Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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0011 In freeze-drying, wet samples are fast-frozen, trans-

ferred to a freeze-drying unit where a low tempera-

ture is maintained (typically 80



Cto90



C) and

the ice is sublimed slowly under low vacuum. Careful

control over the different stages, including initial

freezing, temperature, and vacuum of drying and, in

particular, of the rate of rewarming, has proved to be

important in retaining the microstructural features of

food materials.

0012 Unfortunately, these drying techniques are not

readily adaptable to fatty foods, because solvent

dehydration will remove the fat and freeze-drying

will not adequately stabilize it. Many investigators,

however, have found the techniques successful, for

example, in the study of foods containing carbo-

hydrate or protein gels and ‘fully biological’ tissues,

including meat, vegetable, and fruit.

Low-temperature SEM (Cryo-SEM)

0013 One way to overcome the problems of conventional

drying techniques is to make use of low-temperature

preparation procedures to examine frozen, fully

hydrated food materials. If the temperature of the

sample is sufficiently low (i.e., < 130



C), there

will be negligible freeze-drying in the vacuum

system of the SEM; typically, samples are held at

c. 170



C during imaging. No chemical fixation or

solvent dehydration needs to be involved as the

sample is ‘cryofixed’ (physically preserved). The tech-

nique has been used widely since the early 1980s in

various biological applications, including in the

examination of food.

0014The procedure involves rapid freezing of the speci-

men before transfer on to the precooled stage of a

cryopreparation unit, which may be either dedicated

(attached permanently to the SEM) or nondedicated

(free-standing) vacuum equipment. The sample is often

freeze-fractured in the unit to reveal internal structure.

When particularly high levels of water are present,

fractured and original surfaces can be ‘etched’ by rais-

ing the specimen temperature while it is still under

vacuum so that a thin layer of surface ice is sublimed

away to reveal more of the topographical detail.

0015As with low-temperature preparations for TEM, one

of the most crucial stages is in the initial freezing of the

specimen. It is desirable that the water present is solidi-

fied in a vitreous, noncrystalline state and the

remaining components are preserved in the same form

as they were originally. However, the relatively large

size of samples typically used for SEM prevents vitrifi-

cation of all but a very thin layer at the surface. In

practice, the aim of the freezing process is to render

the size of the resulting ice crystals as small as is prac-

ticable. Low-temperature SEM is particularly suitable

for examination of liquid or semisolid materials such as

margarines, low-fat spreads and dressings, as well as

foods with high levels of crystalline fat such as choc-

olate (Figure 2) and frozen foods such as icecream.

Auxiliary Techniques

X-ray Microanalysis

0016The principle of X-ray microanalysis is related to

the emission of X-rays from a specimen through

1 µm

fig0001 Figure 1 Spoilage yeast (Brettanomyces sp.) isolated from a

soft drink and prepared using critical-point drying. Bar ¼1 mm.

Reproduced from Microscopy: Scanning Electron Microscopy.

Encyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

10 µm

fig0002Figure 2 Fat bloom crystals (form VI) on the surface of a plain

chocolate bar examined using low-temperature scanning elec-

tron microscopy. Bar ¼10 mm. Reproduced from Microscopy:

Scanning Electron Microscopy. Encyclopaedia of Food Science,

Food Technology and Nutrition, Macrae R, Robinson RK and Sadler

MJ (eds), 1993, Academic Press.

3924 MICROSCOPY/Scanning Electron Microscopy

interaction of the electron beam. When any material

with an atomic number of four or more is examined

in an electron microscope, X-rays are generated from

just below the surface. As each element has a unique

X-ray spectrum, the elemental composition can be

determined on the basis of detected X-rays. X-ray

microanalysis systems are of two types: energy-

dispersive X-ray analysis (EDX) and wavelength-

dispersive spectroscopy (WDS). EDX is the more

frequently used technique for biological and food-

type samples. This technique measures the energies

of X-rays and provides analysis of all detectable

elements simultaneously. By contrast, WDS measures

the wavelengths of X-rays with individual crystals,

each of which focuses on one element at a time.

WDS has a better resolution than EDX but is less

easy to use.

0017 In the past, routine EDX detectors have been pro-

tected by beryllium windows that have excluded

transmission of X-rays with an energy of less than

c. 1 kV, i.e., the X-ray energies of elements with an

atomic number below 11 (sodium) and, traditionally,

for the detection of lighter elements, WDS has

been used. However, improved detector design and

the use of plastic windows has enabled the full

range of elements to be detected by routine EDX

systems and WDS is used for specialized analyses.

Through computer interfacing, the X-ray microana-

lytical technique can be used to generate quantitative

data and produce elemental distribution ‘maps’ of

food materials. X-ray mapping techniques have

already provided valuable information relating to

chemical changes in food that have been associated

with their processing, storage, and packaging

(Figure 3).

Cathodoluminescence

0018Certain materials emit long-wavelength photons in

the ultraviolet and visible regions of the spectrum

when they are irradiated with a beam of high-energy

electrons and this phenomenon is termed ‘cathodo-

luminescence.’ Although this technique is widely used

for geological applications, with recent developments

in detector design it has the potential to be applicable

to a wider range of materials, possibly including

foods. Novel treatments could include labeling spe-

cific structures with reagents that exhibit cathodo-

luminescence, much in the same way as fluorescent

labeling is used for light microscopy.

Image Analysis

0019SEM produces electronic/digitized images and as such

they are available for computer manipulation, for

example, in image processing or analysis. Image pro-

cessing involves computer filtration and enhance-

ments to improve the final image quality from the

microscope and this type of facility is often a standard

feature on modern instruments. Image analysis is used

to obtain quantitative data directly from the images,

for example, relating to size and size distribution and

shape of features as well as relative areas and coinci-

dence measurements. Many purpose-built image

analyzers are available commercially with which

analysis of any image source, e.g., objects, photo-

graphs, light microscopy, and TEM images, as well

as SEM images, can be undertaken. The basis for

discrimination of features/areas of black and white

images, such as SEM images, is the difference in gray

levels (differences in black to white) across the image.

SEM images often cause problems because of the very

wide range of gray levels within any one image, al-

though use of high-contrasting techniques, in particu-

lar backscattered imaging, can help eliminate the

problem.

Applications of SEM

0020Many food laboratories have their own scanning elec-

tron microscope or have direct access to one and the

use of the technique is commonplace in the majority

of investigations on food. Applications are many and

diverse, covering practically all of the different food-

stuffs and their ingredients. The strength of SEM in

food studies is its wide magnification range, large

fig0003 Figure 3 (see color plate 108) Digital X-ray map showing

calcium distribution (of calcium phosphate) in frozen hydrated

cheddar cheese.

MICROSCOPY/Scanning Electron Microscopy 3925

depth of focus, and its ease of use. The relatively

straightforward preparation of dry materials has led

to the extensive use of SEM for the characterization

of powders, for example, spray-dried ingredients

(Figure 4), flour and sugars, as well as dried foods

such as coffees and teas. Comparative information is

obtained on the gross size and shape of the particles

and, by breaking them open, details of the internal

microstructure can be determined, for example the

form and interaction of individual components, in-

cluding crystalline inclusions and air bubbles.

0021 SEM images often provide information that can be

directly related to textural properties of food in a

unique way and can also be related to processing

information as well as sensory panel data. Examin-

ation of different types of bakery products, for

example, can reveal the form of the starch, in particu-

lar the intactness of starch granules together with the

levels of association of the protein strands (Figure 5).

Such detail can often be related to the way in which

the product is likely to break down in the mouth and

explain terms such as ‘crumbly’ or ‘brittle.’ (See

Starch: Structure, Properties, and Determination.)

0022 Many food-manufacturing processes involve pro-

duction of a number of relatively unstable, liquid, or

semisolid intermediate products prior to the finished

commercial sample. SEM, frequently in combination

with low-temperature preparation techniques, pro-

vides a way of monitoring the changes taking place

during the development of the final microstructure

and helps to investigate the effects of different pro-

cessing conditions and/or ingredients. Its use has been

particularly valuable, for example, following crystal-

lization of key food components such as sugar or fat

in different foods, including changes in size and shape

of individual crystalline regions.

0023A significant area of food microscopy is concerned

with the identification of contaminants in food, for

example, fragments of glass, metal, paints or insects,

which are introduced accidentally or deliberately

during and after production. The use of SEM, in

combination with EDX, provides a rapid way of

identifying the composition of contaminants, which

can be quite small (< 1 mm). The technique is non-

invasive and thus the evidence is not destroyed.

(See Contamination of Food.)

Recent Developments and Specialized

SEM Techniques

Low-vacuum and Environmental SEM

0024Although conventional SEMs offering the best

resolution operate at high vacuum, they suffer

from limitations in that the specimens intended for

examination have to be rendered stable, so that

they are able to withstand the vacuum, and elec-

trically conductive. Accomplishing this without

altering the structure is difficult and, clearly, delicate

hydrated or volatile samples often present in food

systems are not vacuum-tolerant. Operation of an

SEM at higher pressures opens up the possibilities of

being able to examine a wide range of vacuum-

intolerant specimens without prior preparation. Not

10 µm

fig0004 Figure 4 Spray-dried particles of polydextrose. Bar ¼10 mm.

Reproduced from Microscopy: Scanning Electron Microscopy.

Encyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

10 µm

fig0005Figure 5 Fracture through the center of a semisweet biscuit

showing intact starch granules (S) and fine strands of protein (P).

Bar ¼10 mm. Reproduced from Microscopy: Scanning Electron

Microscopy. Encyclopaedia of Food Science, Food Technology and

Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993,

Academic Press.

3926 MICROSCOPY/Scanning Electron Microscopy

only can hydrated and semisolid samples be exam-

ined without prior preparation, the gas ionization

that occurs with increasing pressure in the sample

chamber effectively neutralizes charge that accumu-

lates on the sample and eliminates charging artifact.

The consequence of this is that a conductive layer of

metal does not need to be applied to electrically non-

conductive samples when they are examined at higher

pressures.

0025 SEMs that operate at higher pressures are now

commercially available and are termed low-vacuum

SEMs (LV-SEMs) or variable-pressure SEMs (VP-

SEMs). A further category can provide (in addition

to variable pressures) a saturated water vapor envir-

onment and this is termed environmental SEMs

(ESEMs). All of these achieve their ability to produce

a source of illuminating electrons from normal elec-

tron gun systems whilst maintaining a sample cham-

ber at low (i.e., poor) vacuum by separating different

regions of the instrument by small, pressure-limiting

apertures.

0026 Because they lend themselves to the examination

of nonconductive samples without the need for

coating, LV-SEMs, VP-SEMs, and ESEMs are now

increasingly being used to examine different food

systems. Examples of specimens suitable for examin-

ation with this type of instrument include finished

products and samples taken from different stages of

food processing as well as for the investigation of

complex structures. Novel approaches to the prepar-

ation of samples can yield further information, e.g.,

exposing a lipid-containing matrix, such as a biscuit,

to osmium vapor which will bind to the lipid phase

preferentially and can then be contrasted by back-

scattered electron imaging. Further applications

include samples immunolabeled with gold or silver

probes and also dynamic experiments, such as

following the crystallization of products in an

ESEM.

0027 Demonstration of elemental composition of un-

fixed, uncoated samples by X-ray microanalysis is

possible, although (because of the higher pressure)

the spatial resolution of the X-ray signal is reduced.

However, mathematical corrections have been de-

veloped by some workers and at least one major

manufacturer of X-ray microanalysis systems offers

such algorithms in its software.

High-resolution and Low-voltage SEM

0028 Resolution is largely dependent on the diameter of the

scanning spot of electrons used to examine the speci-

men. However, with decreasing spot size there is

decreasing signal and, with conventional SEMs, the

signal-to-noise ratio becomes unacceptably poor at

very high magnifications. With decreasing accelerat-

ing voltage there is a concomitant decrease in signal

so, traditionally, relatively high (> 20 kV) accelerating

voltages have been used for SEM examination of

specimens. However, reducing the voltage has a

number of advantages. There is a significant reduc-

tion in the build-up of charge and, with reduced beam

penetration, fine detail on the surface of samples is

revealed.

0029With the increasing availability of instruments

equipped with a field emission gun (FEG) and ad-

vanced electron optics, the opportunity now exists

for the examination of specimens with high-intensity

electron beams with small spot sizes over a wide

range of accelerating voltages (including very low

voltage).

0030High-resolution cryo-stages have been developed

to complement this range of FEG-SEMs so that frozen

samples can be examined at high resolution. There is

now considerable overlap between the information

that can be obtained from fractured frozen samples

examined by high-resolution SEM and that obtained

from traditional freeze-fracture studies made by

TEM. Often, larger areas of a sample are available

for examination by SEM, when compared with

complementary TEM freeze-fracture studies, and

interpretation of the results can be less difficult. How-

ever, in both cases, the quality of freezing is para-

mount and normally it is only a small region close

to the edge of a sample that will have been frozen

sufficiently rapidly to be comparatively free of ice

crystal artifact.

0031Applications that are particularly suitable for

examination with such instrumentation include

emulsions, foams, liposomes, microorganisms, and

food systems where interfaces are of interest.

0032There is an additional category of instruments

(ESEM-FEG) that exists that combines the ability to

operate at high resolution and high gun intensity with

the capability of operating at higher pressures and

relative humidity. These instruments offer an enor-

mous range of different operating modes to suit dif-

ferent sample types. However, this flexibility comes at

a price and the principal limitation is the cost of

purchase.

Specimen Preparation

0033Significant improvements have been made in both

instrumentation and electronics in SEM over recent

years. In applying the technique more widely to

food studies, however, the limitations probably still

lie in the area of presentation/preservation of the

specimen to insure that the microstructure is retained

in its original form when examined by SEM. It is

MICROSCOPY/Scanning Electron Microscopy 3927

this area of the technique where most developments

are likely, particularly in the use of environmental or

LV-SEM or VP-SEM, low-temperature preparation

procedures, and also in high-resolution cryogenic

techniques.

0034 When the first scanning electron microscopes were

available commercially, the appeal of a three-dimen-

sional image led to the rapid production of numerous

micrographs of food materials with perhaps only

limited scientific value. Now that this period is over,

an increasing number of workers are using the fuller

potential of SEM and it has emerged as an important

research tool both in its own right and as a comple-

mentary technique to other microscopical techniques

in microstructural investigations of food and food

processing.

See also: Analysis of Food; Contamination of Food;

Starch: Structure, Properties, and Determination

Further Reading

Cohen SH (1984) Scanning electron microscopy in the

analysis of food microstructure: a review. In: Charalam-

bous G (ed) Analysis of Foods and Beverages: Modern

Techniques, pp. 421–449. Orlando: Academic Press.

Goldstein JI, Newbury DE, Echlin P, Joy DC, Fiori C and

Lifshin E (eds) (1992) Scanning Electron Microscopy

and X-ray Microanalysis. New York: Plenum.

Heathcock JF(1988) Cryotechniques in the examination of

food. Institute of Physics Conference Series no. 93 3(2):

55–57.

Holcomb DN and Kalab M (eds) (1981) Studies of Food

Microstructure. Scanning Electron Microscopy.

Chicago, USA: AMF O’Hare.

Kalab M (1983) Electron microscopy of foods. In: Peleg M

and Bagley EB (eds) Physical Properties of Food, pp.

43–94. Westport: AVI.

Sargent JA (1988) Low temperature scanning electron

microscopy: advantages and disadvantages. Scanning

Microscopy 2: 835–949.

Transmission Electron

Microscopy

J H Holgate and J Webb, Reading Scientific Services

Ltd, Whiteknights, Reading, UK

Introduction

0001 Many investigators have studied the textural behav-

ior of numerous products using physical measure-

ments and showed good correlation with sensory

evaluations of texture and mouth feel. Microstruc-

tural analysis now seems to be playing a key role in

explaining the type of data produced. It is being used

increasingly in the study of a wide range of food

products and ingredients, with the data obtained

being related to a sample’s textural properties and in

some cases to its perceived flavor. Such studies can

help in the overall understanding of the effects of food

processing. (See Analysis of Food; Sensory Evalu-

ation: Sensory Characteristics of Human Foods.)

0002The first commercial transmission electron micro-

scope was available in the 1940s when it was recog-

nized that significant improvements in resolution

could be made over the light microscope through the

use of an electron beam rather than a light beam.

However, the instrument could only operate under

vacuum and required ultrathin specimens for the

beam to pass through. Thus, specialized preparation

techniques had to be developed to insure that micro-

structural and ultrastructural details of specimens

were adequately preserved during each of the pre-

parative stages.

0003In the early years, the application of transmission

electron microscopy (TEM) to the food industry

lagged behind that in metallurgical and biological

fields. This was partly due to problems in specimen

preparation and handling that were specific to food

materials, arising from the lack of homogeneity and

often the presence of high levels of water, sugars, or

fats.

Principles

0004A hairpin tungsten filament of an electron gun, en-

cased in a column at high vacuum, is heated to gener-

ate a cloud of electrons, which is accelerated through

a hole in an anode to produce a narrow beam that is

directed towards the specimen. By using electromag-

netic lenses to focus the resultant beam, an image can

be formed by projecting the electron beam through a

thin specimen. The first instruments produced magni-

fications up to 10 000 but subsequent development

of electromagnetic lens systems and guns capable

of accelerating voltages exceeding 100 kV and up

to 400 kV on standard TEMs has extended the

magnification range up to 1 000 000 or more,

with resolutions of better than 0.2 nm possible on

modern instruments. As the human eye is not sensi-

tive to electrons, the final image is visualized by being

focused on to a fluorescent screen or directly on to a

photographic plate or film.

0005TEM operates under high vacuum (< 10

5

mm Hg

or < 10

4

Pa) in order to minimize the scattering of

the electron beam by gas molecules and allow it to

3928 MICROSCOPY/Transmission Electron Microscopy

travel through the microscope without contamin-

ation. This brings with it the physical problems of

having a specimen which must withstand exposure

to high-vacuum as well as high-voltage electron

beams.

Preparation Techniques

0006 A number of different techniques are available for

the preparation of specimens for TEM. The choice

depends very much on the nature of the food, for

example, whether it is liquid or solid or whether it

contains high levels of water, fats, or sugars, which

may make it difficult to handle during the various

preparative stages. In all cases, however, the basic

requirement is to produce an ultrathin preparation

that allows an electron beam to pass through it,

whilst at the same time being dry and resistant to

the high vacuum and potential high temperature of

a high-voltage beam.

0007 Food often presents particular difficulties because

of its heterogeneity and, in many cases, the presence

of relatively high levels of fats and sugars, often in

combination.

0008 The most frequently used TEM preparation tech-

niques that have been applied fairly successfully to

different food materials are negative staining, thin

sectioning, and freeze-fracture replication, with the

latter two being used most often.

Negative Staining

0009 Negative staining represents one of the easier and

more rapid techniques in terms of specimen prepar-

ation but is limited in food applications to studies of

dilute suspensions or dispersions, for example, food

proteins, liposomes, cellular fragments, liquid crys-

tals, or microorganisms. It cannot be used for large

or more complex food systems.

0010 In this technique the particulate or colloidal com-

ponents are examined directly on a TEM specimen

support grid after they have been surrounded or

embedded in an electron-dense ‘stain.’ The technique

relies on the metallic stain producing an outline of the

structures, rather than reacting positively with them,

thus providing information on overall size and shape

and detail of features such as bacterial flagella. Phos-

photungstic acid (PTA) is one of the negative stains

used most commonly (Figure 1), although some

success has been achieved with uranyl acetate, par-

ticularly for the examination of food protein

macromolecules. A third reagent, ammonium molyb-

date, has been used, but with less predictable results,

and has been shown to be particularly sensitive to

localized pH changes.

0011There was a renewed interest in the use of negative

staining in the mid 1980s as it became possible to use

it in combination with low-temperature TEM prepar-

ation techniques. Fully hydrated, negatively stained

preparations are fast-frozen and then examined by

TEM on a low-temperature (below  150



C) stage.

This approach has been particularly successful in the

examination of viruses and single-cell preparations,

but has been of limited use to date in studying food

components.

Thin Sectioning

0012Probably the most frequently used preparation

procedure for TEM is thin sectioning, where the

specimen is initially preserved chemically or ‘fixed,’

solvent-dehydrated, and then infiltrated and embed-

ded in a resin or other polymeric material. On poly-

merization (usually by high-temperature curing), the

resin hardens so that ultrathin slices or sections

(< 100 nm) of the embedded material can be cut

using an ultramicrotome.

0013Among the various chemical fixatives currently in

use, glutaraldehyde, followed by osmium tetroxide

), remains one of the most effective. However,

a number of different fixative regimes have been

developed for different food systems, often involving

alternative aldehydes or mixtures of aldehydes and

using temperatures, osmolarities, pH values, and

buffers appropriate to the food. Osmium textroxide

has the advantage of being uniquely suitable for fats

and thus has been used particularly for foods contain-

ing high levels of fat (e.g., oil seeds, emulsions,

chocolate). It also functions in its vapor form and,

therefore, is useful as a primary fixative to stabilize

the specimen prior to further treatment. As well as

fixing the sample, chemical bonding of the osmium to

the unsaturated fats produces electron-dense ‘stained’

regions in the preparations. Extended fixation times

are often employed for fatty foods, although these are

often a compromise, as they have sometimes been

shown to produce artifacts in the nonfat areas of the

specimen.

0014Many different types of polymers have been used as

embedding materials over recent years. Epoxy resins

andmethacrylates areperhapsusedmost frequentlyfor

food materials, particularly lower-viscosity polymers.

A number of different resins were developed during

the 1970s and 1980s. Initially they offered particular

advantages of very low viscosity, and thus more rapid

and efficient infiltration, and were thought to be safer

to handle. Unfortunately, a number of the compon-

ents of the resins were still found to be unsafe, having

carcinogenic properties in particular. However, a

range of new acrylates became available in the

1990s, which are not only considered safer to handle

MICROSCOPY/Transmission Electron Microscopy 3929

but have relatively low viscosities. Some of these can

be polymerized at low temperatures by ultraviolet

curing, though care has to be taken to prevent local-

ized heat build-up as the accelerator is activated.

Thus, the problems of exposing materials to high-

temperature polymerization conditions are avoided.

Other polymeric materials available are miscible with

water and thus eliminate the solvent dehydration

stages. However, these have been of only limited use

as they often produce fairly brittle embedded blocks,

which are difficult to section.

0015 When food samples are in liquid or semisolid form,

there is obviously a special requirement to keep the

sample intact during the various preparative steps.

This is achieved by some means of encapsulation

and a number of workers have used a modification

of the Salyaev technique with considerable success in

preparing milks, fruit juices, cell suspensions, and

other liquid food materials. The principle is to

prepare a capillary tube of agar that is filled with

the sample and sealed at both ends with further

agar, before being put into liquid fixative and pro-

cessed as for other materials.

0016 The embedded materials, usually in the shape

of small tubes or blocks, are sectioned using an

ultramicrotome to produce ribbons of ultrathin

sections measuring between 50 and 100 nm. Trad-

itionally, freshly cleaved glass knives are used as

they have very sharp cutting faces. However, dia-

mond knives have increased in popularity over recent

years, as they remain sharp for extended periods of

time and thus are suited to hard materials, e.g.,

woody tissue, bone, and crystalline components.

Their main disadvantage is price, including the high

cost of resharpening.

0017Cut sections are collected on meshed support grids

designed to fit the TEM holders and then examined in

the microscope, usually after staining to enhance the

contrast of the sample and specifically identify

individual components. Contrast in a TEM image is

a result of differential electron opacity and therefore

the most effective stains are based on reaction with

electron-dense materials, in particular those with

the highest atomic numbers, e.g., inorganic stains

containing heavy metals.

0018Traditionally, staining has involved an initial reac-

tion with uranyl acetate followed by lead citrate to

provide a fairly nonspecific staining of the whole

sample. This approach is still used routinely for

many food specimens, with subsequent identification

of components based mainly on their characteristic

morphological features rather than their reaction to

100 nm

fig0001 Figure 1 Negatively stained preparation of material isolated from a comminuted fruit drink showing the presence of hesperidin

crystals (H), chromoplasts (C), and membraneous vesicles (V). Bar ¼100 nm. Reproduced from Microscopy: Transmission Electron

Microscopy, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic

Press.

3930 MICROSCOPY/Transmission Electron Microscopy

stain. However, many different staining regimes have

been developed for specific components, e.g. proteins,

carbohydrates, and polysaccharides, in a wide range

of different foods. Frequently these have been based

on traditional biochemical reactions, but for TEM the

staining compound includes a heavy-metal salt rather

than a chromophore (coloured moiety). (See Carbo-

hydrates: Determination; Protein: Determination and

Characterization.)

Low-Temperature Preparation

Techniques

0019 For specimens which contain high levels of water or

which are liable to change significantly during fix-

ation or embedding procedures, preservation of the

ultrastructure can be achieved with a cryofixation

step, i.e., rapid freezing of small samples. This

preserves the natural three-dimensional structure

and solidifies the material so that it can subsequently

be sectioned or fractured to reveal its internal

structure.

Cryosectioning

0020 Cryosectioning (sectioning at low temperatures)

of frozen materials can be used for biological and

biomedical tissues. It involves the controlled

sectioning of frozen material using an ultramicrotome

at low temperatures. Sections can be examined dir-

ectly on a low-temperature TEM stage or after freeze-

drying. However, there are few published data on the

successful application of this technique to foods be-

cause of the difficulty of cutting ultrathin frozen

sections of food materials and handling them prior

to examination.

Freeze-Fracture/Freeze-Etch Replication

0021The alternative to sectioning frozen material is to

fracture the sample and reveal its internal structure.

Combining freeze-fracture with metal shadowing/

carbon replication techniques has proved a par-

ticularly useful procedure for preparing many differ-

ent foods for TEM, including emulsions, foams, and

fatty confectionery, as well as frozen products such

as icecream (Figure 2). This procedure is referred to

as freeze-fracture or freeze-etch replication. One

of the critical stages in the procedure is achieving

rapid and efficient freezing of the sample to prevent

the formation of visible ice crystals or other arti-

facts and considerable efforts have been made to

optimize this process. Nitrogen ‘slush,’ i.e., a mix of

liquid and solid nitrogen (210



C), is probably the

1 µm

fig0002 Figure 2 Freeze-etch preparation of icecream showing the presence of ice crystals (I), air bubbles (A), and fat droplets (F) within an

aqueous sugar matrix. Bar ¼1 mm. Reproduced from Microscopy: Transmission Electron Microscopy, Encyclopaedia of Food Science,

Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

MICROSCOPY/Transmission Electron Microscopy 3931

most frequently used cryogen, as it is relatively easy

to prepare and use. However, faster freezing rates

are possible with liquid propane and hexane and

these are now being used increasingly, particularly

in combination with purpose-built freezing appar-

atus that are designed to enable safe handling of

these flammable materials. Commercial develop-

ments in this field include specialized apparatus

for cold metal block freezing (‘slamming’) and

spray jet freezing. However, these are relatively

expensive and have been of limited application in

food microscopy.

0022 Some success has been achieved with the use of

cryoprotectants, for example glycerol, which can be

used to infiltrate TEM specimens and inhibit the

development of ice crystals on freezing. However,

the interaction of the chemical has been shown to

change the fine structure of samples and is now used

more as a complementary technique in low-tempera-

ture preparations. Certain foods have been shown to

offer an advantage in achieving fast freezing as they

provide their own cryoprotection with the presence of

high levels of sugars or salts.

0023 Once the frozen samples are transferred on to

the cold stage of a freeze-fracture/etch unit, they are

fractured under vacuum and a carbon/platinum

replica is prepared of the frozen fractured

surface. Replication is achieved by the evaporation

of a thin supporting layer of carbon followed by

platinum deposited at an angle to produce a ‘shadow’

and hence reveal the topographical details of the

surface.

0024 For samples with high levels of water present,

‘freeze-etched’ replicas are made by the sublimation

or ‘etching’ of a thin layer of surface ice before

replication. After replication, samples are removed

from the unit and the replicas are then cleaned in

detergent and/or solvents to remove the food sub-

strate before TEM examination. The choice of

cleaning regime is important in order to clean the

replica adequately whilst at the same time preserving

its integrity. Strong caustic solutions have proved

useful for many food materials, often combined

with detergent or organic solvent washes to remove

the fatty components.

Freeze Substitution

0025 Freeze substitution involves the replacement of water

(ice) in a frozen preparation with an organic solvent

and subsequently with resin. There has been renewed

interest in the technique recently because of its

suitability for preparing biological tissues for

immunolabeling. However, it is still of only limited

application to food materials.

TEM Attachments

Scanning Unit

0026Many modern TEMs have the facility for attachment of

a scanning unit to the main instrument. The unit allows

the beam to be scanned across a selected area of the

specimen and with the addition of suitable detectors

the electron microscope can be used in a surface

imaging mode or in a scanned transmission mode

(STEM). In surface imaging mode, high-resolution

images of the surface of samples can be obtained,

although the small sample chamber restricts the size

of sample that can be examined. The primary applica-

tion of STEM is in imaging thicker samples. Since the

images produced are electronic/digital, they have the

advantage that they can be subjected to image process-

ing and analysis to enhance the information further.

X-ray Microanalysis

0027Interaction of an electron beam with a specimen

produces X-rays which are characteristic of its elem-

ental composition. These X-rays can be collected to

provide qualitative and quantitative chemical data

from a sample. The technique is applicable to both

transmission and scanning electron microscopes and

is dealt with in more detail in the preceding article

Microscopy: Scanning Electron Microscopy. In TEM

its main application is in determining the distribution

and changes in distribution of specific elements

within thin-sectioned preparations. It has been of

limited value for food samples to date, as the fixation

and embedding techniques required for adequate pre-

servation of microstructure frequently result in redis-

tribution of elements within the material.

Applications of TEM

0028Many different food materials have now been exam-

ined using TEM. However, its main use has been in

terms of characterizing the ultrastructure of different

foods and relating this information to process changes

and to textural and other physicochemical properties.

Early studies involved the use of thin-sectioning tech-

niques on foods with relatively high levels of plant or

animal tissue, for example, meat, fruit, and vege-

tables. These studies were able to demonstrate the

cellular changes associated with processing and

cooking fresh tissue and included the use of specific

staining regimes to monitor chemical changes such as

the integrity of plant cell walls, meat fibrils, etc. More

recent studies tend to use a combination of micro-

scopy techniques to study the changes in more detail

and to characterize more complex, composite mater-

ials, such as many confectionery products (Figure 3).

3932 MICROSCOPY/Transmission Electron Microscopy

0029 Studies on liquid or semisolid foods, for example,

milk, emulsions, and fruit beverages, have increased

significantly with the introduction of low-temperature

techniques for handling and preparing these types of

material. It has been possible, therefore, to identify

and characterize the individual components present,

for example, size and shape of fat droplets, form and

level of aggregation of proteins (Figure 4), and to

evaluate changes arising as a result of processes

(heat, shear, pH changes, etc.), ingredients (different

fats, protein source, etc.) or recipes.

0030 The use of TEM for examining dry materials, for

example milk powders, has been mainly superseded

by the introduction of scanning electron microscopy

techniques. However, it has been found that the use of

thin sectioning, and more especially carbon replica-

tion techniques for TEM, has produced valuable

information on the ultrastructural detail of these ma-

terials, in particular, the location and appearance of

protein, which was not possible in any other way.

Future Developments in TEM of Food

Materials

Immunolabeling

0031 Colloidal gold immunolabeling is now probably the

most highly acclaimed technique for electron immu-

nocytochemistry. Gold spheres, bound to selected

antibodies, are incubated with the relevant receptor

to effect high-resolution and highly specific labeling.

The technique is now enhanced by application of

multiple labeling of different antigens using differ-

ent-sized gold particles and quantitative techniques.

However, it has yet to be used extensively for food

materials. This is due to the problems of raising

and isolating antibodies specific to food components,

particularly after the foods have been processed,

combined with the overall difficulties in handling

food for TEM.

Low-Temperature Preparation Techniques

0032The use of low-temperature preparation procedures

has increased significantly over the last few years.

However, considerable advances have been made in

faster freezing methods, alternative ways of handling

frozen materials, e.g., sectioning, complementary

fracturing techniques, and combinations of fractur-

ing/sectioning/labeling, which have mainly been

applied to nonfood materials. It is likely that these

will be applied increasingly to studies on food.

Scanning Probe Microscopy (SPM)

0033This is a developing field and the techniques relevant

to the microscopy of foods are centered on using

scanning tunneling microscopes (STM) and atomic

force microscopes (AFM). They provide high-

resolution visualization of surface morphology and

2 µm

fig0003 Figure 3 Thin section of milk chocolate showing the close association of milk protein (P), sugar (S), and cocoa tissue (C) within a

chocolate particle. Bar ¼2 mm. Reproduced from Microscopy: Transmission Electron Microscopy, Encyclopaedia of Food Science, Food

Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

MICROSCOPY/Transmission Electron Microscopy 3933