being dictated by a number of parameters from the electron source to the sample itself. Image contrast in SEM is created by one of two mechanisms, the creation of back scattered elec-trons (BSE) or secondary elecelec-trons (SE). BSE contrast is sensitive to both local topography and atomic number (Z) variation, the BSE coefficient, η, rising with Z. SE are almost insen-sitive to atomic number but are seninsen-sitive to local topography, the SE coefficient, δ, rises as

Food MiCRostRuCtuRe AnAlYsis

the angle of the incident beam to the surface decreases. BSE have higher energy (up to the energy of the incident beam) and as such can emerge from deeper within the specimen, SE are low energy electrons and can only escape from within a few mean free path interactions of the surface, this means the resolution and surface sensitivity of an SE image is higher than that of a BSE image. Figure 3.6 shows the effect of these two contrast mechanisms.

As η + δ< 1 more electrons are injected into the specimen than are emitted, as such a conductive path must be made from the sample surface to ground to avoid sample charg-ing. Even with a field emission source, that can operate at low accelerating voltage hence

Electron gun

Figure 3.5 Basic components of a conventional scanning electron microscope.

Figure 3.6 SEM images of “honey-roasted” cashew nut showing effect of contrast mechanism on image formation. Contrast in SE image (left) arises predominantly from topography, whereas con-trast in BSE image (right) arises from a combination of topography and atomic number concon-trast. As such the NaCl present (example highlighted by arrow) in the flavoring appears brighter than the sugar crystals (example highlighted by “thumbtack”).

lower electron “dose” to the sample, insulating materials are best imaged with a conduc-tive coating. This is usually a sputter-coated layer of platinum, gold, or gold-palladium a few nanometers thick. The interaction of the beam with the sample does not only generate an electron signal, it can also damage the sample through thermal effects, called “beam damage.” Beam damage occurs during imaging as the beam scans across an area of the sample and can be minimized by lowering the electron dose (by working at lower acceler-ating voltage or lower magnification). Beam damage can be dramatic and fast, and as such easily recognized as an artifact, or more subtle, in which case it is good practice to work at variety of magnifications to check the impact of the beam on the specimen (Figure 3.7).

The need for a high vacuum in the sample chamber has led to various techniques for imaging high vapor pressure food materials, mainly based on fixing and drying. Any dehydration of a specimen will introduce artifacts by the simple act of removing the water.

Shrinkage is the main artifact but the surface tension created as water evaporates can mechanically damage delicate structures. Critical point drying and freeze drying offer alternate routes for sample preparation that introduce less shrinkage and can preserve more delicate features, but still change the structure of the food material considerably.

Figure 3.8 illustrates this with an example of freeze-dried carrot compared to an LM image of the same material. Both CPD and FD usually require the sample to be “fixed” in some way, for example, using glutaraldehyde, to immobilize components.

3.3.1 Cryo-SEM

To avoid the need for drying and fixing, samples can be prepared by rapid freezing and examined in the SEM in the frozen state using a cold stage. This is cryo-SEM and preserves the structure of a sample in a state far closer to the spatial arrangement found in the native Figure 3.7 SEM image of dried uncooked pasta showing beam damage. Beam damage was caused by imaging at higher magnification so the beam only rastered across the indicated area, as such electron dose was high in this area and thermal damage caused the swelling and blistering shown.

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state. Cryo-SEM requires specialized accessories to a conventional SEM, usually consist-ing of an ex situ freezconsist-ing station, a cryo-transfer device to keep the sample under vacuum while moving it to the SEM, and a cold stage able to maintain cryogenic temperatures. Ice crystal formation during the freezing step is inevitable unless special steps are taken to produce glassy ice (high-pressure freezing or introducing cryo-protectants) (Echlin 1992).

The freezing step needs to be as fast as possible to produce micro-crystalline ice and the most common method for this is plunging the sample into liquid nitrogen slush (never simply liquid nitrogen as the cooling rate would be far too slow). Cryo-SEM can preserve very fine features and, when coupled with a Field Emission Gun SEM, is a high-resolution technique (Figure 3.9).

Figure 3.8 Comparison of micrographs from LM and conventional SEM. (a) SEM image of freeze-dried carrot; carrot cubes were freeze freeze-dried and coated with platinum prior to imaging, (b) LM image of fresh carrot; thin sections were hand-cut using a razor blade and stained with toluidine blue (0.05%w/v in 20 mM sodium benzoate buffer, pH 4.4). (Image courtesy of Bronwen Smith, University of Auckland).

Figure 3.9 Cryo-SEM image of apple parenchyma cells. Sample was plunge frozen in liquid nitro-gen slush, fractured under vacuum, sublimed at −95°C for 10 min then sputter coated with gold.

Many cryo-transfer systems include a freeze-fracture capability so that internal struc-tures can be revealed. This is usually located in a chamber off the main sample chamber so that sample temperature can be controlled for limited sublimation of the ice. This is known as “etching” and is used to reveal structures within the water phase. It also reveals the most distinctive artifact of cryo-SEM: ice crystal ghosts. As the micro-crystalline ice grows during freezing, as it will to some extent unless glassy ice is produced, components of the food are excluded from the liquid phase during solidification. The network structure that is revealed on sublimation is very distinctive (Figure 3.10) and care should be taken when interpreting structural information as a result.

All the advantages of conventional SEM are preserved with cryo-SEM, including the high resolution and great depth of field. The disadvantages are also preserved, with beam damage being exacerbated by the presence of water (Walther et al. 1995). One of the main disadvantages of cryo-SEM and conventional SEM with a dried specimen is that the sam-ple is literally or figuratively “frozen,” meaning that dynamic processes need to be inter-rupted to follow progress (Harker et al. 2006; James and Fonseca 2006). The newer variable pressure SEM instruments offer a complementary answer to this last issue.

3.3.2 Variable Pressure SEM

Developments in the 1970s led to new SEMs that could operate with higher pressures in the sample chamber than previously (Danilatos and Robinson 1979; Moncrieff et al. 1979), the first commercial instrument (the Electroscan Environmental SEM or ESEM), becoming available in the 1980s. Due to the fact that this design was purchased and widely commer-cialized by Philips Electron Optics (later FEI) who continued to use the ESEM nomencla-ture these instruments are often collectively referred to as ESEMs. The more generic name is variable pressure SEM or, awkwardly, low vacuum SEM.

Figure 3.10 Cryo-SEM image of banana skin. Sample was plunge frozen in liquid nitrogen slush, fractured under vacuum, sublimed at −95°C for 30 min then sputter coated with gold. Network structure within cells (highlighted with arrow) is a result of ice crystal growth during freezing.

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Variable pressure SEM (VP-SEM) currently refers to instruments that can operate with sample chamber pressures up to around 2500 Pa (20 Torr), whereas conventional SEMs operate at pressures of around 10⁻⁴ Pa (10⁻⁶ Torr). The development and details of these instruments have been thoroughly reviewed elsewhere (Danilatos 1993; Donald 2003;

Stokes 2003) but a brief summary of these details is warranted here so operators in the food engineering space can be aware of any limitations.

The vacuum management that allows the sample chamber (and the sample chamber only) to be at variable pressure is summarized in Figure 3.11. By mounting the sample on a peltier cooled stage the relevant portion of the water phase diagram can be accessed, allowing water to be liquid in the sample chamber. While pressures of up to 2500 Pa (20 Torr) are easily achieved by bleeding some water vapor into the chamber realistically, at these pressures, imaging is challenging so frequently, to keep samples fully hydrated, it is useful to cool them to 4–5° and operate at slightly lower pressure.

The presence of gas in the sample chamber requires alternative detectors to those used in high vacuum. Each manufacturer has used a slightly different approach but in every case the gas is an important part of the imaging mechanism. This is most easily explained when considering the FEI design of “Environmental SE detector.” The primary beam is scattered to some extent by the gas but the majority of the beam still lands in the original

“spot” meaning resolution does not suffer. The SE that are generated interact with the gas, as indicated in Figure 3.12, create an amplification cascade of “environmental” electrons that are subsequently detected by an annular solid state detector. This means that the imaging gas selection is a critical part of imaging in these conditions as the ionization cross-section of the gas will alter the amplification of different parts of the signal (Fletcher et al. 1999). Fortunately, water vapor is an excellent imaging gas for most purposes; nitrous oxide has a similar ionization potential and can be usefully employed for imaging low

High vacuum

pumping Rough vacuum

pumping

Pressure-limiting Apertures

Imaging gas ~~5Torr

Peltier-cooled stage Sample

Figure 3.11 Differential pumping and a series of pressure-limiting apertures allows the sample in a VP-SEM to be at pressures up to 20 Torr, while maintaining the electron column at high vacuum (around 10⁻⁹ Torr).

water activity foods at slightly subzero temperatures, such as chocolate (James and Smith 2009).

Many of the applications of VP-SEM to food structure have been reviewed elsewhere (James 2009) though often details of sample preparation and imaging conditions make some reported studies difficult to interpret. In general, the sample preparation for VP-SEM for food materials is relatively straightforward. Samples are not fixed and not coated for VP-SEM as the positive ions generated during the ion cascade in the imaging gas suppress sample charging. To examine internal structures, a sample section can be prepared by fracturing (which will follow natural lines of weakness) or cutting (which can introduce knife marks and crushing artifacts). This is simple with brittle foods but nearly impos-sible with soft foods, which are better fractured in the cryo-stage and examined using cryo-SEM.

3.3.3 Dynamic and In Situ Studies Using VP-SEM

The sample in VP-SEM is far closer to its native state than in the other iterations of SEM.

As such it can be manipulated to follow dynamic changes in structure. Dehydrating/rehy-drating processes are an obvious area to exploit the technique’s ability to move around the gas–liquid isotherm. Drying studies of apples (Chen et al. 2006) and studies of rehydration behavior of carrots (Smith et al. 2007) have both been followed in real time in the VP-SEM.

SE and electrons from ionization events are

accelerated toward detector

Solid-state electron detector

Further electrons generated from ionization events in imaging gas-amplifying SE

signal

generated from beam-SEs specimen Positive ions from ionization

events are accelerated toward the sample–suppressing

sample charging

Figure 3.12 The gas in the sample chamber of a VP-SEM is part of the imaging system. Secondary electrons ionize the gas amplifying the electron signal and simultaneously suppressing sample charging.

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The scattering of the primary beam in the VP-SEM can introduce artifacts in studies that rely on close measurement of swelling during hydration, through two thorough studies (Tang et al. 2007a, b) give reasons why any introduced errors are negligible.

Heating studies are possible in the VP-SEM, the detectors are not “blinded” by infra-red radiation as are those of conventional SEM and hot stages are available that go to 1500°C. However, the pressure limitation means that to maintain a wet sample temper-atures are not yet achievable above around 20–30°C. As such the idea of watching the changes during, say, baking of bread is not yet possible, whereas the melting of chocolate is quite accessible.

Mechanical deformation is possible with in situ straining stages (Figure 3.13) (Donald et al. 2003; James and Yang 2012; Thiel and Donald 2000). The challenge for these experi-ments is the limited geometry of many SEM sample chambers and substages mean that an overly long working distance limits the pressure that is achievable with good image

Figure 3.13 VP-SEM sequence of images from in situ tensile test of bovine M. semitendinosus.

(Image courtesy of Seo Won Yang, University of Auckland.)

quality. It is also worth remembering that the sample will still need to be in a controlled temperature environment, for example, by resting against a peltier stage, which may intro-duce nonuniform stress conditions.

Contrast variations in images can result from localized electronic properties of the sample, and these can be exploited in the VP-SEM for imaging emulsions. The source of the contrast is a combination of intrinsic SE emission characteristics and extrinsic charg-ing artifacts (Stokes et al. 1998, 2000), which can be deliberately induced by operatcharg-ing with barely enough gas pressure to suppress all sample charging. Figure 3.14 shows this effect exploited to image a sample of mayonnaise.

VP-SEM is not free of artifacts, beam damage will still be an issue (though the mecha-nism is not simply thermal, but includes hydrolysis damage from hydroxyl free radicals (Royall et al. 2001)). In many cases, the major impediment to successful VP-SEM imaging is the one thing that makes VP-SEM an attractive technique in the first place. The ability to keep water in its liquid state is vital to maintaining a sample at close to native state, but water is just as likely to obscure the details of interest. This is simply illustrated by watch-ing NaCl go into solution and recrystallize (Figure 3.15). The liquid phase is not transpar-ent as the imaging energy is not visible light.