Guest Food Microscopists 3

Home Links updated May 19, 2011.

Ann-Fook Yang   Mark Auty

Ann-Fook Yang, M. Sc.

As the chief technician at the Electron Microscopy Unit, Research Branch, Agriculture and Agri-Food Canada in Ottawa, Mr. Ann-Fook Yang is responsible for smooth operations of the laboratory. He takes care of several electron microscopes and all ancillary equipment and ensures that the microscopes are available to agricultural scientists and technicians in disciplines as diverse as entomology, plant science, and biotechnology. In addition, he also teaches electron microscopy techniques to other scientists' technicians. Such procedures may include the preparation of samples for conventional scanning electron microscopy as well as immunogold labeling of various structures for transmission electron microscopy.

Scanning electron microscopy of hydrated specimens
(Environmental SEM)

Two years ago, the laboratory acquired a new environmental scanning electron microscope for work on hydrated specimens in plant science, entomology, and elsewhere. When checking how such a microscope would produce sharp images of fresh samples, Mr. Yang examined also specimens which may be characterized as foods or closely related to foods.

Vegetables such as cauliflower and broccoli are excellent examples where environmental scanning electron microscopy (ESEM) proves useful in visualizing the developing of the miniature florets as shown below.

Environmental SEM (ESEM) was done using small specimens (less than 4 mm in diameter) placed directly in the well of the cooled microscope stage maintained at +1C and a pressure of 0.6 Torr in the chamber. The cut surface of each specimen was sealed with colloidal graphite to prevent water loss.

The image of cauliflower at left was obtained at 0.6 Torr and 1C. The bar indicates 500 µm. Details of the developing florets are shown at a higher magnification at right (bar = 200 µm). There are many macroscopic images of cauliflower available on the Internet, for example as an illustration to the description of this vegetable also providing advice on growing. The internal structure of a cauliflower head is an example of fractals.

Two micrographs of broccoli, another vegetable of the Brassica genus, are shown above. The bar at left is 1 mm long and the bar at right is 500 µm long;. Broccoli is an example of another kind of fractal. Information on various aspects of growing broccoli may be found on the site of the University of Illinois.

The images of cauliflower and broccoli were obtained at relatively low magnifications. However, ESEM may also be used at considerably higher magnifications to study another group of organisms such as fungi. When fungi, commonly called "moulds", appear on foods, they are the sign of spoilage to most North American and European consumers. Aspergillus, Sclerotium, Rhizopus stolonifer (the bread mould), Fusarium, and Penicillium ssp. are only a few of food-spoiling fungi. A characteristic odour which develops in citrus fruits infected by Penicillium italicum or P. digitatum is known to most consumers who have left a lemon, a grapefruit, or an orange in the refrigerator for too long. Transfer of a small piece of the Penicillium digitatum fungus into an ESEM and examination at high humidity and low temperature inside the microscope makes it possible to quickly obtain images of the hydrated hyphae and spores without any additional preparatory steps (green image below at left).

Fungi (moulds) are not common in North American foods and are mostly limited to Camembert and Blue (mould-ripened) cheeses made with Penicillium camemberti and Penicillium roqueforti, respectively. However, since yeasts such as Saccharomyces cerevisiae also belong to fungi, leavened bakery products such as bread may also be included in the list of foods containing fungi.

In Asian foods, fungi are considerably more common as part of the foods. For example, furu is a traditional Chinese food which has a foie gras-like texture and salty taste. It is made by fermenting soy protein curd (tofu) with Actinomucor elegans, Mucor hiemalis, Mucor silvaticus, Rhizopus chinensis and similar fungi. Monascus purpureus, Monascus anka and similar fungi are used to provide pleasing reddish colouring. Furu is also consumed in Japan.
Pear leaf blister mite and eggs (top micrograph, bar: 200 µm);
detail of mite (bottom, bar: 10 µm)

The thick mat of fungal hyphae (the brown image above at right) was obtained by conventional SEM, the first of the two alternative procedures listed below:
1. Conventional SEM: The mould is gently transferred into an osmium tetroxide fixative which contains a detergent (the spores and hyphae of fungi grown on surfaces are naturally hydrophobic; there is no need to use a detergent when fixing immersed fungi such as in Blue cheese or furu). The fixed specimen is dehydrated in ethanol, critical-point dried, mounted on an SEM stub, coated with gold, and examined in high vacuum at ambient temperature. Handling of the specimen increases the risk that the spores will be lost before photography (there are no spores apparent in the image of furu).
2. Cold-stage SEM (cryo-SEM): The specimen is rapidly frozen in nitrogen slush, inserted into the cryo-stage, fractured, and gold-coated (this all is done in vacuo below -80C) and transferred to another cryostage in a conventional high-vacuum scanning electron microscope for examination.

ESEM was also used to examine what at the first glance appeared as rust on pear leaves. The reddish brown spots, several mm in diameter, are also known to appear as defects on the surface of pears. The pest causing the damage, however, is not a fungus but the pear leaf blister mite. Lifting the blister gently under a dissecting microscope reveals minute mites foraging on the leaf tissue, and also unhatched eggs (top image at left). At a higher magnification, ESEM shows details of the mite's front end (bottom image at left).

Although the micrographs shown do not strictly show the internal microstructure of foods, they demonstrate the potential which this kind of microscopy has for agricultural research, foods including. The original black and white micrographs, obtained in digital form, have been coloured.

The micrographs are protected by copyright. For conditions on their use please contact the author.

A.-F. Yang 2000

Mark Auty, Ph.D.

Mark Auty is a food microscopist with 34 years of experience and is currently a Research Officer at the Dairy Products Research Centre, Moorepark, (a division of Teagasc Co. Cork, Ireland. He has studied a wide variety of foods, mainly for industrial clients, using light and electron microscopy techniques. Recently he has specialized in confocal microscopy.

Confocal Laser Scanning Microscopy (CLSM) of Dairy Products

Milk powder
Whole milk powder (WMP) Dual-stained image showing fat (yellow) and protein (blue). Fat droplets within the particle are typically <2 m.
The microscopic structure of food significantly affects its processing characteristics, flavour properties and texture. Food microstructure studies thus provide a key to understanding, and therefore controlling, food behaviour. Confocal laser scanning microscopy (CLSM) is one of the most useful microscopy techniques for studying the microstructure of a wide variety of foods, in particular dairy products. A primary feature of the CLSM is its ability to "optically section" through samples, giving a 3-dimensional view of a food product with minimal sample disturbance. This feature is of great benefit when examining shear-sensitive samples such as dairy spreads and soft cheeses, where other microscopy techniques would necessitate physical disruption of the sample.

Powdered ingredients are desirable for their ease of use and storage stability. The micro- structure of the individual constituents of the powder will determine it's keeping qualities. For example, many fats are prone to oxidation and therefore need to be incorporated into the powder as very small droplets. CLSM was used to study protein and fat distributions in micro-encapsulated fish oils and milk powders for use in chocolate.

Results indicated that powders with high free fat contents (as determined by solvent extraction) did not necessarily have high levels of surface fat but had large fat droplets within the powder particle. Powders with high surface fat contents dispersed poorly in water, but were ideal for chocolate manufacture, where fat is the continuous phase.

Chocolate Cheddar cheese Probiotic Cheddar cheese
Chocolate Chocolate Chocolate
Milk chocolate Dual-stained image showing protein (green), fat (blue), cocoa solids (red). and sugar crystals (dark green/black). Sugar crystal sizes range from 5 to 25 µm. Cheddar cheese Dual stained image showing continuous protein phase (red) and fat phase (blue). Probiotic cheese A live/dead stain has been applied to determine the proportion and distribution of viable organisms in situ. The cheese is stained pale green, live bacteria are green and dead organisms are red. Bar = 10 µm.

Milk chocolates made with milk powders containing varying levels of free-fat were examined by CLSM. Chocolates made with low free-fat powders had a high viscosity and CLSM showed discrete fat droplets surrounded by protein. High free-fat powders resulted in a more homogeneous distribution of fat as seen by CLSM. The major components of milk chocolate (sugar, fat, protein and cocoa solids) could be simultaneously visualised using the dual staining technique.

Additional information about chocolate may be found at these links:
A history of chocolate
Chocolate through the years
Facts about chocolate
Chocolate university online
Making chocolate at home requires some knowledge of the ingredients and their processing but it is highly rewarding.
Books on sugar confectionery

Cheddar cheese
The microscopic structure of cheese, in particular the protein network, greatly influences physical properties and sensory attributes. CLSM was used to help relate cheese processing conditions to the final product quality.

Cheddar, cream cheese, analogue Mozzarella and Mozzarella cheeses were examined using the new dual staining technique developed for powders. The distribution of protein and fat in each of the cheeses varied depending on the variety. Cheddar cheese consisted of irregular fat droplets surrounded by a protein matrix. Cream cheese consisted of aggregates of small fat droplets and protein. Analogue Mozzarella was an emulsion of rounded fat droplets in a homogeneous protein matrix containing crystals of emulsifying salts. Mozzarella consisted of elongated protein fibres containing small fat droplets with some fat droplet clusters entrapped between protein fibres.

CLSM is particularly useful for microbiological studies, since microorganisms may be examined in their natural state. Viability studies can help to assess changes in microbial populations during ripening. The micrograph of probiotic Cheddar cheese above shows cheese supplemented with probiotic bacteria (Bifidobacterium lactis) to enhance the nutritional properties of the product. Differential staining made it possible to determine the distribution of live (green colour) and dead (red colour) bacteria.

Dr. Mark E. A. Auty
Manager, National Food Imaging Centre
Food Chemistry and Technology Department
Teagasc Food Research Centre
Moorepark, Fermoy, Co. Cork, Ireland

tel: +353 25 42442
fax: +353 25 42340

© Mark Auty 1998