Linda L. Leake

Many analytical tools used for years by other industries are now being employed by or adapted for use in the food industry. Here are three emerging techniques that are revolutionizing how food scientists analyze products and ingredients.

Microbiologists observe the display of a confocal microscope being used to examine an alfalfa sprout root that has been experimentally contaminated with Salmonella. The microbes are identified by green or blue dots on the computer screen.

Atomic Force Microscopy
The atomic force microscope (AFM) is a type of scanning probe microscope that has a high resolution of fractions of an Angstrom. Invented in 1986, it is considered one of the foremost tools for imaging, measuring, and manipulating matter at the nanoscale level.

"We have probably created or utilized nanostructures for a long time when producing foods, but we simply didn’t have the tools to analyze and observe them," says Jochen Weiss ([email protected]), Assistant Professor, Food Biophysics and Nanotechnology, Dept. of Food Science, University of Massachusetts, Amherst. "Now, with atomic force microscopy, we know what they are and we can look at them."

The AFM generates images by "feeling" the surface of the sample; it senses the changes in force between the sample’s surface and a probe attached to the microscope as the sample is scanned.

"Much as a visually challenged individual can generate images of surface shape, hardness, and stickiness by touch, so the AFM can produce images of surface topography, adhesion, elasticity, or charge," Weiss explains.

Commercial AFMs can produce submolecular resolutions of samples in gaseous or liquid environments, and under favorable conditions it is even possible to image molecular processes in real time.

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The instrument consists of a microscale cantilever with a sharp tip (probe) at its end that is used to scan the specimen surface. The cantilever is typically made from silicon or silicon nitride and has a tip radius of curvature on the order of nanometers. When the tip is brought into proximity of a sample surface, forces between the tip and the sample lead to a deflection of the cantilever.

Typically, the deflection is measured using a laser spot reflected from the top of the cantilever into an array of photodiodes.

"Atomic force microscopy is a unique tool that allows us to see structures, surface properties, and interactions we can’t normally see," Weiss says. "One example is nanoencapsulation systems for bioactive compounds whose sizes are too small to visualize. Because they are hydrated, electron microscopy cannot be used. Another application is the identification of growth and transfer of biofilms and microbiological colonies from processing to food surfaces."

Weiss and his colleague, Lynne McLandsborough, use the CP II AFM manufactured by Veeco Instruments Inc., Woodbury, N.Y. (phone 516-677-0200,, to investigate how biofilms can be removed from surfaces using combinations of antimicrobials and surfactants.

AFM is also being used in the development of new food packaging materials. "Thanks to this technology, the properties of nanocomposite polymers that have improved barrier properties can be optimized," Weiss points out. "These new materials could be used to make containers microwavable by eliminating the usually needed aluminum layer."

While AFMs originally cost about $250,000, they have become more affordable, with some costing $80,000–85,000, Weiss says.

"Because of its tremendous usefulness and increasingly competitive pricing, the use of atomic force microscopy will become more widespread in the food industry in the not-too-distant future."

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Confocal Laser Scanning Microscopy
Another emerging technique, confocal laser scanning microscopy (CLSM), was developed in 1953 and became a standard analytical technique in the late 1980s. However, only recently has it been used in the food industry.

Michael Tunick ([email protected]), a research chemist with the U.S. Dept. of Agriculture’s Agricultural Research Service, employed CLSM to determine the effects of pressing procedure and storage conditions on the rheology and microstructure of queso blanco, a popular Hispanic-style cheese.

"With CLSM, it’s possible to see structure changes created by changes in abuse and the time," he says. "With cheese, one can tunnel through layers and visualize the size and arrangement of fat globules."

Using a confocal microscope manufactured by Leica Microsystems, Wetzlar, Germany (phone +49-6441-29-0,, Tunick determined that queso blanco’s protein matrix became less continuous and the distribution of fat droplets became more extensive during storage.

"These results were consistent with microstructural breakdown due to proteolysis," he says.

Additional applications of CLSM in the food industry include analyzing heterogeneous emulsion mixtures such as gravy, thickeners, and chocolate, as well as plant structures, including tissues and skin.

The key benefit of CLSM, Tunick says, is its ability to produce blur-free images at various depths in thick specimens. The images are taken point-by-point and reconstructed with a computer, rather than projected through an eyepiece.

First, a fluorescent dye is added to the sample to be analyzed. Fluorescent dyes can be selected to produce a color contrast between component of a specimen. For example, one might, use green for protein and yellow for fat. Then a laser beam is passed through an aperture and is focused by an objective lens into a small volume of the sample. A mixture of emitted fluorescent light and reflected laser light from the illuminated spot is then collected by the objective lens.

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"As the laser scans over the plane of interest, a whole image is obtained pixel by pixel and line by line, while the brightness of a resulting image pixel corresponds to the relative intensity of detected fluorescent light," Tunick says. "The beam is scanned across the sample in the horizontal plane using one or more oscillating mirrors."

This scanning method usually has a low reaction latency, and the scan speed can be varied, he points out. Scans conducted at slow speeds provide a better signal-to-noise ratio, resulting in better contrast and higher resolution. Information can be collected from different focal planes by raising or lowering the microscope stage. The computer can generate a three-dimensional picture of a specimen by assembling a stack of these two-dimensional images from successive focal planes.

"Confocal microscopy provides a significant improvement in lateral resolution and the capacity for direct, non-invasive serial optical sectioning of intact, thick living specimens with a minimum of sample preparation," Tunick adds. "The ability to visualize heterogeneous samples at the sub-micron level allows food researchers to better understand the physical processes taking place in food."

Liquid Chromatography–Mass Spectrometry
A third technique, liquid chromatography–mass spectrometry (LC–MS), combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. It separates compounds chromatographically before they are introduced to an ion source and mass spectrometer. The mobile phase is liquid, usually a combination of water and organic solvents. The sample is ionized and the ions are separated according to their mass. The relative abundance of the ions is recorded by measuring the intensities of the ion flux. Finally, mass spectrometry measures the mass-to-charge ratio of the ions as a means of identifying the compounds.

Liquid chromatography with ultraviolet detection has been used as an analytical technique in the food industry for many years, but the addition of detection capabilities of mass spectrometry is relatively recent, says Navindra Seeram ([email protected]), Assistant Director, Center for Human Nutrition, University of California–Los Angeles and Adjunct Assistant Professor at UCLA’s David Geffen School of Medicine.

"Foods contain multiple compounds in a complex matrix," he says, "and while some compounds don’t respond to some detectors, LC–MS effectively fragments each compound into its own molecular mass. This capability has enabled us to detect a larger number of compounds, as almost all compounds will respond to MS."

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An LC–MS analysis can be run with a sample of just 20 μL or less. Other benefits include increased sensitivity and greater detection power.

"We can extricate complicated food matrices with LC–MS," Seeram says. "The technology can create detailed fingerprints of the phytochemical components of foods."

This information offers the ability to establish a library of food components that explains how their characteristics change over time and in the presence of various factors, including temperature variations and processing techniques, he adds.

"MS has revolutionized our understanding and knowledge of what’s in food. Clearly, LC–MS is a powerful technique offering high sensitivity and specificity."

Seeram uses the LCQ Classic and Advantage Finnigan Thermo-Electron LC mass spectrometers manufactured by Thermo Fisher Scientific, Waltham, Mass. (phone 781-622-1000,, for his research on in-vitro and in-vivo evaluation of foods, herbs, and dietary supplements intended for the prevention and treatment of cancer, cardiovascular, and neurodegenerative diseases.

"Generally, LC–MS is oriented toward the specific detection and potential identification of chemical compounds in the presence of other chemicals in a complex mixture," he points out. "Because LC–MS can be used to identify almost anything in the food matrix, it can distinguish foods that may be mislabeled or contaminated."

MS has several applications relative to food, including identifying unknown compounds by analyzing the mass of the compound molecules or their fragments; determining the structure of a compound by observing its fragmentation; quantifying the amount of a compound in a sample; and determining other physical, chemical, or even biological properties of compounds.

"While still expensive, this technology provides a utility, power, sensitivity, and uniqueness no other food analysis tool can match," Seeram says.

by Linda L. Leake,
Contributing Editor,
Food Safety Consultant, Wilmington, N.C.
[email protected]