Fluorescence microscopy is a commonly employed optical analysis technique, utilizing an external light source to excite and initiate fluorescence in a material.
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A three-dimensional image highlighting the location of excited fluorophores can then be constructed in a layer-by-layer manner using a confocal microscope, elucidating the structure and components of the material. The technique is used in the food industry to ensure consistency of quality and detect contaminants and pathogens.
Why analyze the structure of food?
The microstructure of a food product plays a role in every stage of the production, storage, and transport of said product, in addition to affecting the physical and nutritional properties of the goods and ultimately consumer satisfaction and safety.
Properties of interest directly related to the microstructure of food include the size or length of particles and strands, the flexibility and porosity of the food, and molecular interactions that may take place.
These factors are influenced by the ingredients and preparation methods used during food production, and in turn, go on to affect the quality and stability of the final product. Therefore, food structure is analyzed both for purposes of quality control and in the research and development of new production methodology.
Why use fluorescence microscopy?
Many foods contain innately fluorescent molecules and therefore exhibit auto-fluorescence without the need to add extrinsic fluorophores. Pigments and phenolic compounds such as chlorophyll and lignin are commonly present in plants, while fats such as adipose tissue, bone, cartilage, collagen and elastin are fluorophores common to animal products.
Vitamins A, B2, E, and D are also capable of autofluorescence, among several other minerals and supplemental nutrients. Many of these compounds fluoresce at distinct frequencies, allowing a natural ‘fingerprint’ of the food to be constructed.
For example, meat may contain a high number of autofluorescent tryptophan protein, linoleic acid, and tyrosine residues. Each of these is capable of excitement from light at 280 nm and yet emit at 300 nm, 425 nm, and 350 nm, respectively, due to Stokes shift. This allows the quantity and distribution of these fluorophores to be determined from a single observation.
In cases where the compound of interest is not capable of autofluorescence, a growing library of fluorescent probes is available from which the ideal molecule can be selected to bind with the compound and then subsequently initiate fluorescence. For example, fluorophore Nile Blue is fat-soluble and only fluoresces when in a non-polar lipid environment.
Where far greater specificity is required fluorophore proteins and antibody tags can be selected to identify the presence and quantity of particular nucleic acids or other molecules within the sample. Such detailed examination usually entails the destruction of the food product and application to microarray assays, which are usually labeled with fluorophores only activated when in the presence of the target compound.
These fluorophores may be conjugated to the walls of a stationary array through which the sample must pass, or otherwise to beads capable of suspension and therefore better mixing in a liquid sample.
Specific examples include a sandwich assay developed by Gehring et al. (2008), utilizing fluorescein and Cy3 labeled antibodies to detect immunoglobulin G proteins, E. coli, and S. typhimurium in ground beef, and a suspension array for the detection of organophosphorus and carbamate pesticides using Phycoerythrin, developed by Wang et al. (2014).
When checking for the presence of particular pathogenic microbes genotyping or gene expression analysis is commonly employed in conjunction with fluorescence microscopy, and to this end, a wide and varied number of assays have been developed and are employed as a part of the standard quality control process in every area of the food industry.
Many food industries supplement their goods with antibiotics during the production process, and so constant evaluation of the antibiotic resistance developing in their production chain is necessary. Assays have been developed that detect the presence of many hundreds of genes expected to express when resistance is developing, allowing preliminary steps to be taken.
Some foods may naturally contain compounds that must be monitored, for example, soybeans contain phytoestrogens that exhibit estrogen-like actions once consumed, which although not directly harmful in the quantities concerned, may subsequently interact with the body to produce undesirable results.
Combining confocal microscopy with fluorescence microscopy allows 3D images to be constructed without the need to physically dissect the sample. Unlike some similarly well-resolved microscopy techniques such as transmission electron microscopy, the sample need not be freeze-dried and exposed to a vacuum.
Additionally, the method can be used the monitor the structure of a food throughout the preparation procedure, during the baking of a loaf of bread for example.
An early investigation into the use of fluorescence microscopy during food preparation mixed fluorescein isothiocyanate into bread dough, which bound preferentially with gluten proteins in the mixture, allowing details of bread homogeneity in terms of ingredient distribution and air pockets to be examined.
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