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Microscopic techniques, such as light microscopy, have been used extensively to study the microstructure of food systems since the 1980s. Studying these microstructures in relation to their physical properties and processing behavior provide valuable information for the food industry. Due to the limitations of the visible light on which light microscopy depends, the microstructures of food can be visualized only by cutting thin sections of the food sample. The preparation of the food sample for light microscopy and section cutting can also be very time-consuming. In addition to that, there is a risk of introduction of artifacts into the prepared samples during sample processing which involves various chemicals for fixation and dehydration. Newer microscopic techniques, such as Laser Scanning Confocal Microscopy (LSCM), allow researchers to examine the microstructures of food without the need for tedious sample preparation and cutting thin sections of the food specimen.
Laser Scanning Confocal Microscopy (LSCM)
Unlike the light microscopes that use visible light to illuminate the sample, LSCM instead utilizes a focused scanning laser to illuminate a subsurface of a three-dimensional (3D) specimen. The visual information from that focal plane is then passed back through the specimen and is projected onto the confocal aperture, which is otherwise referred to the pinhole, which is located in front of the detector. In addition to that, samples can be probed with different kinds of fluorochromes, such as rhodamine, fluorescein isothiocyanate (FITC) and Nile red to stain proteins or fat. Labeling the sample with multiple fluorochromes that stain specific components would provide incredible details of the food sample.
Application of LSCM in Food Analysis
LSCM facilitates the examination of microstructures and several 3D food specimens like cheese at various focal planes with minimum to little sample preparation. By adjusting the stage of the microscope, information from several consecutive optical sections of the specimen can be obtained with exceptional lateral resolution. Some of the common applications of LSCM in food analysis include:
Description of Fats in Bulk
Optical sections of high-fat food are difficult to prepare using light microscopy techniques since the sample preparation would result in migration of fat globules. For example, milk fat, which is one of the most common ingredients in many food products has about 400 different fatty acids. The fatty acids present in milk fat have broad crystallization and melting ranges. Researchers have utilized LSCM to study the distribution of microstructures and the rheological properties of various fractions of milk fat. By using LSCM that is equipped with special stages that hold the food samples, the behavior of the fats at various conditions such as temperature changes and agitation can be studied.
Studying Biopolymer Mixtures
Proteins and carbohydrates, like polysaccharides, co-exist in most food samples. Processing technologies such as high-pressure processing are used to pack such biopolymer food materials in a way to extend their shelf life without depleting their nutritional properties. LSCM can, therefore, be used to visualize the behavior of commonly used biopolymer mixtures, such as a skim milk-gelatin mixture, by examining the changes in the microstructures present within the food sample when subjected to various rheological conditions, such as changes in viscosity in relation to changes in pressure and shear rate.
Analysis of Emulsion Systems
Emulsions are biphasic systems where the dispersed phase is distributed in the dispersion medium. For example, in an oil in water (o/w) emulsion, small oil droplets, which would be the dispersed phase, are usually coated with an emulsifier and is dispersed in water, which is considered to be the dispersion medium. The composition of such biphasic systems and the microstructures can be visualized and characterized by using specific fluorophores for proteins and lipids using LSCM.
LSCM, alone or in combination with other microscopic techniques such as light microscopy and polarized light microscopy can serve as a valuable tool in food analysis. The ability of LSCM to examine the internal structure of various 3D specimens at various depths of fields and multiple fluorochrome labeling without extensive sample preparation makes the food analysis less time consuming and more efficient.
Sources and Further Reading
- Cardona, J. A. R., Iriart, C. H., & Herrera, M. L. (2012). Applications of Confocal Laser Scanning Microscopy (CLSM) in Foods. Intechopen. DOI: 10.5772.55653.