Microscopy methods are attractive in that they can provide information about the components of food and also evaluate foods for the possibility of food adulteration or fraud. Moreover, they open the possibility to study and investigate the existing relationship between the structure of food components and the physical or functional properties of foods, thus providing means of detecting possible adulteration or any other changes due to either omission or introduction of foreign substances into foods. When conventional microscopy techniques are employed in the analysis of foods, changes in the internal structures of food materials can occur due to the fragility of foods, which is a prohibitive property to the proper analysis using such techniques. To avoid this phenomenon, techniques such as rapid freezing of foods prior to microscopy analysis can be employed to preserve the integrity of foods in terms of their chemical and structural components.
Microscopy methods also play a crucial role in enlarging or magnifying minute structures to make the process of studying them easier. Forensic food microscopy methods are very important in food forensics, because they are not only useful in the identification of foreign substances in foods but also play a significant role in the whole process for the investigation of the food component’s characteristics as well as facts that may be used to determine when and how the suspected fraudulent foreign substances were introduced into the food products. Microscopy methods may be used to determine whether the suspected fraudulent substance was processed together with the food product during the processing or whether it was introduced in post‐production steps in the already finished food product. These methods can also be employed to ascertain whether particular vital procedures in food processing such as pasteurization have been done or performed at the appropriate temperature. Microscopy can be used to identify the match between materials used in a particular food industry, for example, plastics and metal utensils, etc.
In general, food‐based microscopy techniques can provide information about food components and food quality, as well as food adulteration and the presence of potential health hazard ingredients in food.
There are a number of microscopy techniques that are used in food forensics and they include different types of light‐ and electron‐based microscopy techniques. Examples of light microscopy include dissecting microscopy, optical microscopy, bright field microscopy, confocal laser scanning microscopy, fluorescence microscopy, etc. Electron microscopy examples include scanning electron microscopy (SEM) and transmission electron microscopy (TEM), etc. Other types of imaging scanning microscopy techniques used in food forensics are atomic force microscopy (AFM), X‐ray microscopy, microscopy resonance imaging (MRI), positron emission tomography (PET) imaging, etc.
Food samples that are meant for light microscopy analysis normally have to be prepared by employing specific stains and staining techniques to differentiate the various internal microstructures, thus making their identification easier. After staining, the microstructures become more distinct and their morphological characteristics (shape, size and the mutual cell configuration, the presence of crystals, grains, or other elements) can be used for identification and classification. For example, hematoxylin‐eosin (which imparts shades of red coloration) and toluidine blue (which imparts shades of blue coloration) have been used to distinctively reveal microstructures of foodstuffs of both animal and vegetable origin (Pospiech et al., 2011) According to Pospiech et al. (2011), light microscopy techniques can also reveal adulterations due to the presence of one type of tissue into another foodstuff, for example liver tissue in pâtés, salivary glands, head trimming products, natural guts as casings, presence of banned spices, food colors and other food additives.
There are also several staining techniques that are normally employed with food microscopy and they target specific analyses or tissues. For example, Calleja staining (to impart green coloration), blue Trichrome (to reveal re‐processed food products), or using Picrosirius red (Flint and Pickering, 1984), seem to target collagen which forms part of the connective tissue. On the other hand, alizarin red can reveal the presence of bone fragments in foods and Sudan III can indicate the presence as well as the microstructures of adipose tissues.
In the case of plant‐based foods, special staining methods based on polysaccharides have been devised. For example, starches can best be distinctively stained using Lugol‐Calleja stain; also vegetable polysaccharides can be stained using PAS‐Calleja stain (Valchař, 2005). For additives or spices such as black pepper, paprika, caraway seed, marjoram, coriander, allspice, etc., which can suitably be used for verification, purity and identity, can be stained pink by Schiff’s reagent (Pospiech et al., 2011).
The detection of food adulteration or the presence of other health hazard ingredients in food using microscopy methods is possible due to the fact that the basic microstructures of both plant and animal origin foods are known and well documented. Therefore, as long as the microstructures are visible under a light microscope, it is possible to identify the additional or missing microstructures. Microscopy can detect and identify different types of tissues and food components that are characteristic to a certain type or class of foods and this strengthens the technique to be suitably used to detect adulterations or origin of the food.
In the case of the identification of plant‐based components in foods such as the addition of starchy foods, flour. etc., staining can be applied to identify them due to the presence of specific structural features such as polysaccharides, aleurone cells in cereal seed coats, palisade cells included in soya, or reserve protein. Stains that can be used in these cases may include the Lugol‐Calleja stain (which imparts dark purple to black to the starch) or the PAS‐Calleja stain (which imparts a pink color to polysaccharides). The presence of fibers, such as those employed in food industries as additives and which are starch‐based, can be verified by light microscopy based on their distinctive morphological characteristics. They tend to be disordered, lacking any definitive shape. For example, additives based on vegetable protein tend to possess the characteristic shapes of sponges (especially for wheat protein), and sickles or rings due to the presence of saccharides (Flint and Pickering, 1984). It is also important to note that in order to differentiate pure vegetable protein from other proteins, the product basic staining with toluidine blue has to be employed, as it will impart to the vegetable proteins different shades of blue coloration, for example, to wheat protein: light cyan; and to soy protein: dark blue, and this will contrast other microstructures present in the food product (Tremlová and Štarha, 2002; Tremlová et al., 2006). Moreover, other specific microstructures in plant‐based foods, such as palisade cells and cup cells in soybean flour or textured soy protein, or aleurone cells in wheat flour, can be used to indicate any adulteration in food.
It should be noted that when microscopy is coupled to imaging techniques, it can be used for quantitative analyses.
There are different types of light microscopy that are available for food forensics and they are discussed briefly below.
Dissecting microscopes with low‐magnification may be useful in food forensics because they can be used to magnify food structures, thus enabling a better image. For example, they can magnify minute structures in food fibers, additives which are meant to spice the food, contaminants in foods, etc. Dissecting microscopes with high resolution can provide in‐depth information as compared to the low resolution ones, for example, information about the changes on food components that may take place during food processing procedures and information about the cause of such changes.
Like dissecting microscopes, the optical microscopy employs light and lenses to magnify small structures of cells (cell organelles, etc.) or small organisms (single‐celled/acellular organisms). There are different types of optical microscopes that may have applications in food forensics and they are described below.
Polarizing microscopy is a contrast‐inducing microscopy technique mostly used in food forensics to investigate food structure. Polarizing microscopy light that vibrates in a single direction only (polarized light) is directed to bombard the sample specimen. The food sample specimen has to have anisotropic or birefringent structural components that are capable of rotating the light plane. Isotropic structures with only a single refractive index cannot rotate a plane‐polarized light and thus are inactive to polarizing microscopy. For foods with active structures to rotate the plane‐polarized light, a contrast will be obtained due to the fact that the emerging light beam from the sample will be altered, because once it has passed through the sample and rotated, it will be twisted and/or somehow become extinguished to some extent.
In bright field microscopy, species in a food sample that possess chromophores (functional groups having the ability to absorb light that passes through the sample) can be visualized when the sample is bombarded with light (from an incandescent source) and the difference in absorption can be recorded.
Fluorescence microscopy involves illuminating food samples using a specific electromagnetic band of wavelengths and the bands are absorbed by fluorophores in the food sample, causing the absorbing molecules to emit light bands of longer wavelengths. In this type of microscopy, the difference in excitation and emission wavelengths is computed to produce a signal with high contrast between sample and background.
In confocal microscopy, point illumination of the sample is done in various spots in the sample (scanning of the specimen) to enable the creation of an image of the whole sample. Confocal microscopy is thus useful in cases where an understanding of the performance of the food products as well as its ingredients during processing is needed, because it enables a real‐time visualization of even complex microstructures of food samples. It is also the technique of choice for the simultaneous visualization of food in a process that incorporates the addition of specific dyes to enable the identification of the food components’ microstructure in foods such as protein, fat and starch granules. In this case, this technique is suitable for the understanding of the distribution of the ingredients within the microstructure in various food products. Confocal microscopy can also be used to track changes in food microstructure that take place during food processing or even during food eating. This is due to the fact that food processing conditions have some control in the stability of the food products. The technique can also be useful to study the distribution and microstructures of food flavorings and biopolymer additives that enhance the flavor and texture of foods, such as emulsifying additives, stabilizing additives, carbohydrates, or thickeners to detect any possible adulteration or fraud.
Electron microscopy is one of the techniques that has wide applications in many disciplines, including its use in the evaluation of the microstructure of food as well as other biological products. In electron microscopy, electron beams are used to irradiate the sample specimen and this prompts the need for appropriate sample preparation methods for each food type or for the type of information needed. Various electron microscopy techniques use different principles and are useful for particular analyses to reveal different kinds of information needed from the sample. These techniques include transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo‐SEM, cryo‐TEM, and environmental scanning electron microscopy (ESEM).
In electron microscopy, unlike the light microscopy techniques, a beam of electrons (negatively charged particles that behave like radiation with very short wavelengths) is used to illuminate the sample instead of light. In this technique there is thus a requirement for high vacuum inside the microscope. Electron microscopy is more superior and advanced than light microscopy, as it is capable of providing a much higher magnification at far better resolution than with light microscopy. The main limitation of electron microscopy is that it is more expensive, both in terms of procurement of the machine, running costs and costs of sample analysis.
For the purpose of food forensics, SEM and TEM are the major electron microscopy modes mostly needed. The modern scanning electron microscopes can find applications in the investigation of fragile food samples that are hydrated, such as moulds (fungi and their spores), fruits and vegetables, without the need to dry or freeze the samples (Katsaras, 1994).
However, when using traditional electron microscopy, there are certain requirements that must be observed and these include the fact that the food samples in question should not release any gas or vapor when introduced into either the transmission or scanning electron microscope. Moreover, any food sample that contains water (hydrated) must be dried or frozen at a very low temperature (mainly at –100 °C) in order to eliminate any possibility of the food sample releasing gas or vapor. The exceptions to these include powdered foods samples such as maize/wheat flour (or any food flour), sugar and powdered milk.
Conventional scanning electron microscopy (SEM) is a technique that reveals the surface chemistry of the food sample specimen and can thus point to any deviations from the norm in the case of adulterations. Food samples (previously dried) are normally coated with carbon, gold or metal to dissipate charges and provide a path for the electrons, thus enhancing electron absorption by the sample microstructures.
When a beam of electrons has been directed towards the sample specimen, the resultant scanned image that is generated is normally formed step by step and so it is not just a one‐step process. In the first step, the primary electrons that are directed towards the sample are deflected by elastic scattering processes (Bogner et al., 2007). The use of SEM in food forensics is attractive due to the many advantages that SEM offers, which include its relatively simple sample preparation procedures, wide range of magnification, high depth of field and also the image that is generated is a representation of electronic data that enables both image analysis and quantification options to be performed simultaneously (Aguilera and Stanley, 1999; Fazaeli et al., 2012).
Cryo‐scanning electron microscopy (cryo‐SEM) is one of the variants of SEM that is used to observe and investigate ice crystals in frozen samples, even without the need for thawing (Aguilera and Stanley, 1999; Fazaeli et al., 2012). Unlike conventional SEM, analysis involving cryo‐SEM requires more sample preparation steps, such as coating with metal in order to facilitate and enhance the conductance of electrons (Preetz et al., 2010). The use of cryo‐SEM is attractive in food forensics, as it is not associated with the potential presence of artefacts as in other SEM techniques (Preetz et al., 2010).
The environmental scanning electron microscope (ESEM) is another variant of SEM techniques that are used in food forensics. It is attractive because it allows even wet samples to be analyzed, without any appreciable prior sample preparation such as drying or dehydration, but samples can be analyzed in their very original state. This technique is useful in the in situ dynamic testing of foods. For example, it can show ageing of foods to detect any mislabeling of expiry dates, changes of food components with time, etc.
Unlike conventional SEM, ESEM differs, requiring the presence of a gas in the sample specimen chamber such that samples are not viewed under high vacuum but rather under a diminished vacuum (Fazaeli et al., 2012). The presence of the gaseous environment around the sample is vital as it acts as an electrical charge conductor avoiding sample charging and therefore facilitates signal detection and also acts as a conditioning medium, which will not allow the evaporation of liquids from the sample (Thiel and Toth, 2005).
Due to the difficulties in sample preparation of viscous food specimens such as butter, stirred yogurt, different types and forms of creams, mayonnaise, etc. with tiny microstructures for SEM analysis, approaches that involve concentrating the individual specimens in agar gel capsules have been suggested by Salyaev (1968) as the solution to this limitation. The steps for sample preparation of viscous foods specimens for SEM analysis can be obtained from articles published by Sayaev (1968) and Kalib (1988), etc.
In certain foods such as milk, saturated fat that is present in milk fat globules tends to crystallize at refrigerator temperatures and does not interact with osmium tetroxide or stains. In order to distinguish it from unsaturated fats (when using SEM), some techniques need to be followed that from involve the use of osmium tetroxide, which is buffered by imidazole to make it possible to distinguish the two types of fats by light, color and also by the sharpness of crystalline outlines from unsaturated lipids. However, the sample has to be rapidly dehydrated in a graded ethanol series, and some solvents such as chloroform or n‐hexane should not be used.
In conventional transmission electron microscopy (TEM), the operation has to take place under vacuum to prevent the deflection of electrons by gas molecules. When the sample has interacted with a beam of electrons and the electrons are scattered and thereby are creating a contrast, it then results in the generation of an image. The TEM technique is useful in the understanding of internal structures of food samples; tissues, etc. and thus it can be suitable to reveal changes in food samples that can provide evidence of the effects of the treatment processes or adulteration practices that may affect the structure of foods (Katsaras, 1994).
The general protocol for sample analysis using TEM starts with fixing the tissue glutaraldehyde, dehydrating in alcohol, embedding in plastic by immersion in a solution and polymerized by heating, cut into ultrathin sections of approximately 50–10 nm with an ultramicrotome, and staining the sample with heavy metals for contrasting purposes. This technique is applicable for direct analysis of colloids in the vitrified frozen hydrated state and also in those instances where information about the internal structure of the colloidal system is needed (Kuntsche et al., 2011).
The use of TEM for food samples involves a number of sample preparation techniques prior to the introduction of the sample to the instrument. These methods and techniques include:
In this method, the sample specimen of food is embedded in a particular resin, sectioned by means of a microtome, stained using heavy metal salts (e.g. metal salts of lead or osmium) and introduced into a TEM instrument for analysis.
Alternatively, the sample can be subjected to negative staining, a process which involves mixing of very small amounts of food samples such as milk products (e.g. casein micelles, etc.) with solutions of heavy metal salts on thin electron transparent films and then drying and introducing into a microscope.
Another TEM sample preparation procedure that is used for TEM analyses is known as metal shadowing, which involves drying the food samples on a translucent film and then shadowing with platinum vapor under vacuum. It is then introduced into a TEM instrument for analysis.
Another sample preparation technique for samples that are to be analyzed using TEM is freeze‐fracturing and freeze‐etching, which can be performed without the need for chemical fixation alteration or physical alterations (e.g. drying, embedding in a resin, expelling water/dehydration, etc.). This process, however, involves rapid freezing of the food sample and then freeze‐fracture is at low temperatures below –110°C. The sample will then be thawed before analysis with a TEM.
Encapsulation techniques can be employed for food samples in both SEM and TEM. However, much smaller capsules are required for samples that are to be investigated using TEM as compared to the case with samples for SEM. For the latter technique, considerably smaller capsules would be required than for SEM. The details of this procedure can be obtained in other publications (Kaláb and Larocque, 1996; Veliky and Kaláb, 1990).
Fat foods that are to be analyzed using TEM have to be fixed using appropriate reagents. For example, proteins need to be fixed using glutaraldehyde, while post‐fixation using osmium tetroxide followed by glutaraldehyde is suitable for unsaturated lipids present in foodstuffs. In this post‐fixation process of unsaturated lipids in foods, there is a high possibility of the fatty acids unsaturation points having chemical reactions with osmium tetroxide (through the double bonds) and converting the fatty acids to saturated ones. To avoid this, the hydrolysis step is normally carried out immediately to get rid of the osmium tetroxide (Allan‐Wojtas and Kaláb, 1984; Angermüller and Fahimi, 1982; Greyer, 1977).
Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) are useful in food forensics for the investigation of nano‐ and micro‐structures for authenticity and detection of adulterations and contamination cases in foods. EELS can be used in conjunction with transmission electron microscopy (TEM‐EELS).
Other imaging methods, apart from light and electron microscopy, which have found application in food forensics, include the scanning probe microscopy (SPM) techniques (including AFM and SPM), magnetic resonance imaging (MRI) and acoustic microscopy.
Scanning probe microscopy (SPM) is one of the microscopy technique variants useful in creating the images of sample surfaces using a physical sharp probe that scans the surface of the specimen being investigated. SPM comprises of a range of techniques which all employ a scanning sharp probe in close proximity to the surface of the sample, to provide measurements of certain parameters in relation to the distance between the sample material and the probe. The SPM family of techniques include atomic force microscopy (AFM) and scanning tunnel microscopy (STM).
Atomic force microscopy (AFM) is a technique that reveals the surface of the sample (micro‐ and nano‐structures in foods, biological structures such as tissues, cells, bio‐molecules) in three dimensions. This technique is based on the measurement of interactions between a sharp tip and the surface of the sample (Binnig et al., 1986) and in the process, the surface is scanned using a mechanical probe known as a cantilever and the deflection of this mechanical probe is detected by a focused and reflected light beam that strikes a position sensitive detector (PSD).
Unlike optical and electron microscopy techniques, which generate images in two dimensions as they cannot provide a vertical dimension (Z‐direction) measurement for the sample (only height (for particles) and depth (holes, pits)) measurements, AFM can provide three dimensions (horizontal X‐Y plane and the Z‐vertical plane).
In food forensics, AFM is useful for the investigations of microscopic food components before, during and after food processing, to see whether the surface topography properties that influence textural characteristics of food change and the effect of processing on surface topography.
There are three main imaging modes used in AFM operations, which include contact mode, non‐contact mode, and tapping mode.
In the contact imaging mode, electrostatic and/or surface tension forces from the adsorbed gas layer pull the scanning tip toward the surface and the whole process takes place using a tip connected to the end of a cantilever. Since the operation under contact imaging mode applies a lot of force towards the sample, there is a high possibility of resulting in distorted or low‐quality images, especially when dealing with soft samples (Power et al., 1998).
Non‐contact AFM imaging mode has limitations in that it results in images of low resolution due to its mode of operation.
In tapping AFM mode, the cantilever oscillation amplitude and phase is measured while the feedback is maintained to keep the oscillation amplitude fixed such that the amplitude, phase and topography can be imaged simultaneously. Tapping AFM imaging mode is known to provide high resolution and is not associated with the application of destructive frictional forces to the samples, whether in air or fluid medium. The mode is appropriate for even very soft and fragile samples, as well as structural components of the membrane. Thus for food forensics, this will present the most suitable mode of operation, despite its limitation as being somehow slower when compared to a contact mode.
For example, AFM can be used to study polymer and polymer matrix as well as biological structures such as applications in distinguishing proteins and polymers in foods after different types of treatment and how the microstructures change (Gunning et al., 2004; Woodward et al., 2004); monitoring of how macromolecules change with different types of food manipulations (Yang et al., 2006); and analysis of gene location after fixing DNA in polymer‐coated glass substrates (An et al., 2005; Nakao et al., 2002).
AFM has the potential for use in the simulation process of creating nanostructures from individual atoms or molecules in self‐assembly procedures to create what is envisaged at the moment to be nanofoods. AFM can facilitate in the simulation, optimization and characterization of nanofoods to make the whole process more feasible, economical and practical.
In the scanning tunneling microscopy (STM) technique, the current of electrons generated by atoms at the tip of the physical probe are tunneled to the surface of the sample specimen to create an image of the sample’s surface topography. One requirement for STM is that both sample and tip must be good conductors, a condition which prevents the analysis of many samples of biological origin.
Magnetic force microscopy (MFM) is normally used to measure magnetic force associated with magnetic materials.
Scanning capacitance microscopy (SCM) is used to ascertain capacitance developed in the presence of tip near sample surface for conductors and solids.
Stylus profilometer (SP) is used to study and investigate surface properties for conductors, insulators, semiconductors and solids.
Magnetic resonance imaging (MRI) is a technique that is based on nuclear resonance magnetic spectroscopy, which can be used to detect concentrations of nuclei with spins such as that of a proton, phosphorus, nitrogen, etc. This technique can thus be applied to investigate the measure of the distribution of water and lipids in foods.
X‐ray microscopy is a non‐destructive technique that takes into account all the advantages of the magnifying power of optical microscopy and combines it with the penetrating power of X‐rays, to generate high‐quality two‐dimensional images of minute microstructures. It also has advantages associated with the capabilities of 3‐D tomography to generate high resolution 3‐D images of the sample.
A number of microscopy‐based techniques have the potential to provide solutions to food forensics issues; however, care should be taken by the analysts to match correctly and appropriately these techniques to appropriate properties of the food sample composition that is being investigated, the nature of the sample and its matrix environment and other practical requirements. These techniques can be used successfully to investigate the basic structural properties of different kinds of foods and the differences within and between varieties, origin, the effects of processes such as different kinds of drying (hot air drying, spray drying, microwave, osmotic drying, freeze drying and superheated steam drying), freezing, high hydrostatic pressure, pulsed electric fields, and ultrasound on foods microstructure. These techniques can also be used to reveal the relationship between food processing parameters, conditions and morphological changes of the food components. However, in as much as there are a number of state‐of‐art microscopy related techniques that may be applied to provide solution to food forensic cases, this can be of value only if scientists or technicians operating these equipment/instruments are capable of manipulating the techniques and also capable or encoding the results generated (Ramírez and Aguilera, 2011).