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Review Atomic Force Microscopy Imaging of Food Polysaccharides Food Sci. Technol. Res., 16 (1), 1–12, 2010 Review Atomic Force Microscopy Imaging of Food Polysaccharides * Takahiro FUNAMI Texture Design Division, San-Ei Gen F.F.I., Inc., 1-1-11, Sanwa-cho, Toyonaka, Osaka 561-8588, Japan Received May 4, 2009; Accepted June 3, 2009 Atomic force microscopy (AFM) is a type of scanning probe microscopy that generates images (pri- marily topographical ones) by scanning the surface of samples with a sharp tip. AFM is applicable to samples with low electric conductivities, and its operating range spans that accessible to both light and electron microscopes, allowing for molecular resolution. These features of AFM enable soft materials to be visualized under natural conditions without harsh or damaging procedures. For food polysaccharides, AFM is capable of visualizing not only dispersed molecules but also molecular assemblies with an advan- tage over other physical techniques in quantifying the heterogeneity of samples. From these perspectives, AFM is one of the most versatile techniques for obtaining structural information on food polysaccharides, contributing to the progress of this research area. In this article, AFM images of various food polysaccha- rides are presented along with the usefulness and limitations of this microscopy technique. Keywords: atomic force microscopy (AFM), food polysaccharides, dispersed molecule, molecular assembly, supermolecular structure Introduction but also molecular assemblies or supermolecular structures; Atomic force microscopy (AFM), developed by Binning gel precursors, microgels (i.e., local networks and aggre- and Quate in 1986, is a type of scanning probe microscopy gates), and network structures of bulk gels. Here, food hy- that generates images, primarily topographical ones, by scan- drocolloids are defined as a state of edible colloid particles, ning the surface of samples with a sharp tip attached to a 10-1,000 nm in diameter, dispersing in water as a continuous cantilever. This means that AFM generates images by touch- phase, or, alternatively, polysaccharides and proteins them- ing samples rather than observing them. AFM is applicable selves that regulate such a dispersing state in food systems. to samples with low electric conductivities, differing from a Height and width on the AFM images provide information scanning tunneling microscope, and is free from a diffraction regarding the degree of molecular associations in a quantita- limit, allowing for a molecular resolution up to the sub-nano- tive or qualitative manner. Another advantage of AFM over meter level. These features of AFM enable the visualization other physical methodologies is the ability to directly observe of soft materials, including biomaterials and food materials and quantify the heterogeneity of samples. Food hydrocol- under natural conditions without harsh or damaging prepara- loids are from natural sources in most cases and have com- tive and imaging procedures used in conventional electron plex molecular structures with some heterogeneity, involving microscopy methods, including dyeing, drying, and metal various chain lengths and degrees of branching. From these deposition. AFM imaging works both in air and aqueous en- perspectives, AFM is one of the most versatile techniques for vironments, making it possible to visualize samples in a hy- obtaining structural information on food hydrocolloids, con- drated form such that the samples exhibit behaviors similar tributing to the progress of this research area. to those seen in real systems. Many food hydrocolloids, including carrageenan (Gun- As a tool for the study of food hydrocolloids, AFM is ca- ning et al., 1998; Ikeda et al., 2001), gellan gum (Gunning pable of visualizing not only dispersed or isolated molecules et al., 1996; Ikeda, Nitta, Temsiripong et al., 2004; Mor- ris et al., 1999a), xanthan gum (Camesano and Wilkinson, *To whom correspondence should be addressed. 2001), cellulose (Morris et al., 1999b), pectin (Kirby et al., E-mail: [email protected] 2006; Morris et al., 1999b), gum arabic (Cowman et al., 2 T. FUNAMI 2006; Ikeda et al., 2005), soy soluble polysaccharide (Ikeda contact as the scanning proceeds, realizing high resolution. et al., 2005), curdlan (Ikeda and Shishido, 2005), hyaluro- Contact mode is measurement within the repulsive regime of nan (Cowman et al., 1998), amylose (McIntire and Brant, the intermolecular force curve by keeping the deflection of 1999), starch (Ridout et al., 2002), xyloglucan (Ikeda, Nitta, the AFM cantilever constant. One of the drawbacks of this Kim et al., 2004), and gelatin (Mackie et al., 1998), have operation mode is that the sample is subjected to large lateral been imaged by AFM. Adsorption/desorption of milk-based forces as the tip is dragged over it, sometimes damaging and proteinaceous materials (i.e., β-casein, α-lactoalbumin, and displacing molecules, so called “molecular kicking”, of soft β-lactoglobulin) onto/from the interface of O/W type emul- materials even if the spring constant of the tip is extremely sions (Mackie et al., 2001) and also in-situ monitoring of low. Non-contact mode is measurement within the attractive polysaccharides decomposition with enzymes (Thomson et regime of the intermolecular force curve by keeping the non- al., 1994) are novel and interesting targets for the AFM im- contact distance between the tip and sample constant. In this aging recently investigated. operation mode, the cantilever must be oscillated above the In this article, some AFM images of food polysaccharides surface of the sample at a distance in which measurements are presented along with brief descriptions of this micros- are made outside the repulsive regime of the intermolecular copy technique, including the fundamentals, applications, force curve, resulting in relatively poor resolution. It is also usefulness, and limitations, for the best usage in this research difficult to operate this mode in ambient conditions due to a area. thin layer of water molecules on the surface, which may form a capillary bridge between the tip and sample, causing the AFM basics (fundamentals, applications, usefulness, and tip to “jump-to-contact”. Tapping mode, an intermediate be- limitations) tween the contact and the non-contact mode, detects a weak AFM uses a laser beam deflection system in most cases, repulsive force as a result of tapping the sample surface with where the laser is reflected from the back of the AFM canti- an oscillating AFM tip. The oscillation of the tip is gener- lever onto a position-sensitive detector (Fig. 1). The AFM ated by vibrating the AFM cantilever or the cantilever holder cantilever and tip are usually microfabricated from Si or at the resonant frequency, and utilizing a feedback function Si3N4. When the AFM tip approaches a sample, an attractive in the z-direction to keep the amplitude of vibration constant. “van der Waals” force occurs between the tip and sample, The tip taps the sample surface for a tiny fraction of its oscil- followed by a repulsive force based on the Pauli exclusion lation period, making this operation mode the most suitable principle upon closer approach (Fig. 2). AFM relies on the for soft materials, including food hydrocolloids, due to re- forces between the tip and sample, and these forces, either duced lateral force. The tapping mode is also advantageous attractive or repulsive, are calculated by both the deflection in imaging viscous samples, sometimes leading to poor reso- of the cantilever and its spring constant using the Hookean lution due to adhesion of the sample with the tip. law. AFM imaging modes are differentiated by the force For the visualization of food hydrocolloids, concentra- interaction involved, and three major modes are frequently tions of test solutions usually range from 0.1 to 10 µg/ml used, including contact mode, non-contact mode, and tap- prior to deposition. To observe molecular dispersion or ping mode (or intermediate contact mode); each having isolated individual molecules, very dilute solutions at 0.1 µg/ specific advantages and disadvantages. Contact mode is the ml, for example, should be deposited on a substrate. In most primary and most traditional method for AFM imaging. As the name suggests, the AFM tip and sample remain in close Fig. 1. Principal of atomic force microscopy (schematic). Fig. 2. Measurement of forces in the AFM visualization (schematic). AFM Imaging of Food Polysaccharides 3 cases, samples deposited are then dried in air for immobiliza- specified accessories, thus reducing capillary force. The type tion. During air drying, condensation and displacement of of liquid or solvent used is not an issue as long as the tip and the molecules may occur through so called “molecular comb- sample are completely immersed. Under-liquid imaging is ing”, resulting in associations and aggregations forming in also advantageous in avoiding excessive dehydration dur- situ on a substrate that are not originally present in test solu- ing scanning. In imaging food polysaccharides, n-butanol is tions; known as a “drying artifact”. A single molecule on an the most favorable since it works as a poor solvent to retain image does not necessarily represent a dispersed single mole- sample architectures on a substrate. For example, AFM vi- cule because it may, for example, involve end-to-end type as- sualization under n-butanol shows a different result from that sociations. Two types of substrates are used in most cases as in air though the nature of network formation is substantially a surface; mica and graphite. Mica is more frequently used the same for deacylated gellan gum (1 µg/ml) in the pres- to visualize food hydrocolloids than graphite due to its higher ence 0.01 mM NaCl (Fig. 4). This is attributable to a lower hydrophilicity. Combination of two substrates is helpful in degree of intermolecular associations under n-butanol as a imaging some food hydrocolloids with amphiphilic charac- result of preventing dehydration during scanning as repre- teristics or surface activity. Each substrate should be used af- sented by a decreased vertical height on the image.
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