MICROSCOPY RESEARCH AND TECHNIQUE 24:400-422 (1993)

Impact of Freeze Substitution on Biological

Electron Microscopy
SIGRUN HIPPE-SANWALD
Christian-Albrechts-Universitat zu Kiel, Botanisches Znstitut, Biologiezentrum, 0-2300 Kiel 1 , Germany

KEY WORDS Cryofixation, Freeze substitution, Low temperature embedding, Lowicryl, Fine
structural preservation, Extracellular material, Bacteria, Algae, Cyanobacte-
ria, Fungi, Transgenic plants, Wheat, Barley, Tobacco, Conidia, Hyphae, Haus-
toria, Plant pathogen interface, Molecular preservation, Retention diffusible
elements, Microanalysis, Autoradiography, Fungicides, Lipids, Immunocyto-
chemistry, Protein antigenicity, Elicitor, Extensin, Foreign proteins
ABSTRACT Considering the increasing necessity for improved preparation techniques in bi-
ological electron microscopy as a basis for the identification and localization of cellular substances
within the compartments of the cell, this review is focussed on the method of freeze substitution as
a n important link between the cryofixation (ultrarapid freezing) and resin embedding of biological
specimens. The theory and practice of freeze substitution is summarized with particular interest in
the physical and thermodynamic as well a s in the chemical basis of this technique. A survey of
practical aspects of the technical process of freeze substitution concerning the equipment and
various protocols successfully applied in biological systems is also given. The main advantage of
freeze substitution versus conventional chemical fixation is seen in the maintenance of the hydra-
tion shell of molecules and macromolecular structures. This results in a n improved fine structural
preservation, superior retention of the antigenicity of proteins and decreased loss of unbound,
diffusible cellular components. Examples of excellent visualization of the ultrastructure of macro-
molecular complexes (nucleic acids, extracellular material, membranes etc.), small organisms (bac-
teria, algae, cyanobacteria and fungi) and large biological samples such as plant and animal tissue
as well as the plant-pathogen (fungus) interface and infection structures are presented. Recent data
on the molecular characterization of freeze-substituted biological tissue are exemplified with spe-
cial emphasis on the subcellular detection of soluble components (elements, lipids, protleins and
drugs) and the inter-iintracellular localization of proteins including foreign proteins in transgenic
plants. The molecular analysis of freeze-substituted specimens is achieved by the combination of
low temperature preparation techniques in biological electron microscopy with various detection
methods such as X-ray microanalysis, immunocytochemistry and high resolution aiutoradi-
ography. o 1993 Wiley-Liss, Inc.

INTRODUCTION preparation (Bullivant, 1970; Dubochet et al., 1981;

Combining fine structural preservation with the po-
tential of molecular characterization of the cells’ native
state is one goal of modern cell biology. Low tempera-
ture (LT) preparation techniques in biological electron
microscopy are regarded as primary tools to stabilize
the dynamic ultrastructure as i t exists under physio-
logical conditions. The integration of improved fine
structural preservation with a molecular analysis of
the cell is achieved by the development of secondary
probing techniques including X-ray microanalysis, im-
munocytochemistry and autoradiography. The sensi-
tive detection of elements, proteins, nucleotides, lipids,
and drugs is now possible on the electron microscopic
level, which opens new ways for investigations in cell
physiology, molecular biology, animal and plant pa-
thology, and pharmacology.
To reduce the limitations of conventional chemical
fixation and dehydration resulting from its slow action
(seconds to minutes) is the most important aim of LT
Plattner and Bachmann, 1982). Severe ultrastructural
changes, loss of the antigenicity of proteins and extrac-
tion of unbound, soluble components of the cell because
of the breakdown of selective membrane permeability
are typical artefacts introduced by chemical fixation a t
room temperature (Plattner and Zingsheim, 1983).
The main progress in electron microscopy in recent
years was the introduction of preparation techniques
which are ideally performed completely under low tem-
perature conditions in order to avoid such artefacts and
to enable a n analytical evaluation (Livesey and Lin-
ner, 1987). The consideration of two important criteria
as prerequisite for optimum LT preparation is re-
quired. Firstly, the biological tissue has to be cryofixed
in order to maintain the complex integrity of the cell,
Received February 24, 1991; accepted in revised form February 4, 1992

0 1993 WILEY-LISS. INC

 IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM 40 1
trapping all the solutes, macromoleculles and mem- carried out at a temperature low enough to avoid sec-
branes without segregation effects and damage caused ondary ice crystallization. Water is the major compo-
by ice crystallization. Rapid freezing takes place within nent in biological cells. By means of hydrogen bonding
a fraction of a second or even milliseconds, suppressing and ionic Coulomb forces, the water molecules interact
freezing damage in small and even large tissue sam- with each other, and with almost all molecules of bio-
ples. Comprehensive surveys on ultrarapid freezing op- logical interest. In this context, fundamental questions
tions for biological tissue are given in several recent are coming up about the influence of freeze substitu-
reviews (Bullivant, 1970; Gilkey and Staehelin, 1986; tion on the conformation and supramolecular structure
Menco, 1986; Moor, 1987; Plattner and Bachmann, of cellular components as well as on their distribution.
1982; Sitte et al., 1987). Secondly, the sensitive biolog- What happens to the aqueous cellular material when
ical system has to be prepared for a fine structural and water is removed and substituted by an organic sol-
molecular analysis. This can be done in the frozen-hy- vent? In the following section, attention is focussed on
drated state involving cryosectioning and freeze-frac- the fate and function of water, forming water shells
ture techniques or in the resin-embedded state involv- around the molecules and filling most of the spaces in
ing conventional thin sectioning. For the latter the biological tissue. The theoretical basis of the phys-
procedure, freeze substitution (FS) or freeze-drying of ics of water and ice with regard to the fine structure
the frozen material and embedding preferentially un- and retention of biological material, the macromole-
der low temperature conditions is necessary (Feder and cules and the diffusible, aqueous sol-gel constituents
Sidman, 1958; Frederik, 1987; Harvey, 1982; Stein- has been recently reviewed in detail (Bachmann and
brecht and Muller, 1987; Zierold and Steinbrecht, Mayer, 1987; Kellenberger et al., 1986; Kellenberger,
1987). In any case, the transfer of the sample from the 1987; Negendank, 1986; Steinbrecht and Muller,
frozen-hydrated state to the fixed and embedded state 1987).
is regarded as a particular challenge. For thermody-
namic reasons to be discussed later, the transfer has to Cellular Water
be done under low temperature conditions (Kellen- Cellular water exhibits different physicochemical
berger, 1987; Kellenberger et al., 1986). properties as considered in the following discussion.
However, cryofixation and freeze substitution in Water associated with the surfaces of biological mac-
combination with low temperature embedding now ap- romolecules, macromolecular assemblies, or intracellu-
pear to be feasible methods to overcome the disadvan- lar membranes, forms a hydration shell known as
tages and artifacts introduced by conventional meth- bound cellular water. Nearly all biological substances
ods. This paper focuses on the theory and ]practiceof FS such as polysaccharides, nucleic acids, most proteins
in biological electron microscopy: a link between cryo- and even lipid bilayers turn out to be hydrophilic, sur-
fixation and resin embedding by which the cellular wa- rounded by hydration shells of pure water on the sur-
ter is gently removed from a frozen specimen. Results face of the molecules (Killian and deKruijff, 1985;
on systematic studies on the physics and chemistry of Kuntz and Kauzmann, 1974). In pure water, the hy-
FS as well as aspects regarding the technical equip- dration shell of nucleic acids is formed by approxi-
ment are discussed. The progress obtained by the ap- mately 20-50 water molecules per nucleotide (Hearst
plication of FS versus chemical fixation in preserving and Vinograd, 1961). Such surface-modified water is
native cellular structures and antigenicity of proteins particularly important for the cell metabolism, and it is
as well as the retention of diffusible cellular compo- essential for the solubility of the molecules in aqueous
nents are exemplified in detail. A fundamental under- media (Negendank, 1986). The ground plasm of the
standing of the physical and chemical basis of FS is cytoplasm is thought to be composed of different aque-
necessary for the interpretation of research results, ous sol and gel combinations forming globular and fi-
since an increasing amount of novel cytological infor- brous elements. Sols are solutions of biological macro-
mation is being reported on the basis of this LT molecules of fibrous (e.g., DNA) or globular shape.
method. Depending on the viscosity of the sol, the macromole-
cules are freely mobile. Unlike plasmatic sols, most
PHYSICAL AND CHEMICAL BASIS OF biological gels are based on fibers and characterized by
FREEZE SUBSTITUTION local interaction. The meshwork can be filled with a sol
In order to prepare a frozen biological specimen for of globular molecules (Kellenberger, 1987).All cellular
qualitative and quantitative ultrastructural research, organelles, membranes, ribosomes, and the cytoskele-
various methods are possible. Depending on the main ton are embedded in the ground plasm of different
goal of analysis and in particular on the technological aqueous sol-gel constitution. However, the role of the
facilities in the laboratory, cryosectioning in combina- hydration shells in stabilizing and possibly rigidifying
tion with an evaluation of the frozen-hydrated sample flexible structures remains t o be investigated.
by low temperature electron microscopy as well as con-
ventional thin sectioning are recommended. The latter Dehydration
approach requires the removal of cellular water by During freeze substitution, the cellular water is re-
freeze-drying or freeze substitution prior tlo infiltration moved or replaced by an organic liquid. Presumably,
in adequate embedding media. bulk water is more easily removed from the cell than
Freeze substitution dissolves the cellular ice in the water of the hydration shells. Removing the water from
frozen specimen by an organic solvent which usually the hydration shell, as it happens during conventional
contains chemical fixatives. The procedure has to be dehydration at room temperature, leads to serious al-
402 S.HIPPE-SANWALD
terations of the fine structure and loss of 20-70% of the
original cellular volume (Lee, 1984). Globular (e.g.,
proteins) and fibrous (e.g., DNA) sol are randomly dis-
tributed in water but form irreversible networks and
aggregates in organic solvents a t room temperature.
Similar aggregations occur when gel-like material
(e.g., microtubules) is transferred to a n organic solu-
tion; this tendency can be decreased, however, by
chemical cross-linking (chemical fixation) and progres-
sive dehydration. But the smaller the aggregates be-
come, the lower the temperature has to be a t which the
water is substituted (Kellenberger et al., 1986). Sys-
tematic studies on the freeze substitution procedure
consider four important criteria, such a s the effect of
the low temperature (Bank, 1973; Sakai et al., 19681,
the rate of the substitution process, the efficacy of var-
ious organic solvents and fixatives (Humbel et al.,
1983; Bridgman and Reese, 1984; Ikeda et al., 1984)
and the feasibility of combining freeze substitution
with LT embedding in LowicrylB instead of conven-
tional araldite resins (Carlemalm et al., 1986; Hippe
and Hermanns, 1986; Humbel et al., 1983; Hunziker et
al., 1984).
Temperature
Temperature is particularly important when consid-
ering freeze substitution, since all components of a bi-
ological system change their interrelation when the
temperature changes. During freeze substitution, tem-
perature increases above that of the solid, frozen stage
of sample storage, i.e., the liquid nitrogen temperature
of 77 K. Freeze substitution usually starts a t a rela-
tively high temperature of between 188 and 193 K.
This temperature is considerably above the devitrifica-
tion range of solid, amorphous water-150 K (Bach-
mann and Mayer, 1987). However, the conditions of
recrystallization of cellular water appear to be differ-
ent from solid water according to the different status
and complex binding of water in the cell. In some cases,
components of the cell may act as natural cryopro-
tectants. High recrystallization temperatures of 204 K
and even 230 K are reported for frost-hardy plant cells
(Sakai et al., 19681, yeast cells (Bank, 1973), human
red blood cells (Nei, 1973) and rapidly frozen silk moth
antennae (Steinbrecht, 1985a). These data are also
compatible with successful electron microscopic studies
on the ultrastructural preservation of biological cells
and tissue prepared by freeze substitution around 190
K. Some aspects of fine structural results are discussed
in the Applications section.
Rise of temperature also activates the movement of
molecules and removes parts of the hydration shell,
promoting hydrophobic interactions between the mol-
ecules. The aggregation of proteins with increasing
temperature seems to contradict the laws of entropy
which state that the order decreases with increasing
temperature. Thermodynamic measurements, how-
ever, explain this paradox: the decrease of order that
accompanies the removal of the hydration shells has
more weight than the increase of order by the polymer-
ization of protein subunits into regular arrays (Lauffer,
1975; Kellenberger, 1987, 1991).
In this context, the melting temperature being inter-
preted as a sort of measure for the degree of hydro-
philicity of molecules, such as cellular compounds, or-
ganic solvents and fixatives, rlespectively, is
particularly important regarding freeze substitution.
Depending on the melting temperature of the cellular
compounds, molecular aggregations are diminished
with decreasing temperature, because the hydration
shells are presumably more stable under such condi-
tions. However, there are no quantita.tive data avail-
able on the role and fate of the hydration shells during
freeze substitution. Electron microscopic results might
support this interpretation, since the lower the temper-
ature, the finer are the aggregates of macromolecules
and supramolecular structures in organic solvents
(Carlemalm et al., 1986; Humbel et a].., 1983).
With regard to immunocytochemistry, preservation
of the hydration shell of proteins is particularly signif-
icant. Presumably, the hydration shell stabilizes the
tertiary structures of proteins, and the antigenic sites
remain preserved. Removal of the hydration shells
would promote conformational changes of the proteins,
resulting in a reduced possibility for immunolabeling
(see Applications).
Chemicals
The lowest temperature at which freeze substitution
can be performed is determined by the melting point of
the organic solvent used. Treatment of the specimens
with organic solvents such a s acetone or methanol is
required for dissolving the cellular water prior to infil-
tration in adequate embedding media. In freeze substi-
tution, the dehydration speed depends on the temper-
ature and on the water capacity of the solvent a t that
temperature. The water capacities of organic solvents
most widely used in freeze substitution differ consider-
ably: as for methanol, 29% (v) water can be dissolved a t
183 K, whereas the water capacity of ethanol and ace-
tone is much lower: 13%for ethanol and less than 2%
for acetone a t 183 K FS temperature. According to
quantitative measurements on the substitution of 3H-
ice by various solvents as a function of time and tem-
perature, methanol is the fastest substituent and ace-
tone is considerably slower. At 183 K , the substitution
rate of acetone is extremely reduced by only 1%water,
but methanol will substitute specimens fairly rapidly
even in the presence of 10% water (data from Stein-
brecht and Muller, 1987). The substitution in acetone
can be considerably improved if the process is per-
formed over a longer period of time (7 days), followed
by rapid warming of the specimens (Steinbrecht, 1982).
Ice recrystallization occurs only in those cases where
the substituting acetone contains 5% water. By com-
parison, diethylether is by far the slowest medium
(Humbel et al., 1983). Details concerning the success-
ful application of various organic substitution fluids
and the choice of fixatives are given in the next section,
with the discussion of general methods and protocols
for freeze substitution.
In order to avoid collapse of the fine structure during
dehydration, additional cross-linking by chemical fix-
atives is usually necessary. Since the fixatives are
added to the organic solvent, cross-linking starts a t the
lowest temperature of freeze substitution. Relatively
 IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM
little information about the temperature dependence of
fixatives during freeze substitution is available in the
literature (Steinbrecht and Muller, 1987). Humbel et
al. (1983) found that the addition of uranyl acetate and
glutaraldehyde, respectively, to methanol reduces the
extraction of phospholipids and results in cross-linking
even at around 243 K. Several studies have shown the
feasibility of combining freeze substitution with LT
embedding techniques. This is now possible with the
application of various methacrylate resins (LowicrylB
HM20, K4M, HM23, K11M; Carlemalrn et al., 1985).
Lowicryl allows the infiltration under low temperature
conditions because the embedding medium remains
liquid. In all cases reported so far, improved stabiliza-
tion of the biological structure (membranes, proteins,
etc.) can be attained when freeze-substituted speci-
mens are embedded at LT conditions. The most impor-
tant advantage of LT embedding of freeze-substituted
samples lies in the improved preservation of the anti-
genicity of proteins. A selection of excellent results on
the preservation of the fine structure a13 well as exam-
ples of LT-prepared and immunostained biological ma-
terial are presented in the Applications section.
TECHNICAL PROCESS
The most crucial step of any cryotechnique is a good
cryofixation by ultrarapid freezing. Secondary tech-
niques like freeze substitution are only suitable in case
the primary artefacts of freeze damage are reduced to a
minimum. The successful application of ultrarapid
freezing techniques to the cryofixation of biological ma-
terial has been comprehensively revievved in the liter-
ature and will not be discussed in this paper (Gilkey
and Staehelin, 1986; Menco, 1986; Moor, 1987; Platt-
ner and Bachmann, 1982; Sitte et al., 1987).
Equipment
Different equipment for performing freeze substitu-
tion followed by low temperature embedding has been
described in detail. Humbel and Miiller (1986) have
built a special cryostat consisting of a cylindrical brass
container to be placed in a freezer equipped with ultra-
violet lamps for LT polymerization. A two-point tem-
perature control unit regulates cooling and heating
which is achieved by liquid nitrogen. The regulator can
be programmed for keeping four direrent tempera-
tures constant during different time intervals. Small
Eppendorf tubes are used as substitution vials.
Whereas the handling of tissue for media change is
very simple, suspensions have t o be pelleted in a mi-
crofuge also placed in the freezer. Based on this facility,
an apparatus is now commercially available by Balzers
Union (Balzers, Liechtenstein).
According to the technique describeld above, a simi-
lar method has been applied in our own experiments
(During et al., 1990; Hippe, 1985,1987;Hippe and Her-
manns, 1986, 1988; Hippe et al., 1989; Hippe-Sanwald
et al., 1992a,b). Instead of employing a self-built cry-
ostat or a simple cooling bath, a commercial LT freezer
operating a t 183 K was used. In order to maintain con-
stant temperature conditions during the substitution
process, the vials were placed into a large copper block.
In such a device the temperature asymptotically ap-
403
proaches ambient temperature, the halftime depending
on the volume of the metal block and its insulation
(Steinbrecht and Muller, 1987). Eppendorf tubes, as
well as jars made of steel, each screw-capped and
tightly sealed, are equally recommended as substitu-
tion vials. In this context, Howard and O’Donnell
(1987) prefer 120 ml jars containing a 50 ml polypro-
pylene beaker and a surrounding layer of indicating
desiccant (e.g., anhydrous calcium sulfate).
More versatile equipment is recommended in case
the freeze substitution and LT embedding in Lowicryl
are to be performed for complex substitution schedules
and systematic studies under controlled and reproduc-
ible conditions. Suitable equipment has been developed
which permits choice of temperature and of any desired
warming schedule (Humbel and Muller, 1986; Muller
et al., 1980; Sitte, 1984; Sitte et al., 1986). Instrumen-
tation for automated freeze substitution in combina-
tion with subsequent Lowicryl embedding is now com-
mercially available from Balzers Liechtenstein (FSU
010 Freeze Substitution Unit) and from Reichert/Leica,
Germany (CS-Auto Cryosubstitution Device).
General Methods
The schedule for a combined freeze substitution and
embedding procedure of biological material generally
depends on the research goal. For preservation of the
fine structure, a number of organic solvents containing
fixatives in different concentrations have been success-
fully applied. Acetone as well as methanol are the most
widely used substitution solvents containing fixatives
in various concentrations and combinations: OsO, (2-
4%), glutaraldehyde (1-3%), uranyl acetate (0.5%),as
well as tannic acid, permanganate, hafnium chloride,
and acrolein (Akisaka and Shigenaga, 1983; Bridgman
and Reese, 1984; Bullivant, 1970; Harvey, 1982; Heath
et al., 1985; Hippe, 1985, 1991; Hippe and Hermanns,
1986; Hoch, 1986; Howard and Aist, 1979; Howard and
O’Donnell, 1987; Hunziker et al., 1984; Ikeda et al.,
1984; Knauf et al., 1989; Robards and Sleytr, 1985;
Steinbrecht and Muller, 1987; Zalokar, 1966). In most
cases, substitution is carried out at dry ice temperature
(194 K) for several days followed by slow warming and
chemical fixation over 1to 2 days. At about 275 K, the
organic solvent-fixative mixture is replaced by pure
substitution fluid, followed by infiltration with resin
and polymerization as usual.
Subsequent embedding in conventional resins,
mostly Epon-Araldite mixtures (e.g., Spurr’s low vis-
cosity resin), or in LR White or methacrylate resin
(Lowicryl resin), respectively, is equally possible and
gives excellent resolution of the fine structure (Carle-
malm et al., 1985, 1986; Hippe, 1985; Hippe and Her-
manns, 1986; Hoch, 1986; Horowitz et al., 1990; Hun-
ziker and Schenk, 1984; Nicolas et al., 1989).
Employing certain epoxy resins may result in severe
contrast problems for the fine structural analysis
which can, however, be overcome by mixing Araldite
with Polybed 812 or using Quetol 651 resin (Howard
and O’Donnell, 1987).
Regarding the localization of bound and unbound
proteins and lipids or of other divers chemical com-
pounds and cellular ions by immunocytochemistry, by
404 S. HIPPE-SANWALD

elemental microanalysis or by high resolution autora- 1 : l for 4 hours, followed by infiltration of pure ERL
diography, the choice of the fixative, the dehydration (100%) at 238 K for 16 hours, and at 258 K for 4 hours.
solvent and the embedding procedure is critical and The infiltration solution is replaced by a mixture of
needs careful evaluation. In general, the conditions for ERL:complete Spurr’s LV resin = 1:l at 258 K for 5
optimum analysis must be worked out for each system hours, by a n 1:3 mixture a t 277 K for 16 hours, and by
separately. This means that no single procedure can be pure Spurr’s LV resin for 24 hours. The embedding is
recommended as the best approach (Bendayan et al., performed in flat molds and the samples are polymer-
1987; Edelmann, 1991; Hippe, 1987; Hippe and Her- ized at 333 K for 12 hours (Hippe, 1985).
manns, 1988; Knoll et al., 1988; Monaghan and Rob- (2) A similar preparation protocol in combination
ertson, 1990; Weibull et al., 1984; Weibull and Chris- with the LT embedding in Lowicryl K4M and HM20,
tiansson, 1986; Zierold and Steinbrecht, 1987). respectively, has been used for the fine structural study
The use of diethylether containing 20% acrolein as a of sporidia of Ustilago auenae in comparison to conven-
substitution medium in plant material has formerly tionally embedded material (Hippe and Hermanns,
been recommended for retaining the original distribu- 1986). As opposed to the procedure mentioned above,
tion of water-soluble substances and electrolyte ele- only 3% glutaraldehyde is applied as fixative. Addi-
ments (Harvey, 1982). Acetone and ethanol containing tionally, Lowicryl embedding permits lowering the
acrolein (5-10%) and/or glutaraldehyde plus parafor- temperature considerably during the infiltration and
maldehyde in various concentrations have been docu- polymerization process. The infiltration in Lowicryl
mented a s substitutionifixation media for the purpose K4M is performed a t 238 K, whereas the impregnation
of microanalysis and immunocytochemistry (Bridgman and polymerization in Lowicryl HM20 is done a t about
and Reese, 1984; Harvey, 1982; Murata et al., 1985; 213 K. This protocol results in a n improved visualiza-
Ornberg and Reese, 1981). tion of fungal fine structures. Secondly, in combination
During the last few years, it has become evident that with high resolution autoradiography, the intracellu-
the use of fixatives (e.g., aldehydes, OsOJ in relative lar localization of unbound chemical compounds (e.g.,
high concentrations alters the conformation of cellular fungicides) is possible on the basis of this method
macromolecules and decreases antigenic sites detect- (Hippe, 1987).
able by immunocytochemistry. A superior localization This procedure has been proven to sufficiently pre-
of fixation-sensitive antigenic sites can be achieved, serve the antigenicity of various proteins in plant ma-
however, by the reduction of aldehyde concentration or terial. The schedule, characterized by a three temper-
even by completely omitting the fixative during freeze
substitution. Additionally, the deleterious effect of or-
ganic solvents (e.g., methanol, acetone) can be reduced
by lowering the temperature during the freeze substi-
tution process and the final embedding. In this context, Abbreviations
embedding in Lowicryl resin is recommended, which C chloroplast
yields a n increase in the intensity of the labeling of ch chondrocyte
cw cell wall
antigenic sites. The subcellular lifelike preservation of cytoplasm
CY
soluble, unbound proteins, of organic compounds and em extrahaustorial membrane
even of mobile ions in various biological specimens is ema extrahaustorial matrix
another advantage of this low temperature method ex extensin-like epitopes
er endoplasmic reticulum
(Edelmann, 1991; Hippe, 1987; Hippe et al., 1989; ERL vinyl cyclohexene dioxide
Hippe-Sanwald et al., 1992b; During et al., 1990; FS freeze substitution
Kellenberger et al., 1986; Knoll et al., 1988; Marticke g glycogen
et al., 1992; Monaghan and Robertson, 1990; Nicolas e t go Golgi body
H Haustorium
al., 1989; Van Leeuwen, 1986). HB haustorial body
HF haustorial finger
Protocols HMC haustorial mother cell
As a n example, three different schedules will be pre- HP High pressure
sented. They have been proven as reliable protocols for hCY host cytoplasm
IC intercellular space
the freeze substitution and embedding of bacterial, fun- LV low viscosity
gal, plant, and animal specimens. LT low temperature
(1)According to Muller et al. (1980) and Hunziker e t 1Y lysozyme
m mitochondrion
al. (19841, a stepwise substitution starting a t 178 K me mesosome
and continuing a t 213 K and 243 K is recommended. mf microfilament
Methanol is chosen a s organic solvent because of the mve multivesicular body
high water capacity in contrast with other media. n nucleus
Methanol contains a mixture of fixatives: 0.5% uranyl nc nuclear crystal
nb neck band
acetate, 1% OsO,, 3% glutaraldehyde and up to 3% Pl plasmalemma
water being introduced by using a 50-70% aqueous PLT progressive lowering temperature
glutaraldehyde solution. Subsequent embedding in r ribosomes
Spurr’s low viscosity (LV) resin (Spurr, 1969; Lauchli S sheath
se extracellular slime
et al., 1970) starts a t 238 K by initial impregnation in THRGP threonine-rich glycoprotein
ERL (vinyl cyclohexene dioxide = VCD):methanol = V vacuoles
 IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM
ature step substitution process, is taken as a basis for
the immunocytochemical analysis of high pressure
(HP) and propane jet-frozen transgenic and nontrans-
genic plant tissue. However, glutaraldehyde is omitted
as a fixative and replaced by 0.5% uranylacetate
(Hippe et al., 1989; During et al., 1990; Hippe-Sanwald
et al., 1992b; Marticke et al., 1992).
(3) Alternatively, HP-frozen bacteria (Figs. 1and 2),
fungi (Figs. 3 and ll),and plant material, which in
some cases had been infected by the phytopathogenic
fungus Puccinia graminis f.sp. tritici (Figs. 24-27),
have been freeze-substituted automatically at 188 K by
the CS-Auto instrument for 3 days (compare Applica-
tions section). After the substitution in methanol/0.5%
uranyl acetate, the temperature is slowly increased ac-
cording to the programmed schedule: one degree per
hour to the final temperature of 223 K. Washing and
subsequent stepwise infiltration in Lowicryl HM20 is
performed over a period of two days. The final flat em-
bedding, the transfer of the samples into the moulds,
and the UV polymerization is done in the CS-Auto un-
der controlled conditions at 223 K for 24 hours. Prior to
removing the polymerized samples from the moulds,
the temperature is set to room temperature. This pro-
cedure results in improved fine structural preservation
of HP-frozen biological material, such as plant tissue
infected by fungi. The immunocytochemical localiza-
tion of various proteins (elicitors, THRGP-like epi-
topes, etc.) at the host-parasite interface of biotrophic
pathogens on monocotyledonous host plants has been
achieved on the basis of this protocol for the first time
(Hippe-Sanwald et al., 1992; Marticke et al., 1992).
APPLICATIONS
Theoretical considerations and experimental results
obtained with low temperature preparation techniques
in biological electron microscopy support the view that
these modern techniques are a prerequisite for main-
taining native cell and tissue properties.
Improved preservation of the fine structure and an-
tigenicity of proteins as well as the retention of mobile
compounds of the cell can only be attained by low tem-
perature preparation, which minimizes molecular dis-
tortion and movement. The data published to date fall
into two categories: those that explore the potential of
freeze substitution (in combination with adequate
freezing techniques) for the preservation of the struc-
tural integrity of various biological systems, and those
that employ freeze substitution as a tool for stabilizing
soluble components and sensitive antigenic sites. The
majority of studies fall into the first category, i.e., im-
proved visualization of the fine structure including the
detection of novel features of cellular organization. De-
tailed data are available on macromolecules, e.g., ex-
tracellular components, on small objects as bacteria,
algae and cyanobacteria, and fungi, as well as on large
specimens like plant and animal tissue samples. In this
context the plant-pathogen interaction of fungi grow-
ing on plant tissue has been analyzed in particular.
The progress in preserving the fine structure as
achieved by freeze substitution versus chemical fixa-
tion is exemplified in the following section. The second
405
part of the discussion deals with the advantages of
freeze substitution regarding the detection of bound
and unbound cellular components based on electron
probe analysis, high resolution autoradiography and
immunochemistry .
Fine Structure
Macromolecules and Extracellular Material.
Systematic studies on the preservation of the fine
structure of freeze-substituted macromolecules, such as
chromatin, as well as extracellular material like insect
cutin, animal dentin and predentin, proteoglycan in rat
cartilage, and the extracellular matrix in early chicken
embryos, are available in the literature (Allenspach et
al., 1988; Carlemalm et al., 1986; Horowitz et al., 1990;
Hunziker et al., 1984; Hunziker and Schenk, 1984;
Goldberg and Escaig, 1987; Steinbrecht, 1985b).
During dehydration, aqueous solutions of DNA,
RNA, of nucleoproteins of all types, as well as of pro-
teins, are precipitated in a certain medium of a water-
miscible organic solvent, e.g., ethanol, methanol and
acetone. Depending on the initial concentration of the
solvent and its water content, and on the temperature
and the presence of fixatives, the sizes and shapes of
the aggregates differ (Carlemalm et al., 1986; Kellen-
berger et al., 1981, 1986). The bacterial chromatin of
the Gram-positive bacterium Nocardia corallina is
coarse when prepared conventionally a t room temper-
ature (Fig. l),less coarse when submitted to a progres-
sive decrease of temperature (PLT technique) and least
coarse when ultrarapidly frozen and cryosubstituted at
193 K (Fig. 2). In the latter case, the DNA of Nocardia
corallina is homogeneously distributed in a distinctly
resolved bacterial cytoplasm containing numerous ri-
bosomes.
Although heavy metal fixatives (e.g., uranyl acetate)
reduce the aggregation artefacts by cross-linking non-
eukaryotic chromatin in conventionally prepared spec-
imens, ultrarapid freezing followed by freeze substitu-
tion has been proven as the method of choice for a
superior preservation of both noneukaryotic (Fig. 2)
and eukaryotic chromatin (Kellenberger et al., 1986).
As recently published by Horowitz et al. (1990), spray
freezing followed by freeze substitution and LT embed-
ding are particularly appropriate for studying eukary-
otic nuclear structures (chromatin fibres, nucleosomes,
etc.) in situ. Eukaryotic nuclear structures such as het-
erogeneous chromatin, bundles of microfilaments, the
nuclear membrane and noncollapsed nuclear pores of a
tobacco stem tissue cell (HP-frozen, freeze-substituted
and Lowicryl-embedded; Hippe et al., 1989) are shown
in Figure 15 as an example.
Complex extracellular material that is usually col-
lapsed and aggregated after conventional preparation
is well preserved by this method in the bacterium No-
cardia corallina (extracellular slime matrix; Figs. 1
and 2), in plant tissue (intercellular space components;
Figs. 14 and 16), in various plant-pathogen infections
(fungal extrahaustorial matrix; Figs. 21, 25 and 261,
and in animal specimens (insect cuticle, rat dentin,
proteoglycan in cartilage tissue; Fig. 18). Novel fea-
tures of extracellular material in fungi and plants are
discussed in the following.
406 S. HIPPE-SANWALD
Freeze-substituted butterfly cuticles have been in-
vestigated with particular interest in the arrangement
and formation of extracellular microfiber layers during
pupal development (Steinbrecht, 1985b). Rapid freez-
ing in combination with freeze substitution using mal-
achite green, acrolein and osmium tetroxide staining is
another interesting example for the study of extracel-
lular matrix in rat incisor tissue (Goldberg and Escaig,
1987).By this method, the detection of phospholipids as
extracellular matrix compound in rat incisor predentin
and dentin is allowed. More evidence for the retention
of soluble lipids and other mobile cellular components
is given by microanalysis and autoradiographic studies
on freeze-substituted material (compare Fig. 5; Hippe,
1987; Hippe and Hermanns, 1988; Knoll et al., 1988;
Zierold and Steinbrecht, 1987).
The intercellular substance of cartilage tissue con-
sists of a proteoglycan phase trapped within a network
of collagen fibrils (Fig. 18; Hunziker et al., 1984; Hun-
ziker and Schenk, 1984). Successful attempts in pre-
serving the native structural integrity of proteoglycan
and collagen macromolecules (compare Fig. 18, inset)
and its relationship to epiphyseal chondrocytes in rat
cartilage tissue after LT processing have been made by
Arsenault et al. (1988), Hunziker et al. (1984), and
Hunziker and Herrmann (1987). It is important to em-
phasize that this recent progress in LT methods, lead-
ing to improved preservation of cartilage fine struc-
ture, is seen as a prerequisite for an understanding of
cartilage function and development (Arsenault et al.
1988; Hunziker et al. 1984; Hunziker, 1993).
Bacteria. Due to freeze substitution, new informa-
tion has been obtained on the general anatomy and fine
structure, e.g., cell wall and chromatin, in different
bacterial species. One of the structural features, ob-
served about thirty years ago in bacillus species and
other Gram-positive bacteria, are mesosomes. Meso-
somes cannot be detected in Bacillus subtilis and other
bacteria after cryofixation. It is quite clear now, that
mesosomes are artefactual structures formed during
conventional fixation (Hobot et al., 1985; Ryter, 1988).
Additionally, no difference in the shape of the chroma-
tin (bacterial “nucleus”) of Gram-positive and Gram-
negative bacteria is observed when LT preparation is
performed. As already documented above, freeze-sub-
stituted DNA exhibits homogeneous fine structure and
is distinctly resolved (Fig. 2). The DNA is probably
directly bound to the cytoplasmic membrane since no
mesosomes occur in the living state of bacteria (Ryter,
1988). The fundamental differences in the cell wall or-
ganization of Gram-positive and Gram-negative bacte-
ria are also confirmed by the cryosubstitution method.
It enables a higher resolution of the bacterial surface
and of the cell wall layers, as well as a superior pres-
ervation of cell surface slime material and retention of
surface antigens, like capsular polysaccharides and li-
popolysaccharides (Acker, 1988; Ryter, 1988; Acker
and Kammerer, 1990).
Algae and Cyanobacteria. With respect to the ul-
trastructural organization of algae and cyanobacteria,
freeze substitution has also been used in comparison to
conventional methods. New information has been ob-
tained on the dictyosomes in Scenedesmus acuminatus
(Ueda and Noguchi, 1986), on the fine structure of cel-
lular division (autosporogenesis) in the coccoid green
alga Trebouxia aggregata (Sluiman and Lokhorst,
1988), and the internal organization of the blue-green
alga Anabaena variabilis (Frosch and Westphal, 1989).
In the latter case, frozen cyanobacteria are freeze-sub-
stituted in pure acetone and subsequently embedded in
the water soluble melamine resin NanoplastB at 190
K. Compared to conventional preparation at room tem-
perature, more details of the cytoplasm (DNA contain-
ing chromatin, polyhedral bodies, phycobilisomes, ribo-
somes, thylakoid membranes and rows of fine-grained
ellipsoid glycogen granules) are resolved after the LT
procedure. The applicability of this method on the prep-
aration of plant tissue and other biological objects
containing a high amount of water remains to be
investigated. Furthermore, the suitability of freeze-
substituted and Nanoplast-embedded material for im-
munocytochemical analysis has to be confirmed in the
future (Frosch and Westphal, 1989).
Fungi. In recent years the utilization of freeze sub-
stitution techniques for studying fungal cells at the
ultrastructural level has become increasingly impor-
tant. Numerous fine structural investigations are
available on the cytoplasmic organization of different
yeast species (Baba and Osumi, 1987; Hippe and Her-
manns, 19861, hyphal tip cells of septate filamentous
fungi (Hoch, 1986; Howard, 1981; Howard and Aist,
1979; Newhouse et al., 1983; Roberson and Fuller,
19881, fungal mitosis including cytoskeleton features
(Heath and Rethoret, 1982; Heath et al., 1984; McKer-
Figs. 1 and 2. Longitudinal sections of the Gram-positive bacte-
rium Nocardia corallina after conventional preparation (Fig. 1 ) ac-
cording to Volker et al. (19771 and low temperature preparation (Fig.
2) using HP freezing, freeze substitution and Lowicryl HM20 embed-
ding according to section entitled Technical Process; Protocols.
Fig. 1. Coarse fine structural visualization of Nocardia corallina
showing aggregated chromatin fibrills (arrowheads), mesosome (me1
artefacts, and collapsed, extracellular slime matrix be). Bar = 0.25
pm.
Fig. 2. Improved ultrastructural preservation of the bacterium
Nocardia corallina exhibiting nonaggregated chromatin fibrills ho-
mogeneously embedded in a distinctly resolved cytoplasm. Numerous
ribosomes are clearly visualized. Superior retention of the noncol-
lapsed, extracellular slime coat (se) appears characteristic for low
temperature processing. Bar = 0.25 pm.
Fig. 3. Cross-sectioned germ tubes ofPuccinia graminis f.sp. tritici
processed according to section entitled Technical Process; Protocols.
The germ tube contains dense cytoplasm. Mitochondria, vacuoles and
ribosomes are visible. The outer cell wall is characterized by a dis-
tinctly resolved extracellular matrix (arrowheads). Bar = 0.5 pm.
Fig. 4. Overview over the fine structure of yeast-like sporidia of
Ustilago auenae processed according to Hippe and Hermanns (1986).
Homogeneously preserved cytoplasm composed of fibrillous cytoplas-
mic matrix, a high amount of ribosomes, and smoothly contoured
mitochondria (m1. Bar = 0.2 pm.
Fig. 5. Electron microscopic autoradiography of low temperature
processed sporidia of Ustilago auenae for the localization of systemic
fungicides according to Hippe (1987). Accumulation of silver grains
visible over lipid droplets of a fungicide resistant strain of Ustzlago
auenae. Bar = 0.25 pm.
IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM 407

Figs. 1-5.
408 S. HIPPE-SANWALD
racher and Heath, 19851, reproductive structures, i.e.,
spores, conidiophores and. conidia (Mims et al., 1988,
1990; Newhouse e t al., 1990) and fungal infection
structures (Dahmen and Hobot, 1986; Hippe, 1985;
Hippe-Sanwald et al., 1992; Knauf et al., 1989; Mend-
&;enet al., 1991; Studer et al., 1989; Welter, et al. 1988).
Conventional electron microscopy on yeast speci-
mens, e.g., Sacharomyces spec., Kloeckera spec. etc.,
has been a problem in the past because thick cell wall
layers prevent penetration of fixatives into the cells.
According to recently published data, this problem is
now solved by using freeze substitution on ultrarapidly
frozen cells and consecutive embedding in Lowicryl LT
resin HM20 and Quetol 812, respectively (Hippe and
Hermanns, 1986; Baba and Osumi, 1987). The fine
structure of yeast-like sporidia of the phytopathogenic
fungus Ustilago auenae, which is shown in Figure 4,
appears well resolved and smoothly contoured. The
fungal membrane system, the mitochondria and the
ribosomes exhibit a distinct profile (Fig. 4).Glycogen
appears to be common storage material in fungi since i t
is also found in other fungi, like Erysiphegraminis f.sp.
hordei during different stages of development (Figs. 10,
11, and 22) as well a s in axenic cultures of Puccinia
graminis f.sp. tritici (Hippe-Sanwald; unpublished
results).
Apical growth in filamentous fungi (i.e., Asco-
mycetes, Basidiomycetes) is a highly polarized cellular
process that involves many cytoplasmic activities.
Thus, cytological research is particularly focussed on
the hyphal tip cell and its ultrastructure. Freeze-sub-
stituted Spitzenkorper are now described as apical, cy-
toplasmic regions containing dense aggregation of api-
cal vesicles and microvesicles surrounded by a vesicle-
free zone in hyphal tip cells of various fungi: Fusarium
acuminatum (Howard and Aist, 1979); Laetisaria arua-
lis (Hoch and Howard, 1980);Endothiaparasitica (New-
house et al., 1983); Sclerotium rolfsii (Roberson and
Fuller, 1988). Another advantage of freeze substitution
in filamentous hyphae is expressed in the superior vi-
sualization of labile cytoskeletal elements. It enables a
better understanding of the functional role of microtu-
bules in organelle positioning, of the mechanisms of
mitosis and of other dynamic processes in the cell. In
this context, ultrastructural studies on mitosis in fungi
like Zygorhynchus moelleri (Heath and Rethoret,
19821,various Saprolegnia isolates (Heath et al., 19841,
and Basidiobolus haptosporus (McKerracher and
Heath, 1985) are focussed on the arrangement of spin-
dle and kinetochore associated microtubules.
As a n example, some aspects of the fine structure of
freeze-substituted hyphae of powdery mildew, Erysiphe
graminis f.sp. hordei, grown on the leaf surface of bar-
ley plants as well as cross-sectioned germ tubes of Puc-
cinia graminis f.sp. tritici are shown in Figures 3, 6-9.
The cross-section in Figure 6 exhibits typical compart-
ments of a fungal hypha: the nucleus is surrounded by
a distinctly preserved nuclear membrane; the ribo-
somes are arranged in chains; small vacuoles and Golgi
bodies are visualized in the smoothly contoured cyto-
plasm. Mitochondria and the rough endoplasmic retic-
ulum are strikingly organized in the periphery of the
cell. At the sites of hyphal branching and in the vicin-
ity of the nucleus, stacks of rough endoplasmic reticu-
lum, multivesicular bodies and Golgi cisternae have
been observed (Figs. 7-9). The demonstration of Golgi
bodies in freeze-substituted fungi is regarded as a spe-
cial advantage since Golgi bodies of different members
of Ascomycetes and Basidiomycetes are notorious for
being poorly preserved with conventional procedures
(€loch,-1986).
Only a limited number of detailed data on ultrastruc-
tural aspects of the organization of fungal reproductive
structures, on the maturation process of conidia and on
various types of spores are available in the literature.
The poor preservation of the samples obtained with
conventional chemical fixation protocols can now be
overcome by using freeze substitution methods. Re-
cently, promising results have been published on a va-
riety of fungal reproductive structures and spores
(Mims e t al., 1988,1990; Newhouse et al., 1990). In all
cases, freeze-substituted specimens yielded informa-
tion not available in conventionally fixed material
about endomembrane systems, multivesicular bodies
and cytoplasmic and nuclear microtubules as well as
about the Golgi cisternae.
Unlike the fine structure of conidia of Aspergillus
nidulans (Mims et al., 1988) and other fungi (Mims et
al., 1990) freeze-substituted conidia of powdery mildew
Erysiphe graminis f.sp. hordei appear to be organized
differently (Figs. 10 and 11). Conidia are investigated
shortly after formation and separation from the fungal
mycelium on the plant leaf surface as described by
Hippe (1985). At this stage of development the conidia
contain a large amount of storage material (glycogen)
and ribosomes, whereas only small amounts of cyto-
plasm and a few mitochondria are found in the periph-
ery of the conidia (Fig. 11). Bundles of microfilaments
are commonly preserved just beneath the plasmale-
mma and the cell wall. The thick cell wall is charac-
terized by a n electron-dense, surface-structured outer
layer and a translucent inner layer (Figs. 10 and 11).
Plant Tissue. The quality of freeze-substituted
plant tissue depends strongly on the freezing method
Figs. 6-9. Freeze-substituted hyphae of powdery mildew, Ery-
siphe graminis fsp. hordei, grown on the surface of barley leaves; low
temperature preparation according to Hippe (1985).
Fig. 6. Overview over a cross-sectioned hypha of powdery mildew.
The following fine structural details are visible: the distinctly pre-
served plasmalemma (pl) beneath a relative thin cell wall (cw); a
homogeneously structured cytoplasm containing the nucleus (n), ri-
bosomes, peripherally arranged mitochondria (m) and endoplasmic
reticulum (er), Golgi bodies (go),vacuoles (v) and multivesicular bod-
ies (mve). Bar = 0.5 pm.
Fig. 7. Section of a branched hypha of powdery mildew. At the
branching site an accumulation of rough endoplasmic reticulum (er)
near the nucleus (n) is visible. Bar = 1 pm.
Fig. 8. Enlarged area of hyphal cytoplasm of Erysiphe graminis
f.sp. hordei containing several multivesicular bodies (arrowheads).
Bar = 0.25 pm.
Fig. 9. Section of hyphal cytoplasm of Erysiphe graminis f.sp. hor-
dei showing circular Golgi cisternae (go) in the vicinity of the nucleus
(n) and a large vacuole (v). Bar = 0.75 pm.
IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM 409

Figs. 6-9.
410 S. HIPPE-SANWALD
used and is influenced by the size and the properties of
the plant material, i.e., the water content, the degree of
tissue vacuolization, and probably the amount of nat-
urally occurring cryoprotectants. The effectiveness of
freeze substitution has been particularly proven in
small plant specimens like tomato and legume root
hair cells, chloroplasts, as well as pollen grains, pollen
tubes, stamen hairs and anthers of various plant spe-
cies (Lancelle et al., 1986; Lancelle and Hepler, 1989;
McCully and Canny, 1985; Nishizawa and Mori, 1989;
Ridge, 1988; Rother et al., 1988). The majority of in-
vestigations on the fine structure of large plant tissue
samples are mostly done with high pressure frozen ma-
terial. However, barley, wheat and tobacco leaves ap-
pear difficult to preserve, whereas apple, clover, and
bean leaves as well as root tissue of garlic, soybean,
Arabidopsis and tobacco seedlings, and Drosera tenta-
cles are suitable material (Kiss et al., 1990; Licht-
scheidl et al., 1990; Mendgen et al., 1991; Staehelin et
al., 1990; Studer et al., 1989). Concerning freeze-sub-
stituted apple leaves infected by the apple scab fungus
Venturia inaequalis, even Escaig’s slam-freezing tech-
nique gives good results (Dahmen and Hobot, 1986).
Presumably, naturally occurring cryoprotectants (e.g.,
sugars) suppress ice crystallization and facilitate the
freezing procedure. According to systematic investiga-
tions on root tissue of Dahlila variabilis and highly
vacuolated storage parenchyma cells of Helianthus tu-
berosus, a modification of the substitution solvent, usu-
ally methanol and acetone, is recommended by adding
dimethoxypropane (Kaeser et al., 1989).
In the following section, four different examples of
HP-frozen and freeze-substituted plant material are
given. Leaf tissue of Arabidopsis thaliana is presented
in Figures 12 and 13. Transgenic tobacco stem tissue
and tobacco callus culture are shown in Figure 14-17
(compare also Hippe et al., 1989).After LT preparation,
the plant cytoplasm remains, for the most part, at-
tached to the cell wall (Figs. 12-14). The well pre-
served stomata1 cells of Arabidopsis epidermal tissue
contain homogenous cytoplasm, small chloroplasts, a
nucleus in each cell, mitochondria and vacuoles (Fig.
13). A large tobacco nucleus containing heteroge-
neously structured chromatin and nuclear crystals is
visualized in detail (Fig. 15). In the intercellular space
of transgenic tobacco, fibrillar material is identified as
accumulations of secreted foreign protein T4 lysozyme
by immunocytochemistry (Fig. 16; see also section en-
titled Molecular Characterization of the Cell). An en-
larged section of tobacco callus culture is presented in
Figure 17. The irregular morphology of chloroplasts
and strikingly small mitochondria are characteristic
features for tobacco callus cells. As a fourth example, in
Figure 24 a section of parenchymal wheat leaf tissue
infected by Puccinia graminis f.sp. tritici is presented.
The fungal haustorium is surrounded by smooth plant
cytoplasm. Strikingly, the fungal cytoplasm is darker
stained than the plant cytoplasm. The plant-pathogen
interface characterized by a fibrillar, extrahaustorial
matrix and extrahaustorial membrane is clearly de-
fined (Fig. 25) resembling the host-parasite interface of
Uromyces appendiculatus and Phaseolus vulgaris
(Knauf et al., 1988; Mendgen et al., 1991).
Animal Tissue. Numerous studies have focussed on
animal cells and tissue difficult to preserve in a satis-
factory manner by conventional fixation. However, the
quality of preservation obtained in botanical samples
by LT preparation including freeze substitution as de-
scribed in this paper varies more than with animal
tissues. Among these data, investigations on the devel-
opment and molecular organization of growth carti-
lage, secretion processes of growth hormone by anterior
pituitary cells, liver tissue, muscle fibers, visual cells,
nerve fibers and blood cells as well as dipteran anten-
nae have contributed to a better understanding on the
structure and function relationships (Arsenault et al.,
1988; Dahl and Staehelin, 1989; Edelmann, 1989; Hun-
ziker et al., 1984; Steinbrecht, 1982; Studer et al., 1989;
Van Harreveld and Crowell, 1964; Verna, 1983). Con-
cerning the secretion of growth hormone by pituitary
cells, high pressure freezing in combination with freeze
substitution is sufficiently fast and careful to permit
the stabilization of secretory vesicles in the act of dis-
charging their contents to the external medium (Dahl
and Staehelin, 1989). As already mentioned, another
advantage of freeze substitution is the preservation of
extracellular material in rat avascular tissue consist-
ing of chondrocytes and intercellular matrix as well as
in other biological samples (Fig. 18; Hunziker et al.,
1984). The improved retention of mobile ions during
muscle contraction is taken as another evidence for the
sensitive preparation conditions of biological speci-
mens during freeze substitution (Edelmann, 1989).
These data have substantially supported previous re-
sults on the localization of soluble, cellular compounds
in freeze-substituted biological specimens discussed be-
low.
Plant-Fungi Interactions. During recent years ex-
tensive new information has been obtained about
freeze-substituted fungal infection structures inside
the plant tissue. These studies have led t o a better
understanding of the physiology and fine structure of
the host-parasite interface. As an example, the im-
Figs. 10 and 11. Young stages of conidia of powdery mildew pro-
cessed according to Hippe (19851. Compare low temperature prepara-
tion protocol described in Technical Process section.
Fig. 10. Enlarged view of the cytoplasm of a conidium of powdery
mildew. Numerous glycogen particles (g) are seen. Bar = 1 km.
Fig. 11. Enlarged section of the periphery of the conidial cyto-
plasm of Erysiphe graminis f.sp. hordei exhibiting bundles of micro-
filaments (mf) and mitochondria (m). Bar = 0.25 km.
Figs. 12 and 13. High pressure frozen, freeze-substituted and Low-
icryl HM2O-embedded leaf tissue of Arabidopsis thaliana. Bars = 5
km.
Fig. 12. Homogenousiy structured mesophyil tissue of Arabidop-
sis thaliana. Large areas of intercellular space (IC) are seen in be-
tween the cells. The mesophyll cytoplasm containing many chloro-
plasts (C) is closely attached to the cell wall.
Fig. 13. Section of epidermal tissue ofArabidopsis thaliana exhib-
iting excellently preserved stomata and epidermal cells. Chloroplasts
(C), nuclei (n) and vacuoles (vj are seen in the cytoplasm of the sto-
mata cells.
IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM 411

Figs. 10-13.
412 S. HIPPE-SANWALD
proved ultrastructure of freeze-substituted haustoria of
Erysiphe graminis f.sp. hordei grown on compatible
barley epidermal tissue is described in detail (Figs. 19-
23). Bipolar, finger-like haustoria of Erysiphe graminis
fsp. hordei being surrounded by a sheath (extrahaus-
torial sheath membrane and sheath matrix) are char-
acteristically formed only in epidermal tissue (Fig. 19).
The extrahaustorial membrane as well as the profile of
the plasmalemma and the organelles (mitochondria,
nucleus, etc.) appear sharply resolved. The cytoplasm
including ribosomes, glycogen, etc. is smoothly con-
toured. Aggregations of haustorial glycogen resemble
in size and shape the particles found in conidia (Figs.
10, 11, and 22). The haustorial cell wall is character-
ized by an outer electron-dense layer (Figs. 20 and 21).
Occasionally, the sheath matrix contains fibrillar ma-
terial accumulated around the haustorium (Fig. 21).
Recently, polyclonal rabbit anti-THRGP antibodies of
Zea mays (i.e., antibodies against monocotyledonous
maize extensin, a threonin-hydroxyproline-richglyco-
protein; Kieliszewski and Lamport, 1986) have been
proved to be cross-reactive against interfacial material
of the extrahaustorial matrix as well as against com-
ponents of the outer cell wall of haustoria of powdery
mildew on barley in the compatible and incompatible
interaction. So far, the existence of THRGP-like
epitopes in the extrahaustorial matrix, which may
originate from the host plant, cannot be excluded
(Hippe et al., 1991b). Similar results on an accumula-
tion of THRGP-like epitopes at the host-pathogen in-
terface have been obtained in various rust systems
such as in Puccinia graminis f.sp. tritici and in Puc-
cinia recondita on compatible and incompatible wheat
leaf tissue (Hippe et al., 1991b). An example of dense
immunogold labeling of the extrahaustorial matrix of
HP-frozen, freeze-substituted and Lowicryl HM2O-em-
bedded stem rust haustoria developed on compatible
wheat tissue is shown in Figure 23. Thus, freeze sub-
stitution in combination with adequate freezing tech-
niques permits both: a more satisfactory evaluation of
cytological as well as molecular events occurring in the
compatible and incompatible plant pathogen interac-
tion (Hippe et al., 1991a,b).
However, the data so far available in literature are
mostly focussed on the compatible host-parasite inter-
action. Freeze-substituted fungal infection structures
like appressoria, intercellular hyphae and haustoria
are very clearly visualized. Ultrastructural studies
have been performed on appressoria of Magnaporthe
grisea (Howard et al., 19911, on intercellular hyphae of
various rust fungi (Harder et al., 1989) and fungal
haustoria: e.g., the apple scab fungus Venturia inae-
qualis on apple leaves (Dahmen and Hobot, 1986;
Studer et al., 1988, 1989), the bean rust Uromyces ap-
pendiculatus on bean (Welter et al., 1988; Knauf et al.,
1989; Mendgen et al., 19911, and wheat stem rust Puc-
cinia graminis fsp. tritici (Figs. 24-27).
A comprehensive description of rust haustoria by
Harder and Chong (1991) and Mendgen et al. (1991) is
based on modern methodology. The reviewers appreci-
ate the progress of the fine structural preservation of
the interface between the two organisms as a prereq-
uisite for understanding host-parasite interactions. It
has to be emphasized that for satisfactory preservation
of fungi growing deeper in the parenchymal plant tis-
sue, freeze substitution is only worthwhile using high
pressure frozen material (Studer et al., 1988, 1989;
Welter et al., 1988; Knauf et al., 1989; Mendgen et al.,
1991). Freeze substitution shows the extrahaustorial
matrix and the haustorial cell wall of biotrophic rust
fungi to be clearly differentiated from the host cyto-
plasm (Knauf et al., 1989; Mendgen et al., 1991; com-
pare also Figs. 23 and 25). The extrahaustorial matrix
of rust fungi contains a variety of substances, including
polysaccharides and glycoproteins, being visualized by
immunogold labelling (Harder and Chong, 1991).
Preliminary results on small areas of adequately
preserved fungal infection structures are available for
wheat leaves infected by the wheat stem rust fungus
Puccinia graminis f.sp. tritici (Figs. 24-27). In Figure
24, an infected mesophyll cell of a wheat leaf is shown
containing a round-shaped haustorium and a section of
the haustorial mother cell. The haustorial neck is char-
acterized by an electron-dense neck band. The fungal
nucleus has not yet penetrated into the young hausto-
rium. As mentioned above, the host cytoplasm and the
rust haustorium are separated by the extrahaustorial
membrane and the extrahaustorial matrix. Similar to
Uromyces appendiculatus (Knauf et al., 1989) the ex-
trahaustorial membrane of Puccinia graminis fsp. trit-
ici is not undulated and the side facing the plant cyto-
plasm is lined by a delicate fringe of well stained
fibrillar material (Fig. 25). The extrahaustorial matrix
in between the extrahaustorial membrane and hausto-
rial cell wall is composed of fibrillar material homoge-
Figs. 14-16. Low temperature processed transgenic tobacco stem
tissue and tobacco callus tissue for comparison (Hippe et al., 1989).
Fig. 14. Overview over freeze-substituted tobacco stem tissue. The
plant cytoplasm (cy) and the chloroplasts (c) appear closely attached
to the cell wall. The intercellular space (IC) contains divers fibrillous
material. Bar = 2 pm.
Fig. 15. Plant cell nucleus (n) containing heterogeneously struc-
tured chromatin and several nuclear crystals (nc). Improved preser-
vation since no artefact-like aggregations are visible. Bar = 1 pm.
Fig. 16. In situ localization of the foreign protein T4 lysozyme (ly)
in transgenic tobacco stem tissue. Synthesized and secreted T4
lysozyme is found predominantly in the intercellular spaces of freeze-
substituted tissue by using a polyclonal rabbit anti-T4-lysozyme an-
tibody and the protein A gold technique. Bar = 0.5 pm.
Fig. 17. Freeze-substituted tobacco callus tissue (processed accord-
ing to Hippe et al., 1989) exhibiting irregular shape of the chloro-
plasts (c) as well as several mitochondria (m); the rough endoplasmic
reticulum (er) and a Golgi body (90) are visualized. Bar = 0.5 pm.
Fig. 18. Electron micrograph of growth plate cartilage of rat
epiphyseal tissue processed by high pressure freezing, freeze substi-
tution, and Lowircyl embedding (Hunziker and Herrmann, 1987). The
plasmalemma (PI) of a chondrocyte (ch) is cut perpendicularly. The
proteoglycan network forms the pericellular matrix. Intimate contact
between the pericellular matrix and the cell is established. The pre-
cise localization and dense distribution of proteoglycan based on a
high immunosensitivity of the freeze-substituted material is shown
by immunocytochemistry. Bar = 0.1 pm. The inset shows an en-
larged region of improved preserved fine structure of proteoglycan
homogeneously immunogold labeled. Bar = 0.025 pm.
IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM 413

Figs. 14-18.
414 S. HIPPE-SANWALD

neously distributed. Preliminary studies on the local- occurs to a comparatively high amount in vacuoles of
fungicide-resistant strains of Ustilago auenae (Hippe,
ization of a n elicitor-active glycoprotein that elicits the
resistance reaction in wheat indicate labeling of the 1987). The intracellular localization of the unbound,
haustorial cell wall and the extrahaustorial matrix diffusible organic agent is achieved by performing
(Marticke et al., 1991). However, the density of detect- freeze substitution and embedding in Lowicryl HM20
able antigenic sites is considerably increased by using a t 213 K in combination with high resolution auto-
freeze substitution instead of conventional fixation pro- radiography. In a similar study, characteristic accumu-
cedures (Figs. 26 and 27). The advantages of freeze lations of [3H]Triadimenol a t the infection sites (pa-
substitution in combination with immunocytochemis- pillae) of powdery mildew Erysiphe graminis f s p .
try are discussed below. hordei on barley leaves have been found (Hippe and
Hermanns, 1988). Since the molecular basis of the
Molecular Characterization of the Cell resistance mechanism against sterol biosynthesis-in-
Apart from the aim of improving fine structure by hibiting fungicides in fungi is still under discussion,
freeze substitution, the question arises a s to whether localization studies on the distribution pattern of mo-
this technique can be used for a molecular evaluation bile chemical agents are particularly important and
of biological material. In this section, the discussion is permit a deeper understanding of cellular detoxifica-
focussed on the effectiveness of freeze substitution in tion processes. With respect to lipid retention in Usti-
combination with LT embedding for the retention of lago auenae sporidia, the results on the improved visu-
diffusible substances such as various organic com- alization of lipid droplets containing most of the
pounds, lipids, ions and proteins by autoradiography, fungicidal agent are supported by data on the preser-
microanalysis and immunocytochemistry, respec- vation of pure lipid vesicles (Verkleij et al., 1985) and
tively. the retention of liposomal [3H]dipalmitoyl-phosphati-
Retention of Diffusible Components. For the in- dylcholine over epithelial plasma membrane monolay-
tracellular localization of water-soluble and diffusible ers (Knoll et al., 1988).
components of the native cell, different combinations of Even though the suitability of freeze substitution fol-
methods are known. Electron-probe microanalysis and lowed by X-ray analytical electron microscopy has been
autoradiography including adequate preparation tech- successfully proven for the identification of water-
niques like dry-mount and thaw-mount techniques, soluble ions and salts in various plant and animal spec-
frozen hydrated cryosections as well as plastic sections imens, controversial opinions on this method exist
of freeze-dried a s well as ultrarapidly frozen, freeze-
substituted and Lowicryl-embedded material have suc-
cessfully been applied to evaluate the lifelike distribu-
tion of mobile substances on the electron microscopic Figs. 19-23. Ultrastructure of haustoria of Erysipphe graminis
level (Edelmann, 1991; Frederik, 1987; Fisher and f.sp. hordei preserved by freeze substitution (Hippe, 1985).
Housley, 1972; Harvey, 1982; Hippe, 1987; Knoll et al.,
1988; Stumpf, 1976; Verkleij et al., 1985; Wroblewski, Fig. 19. General organization of a longitudinally sectioned haus-
1989; Zierold and Steinbrecht, 1987). torium (H) located in a barley epidermal cell. In the compatible in-
teraction, most of the host cytoplasm (hcy) remains attached to the
In situ localization while avoiding translocations and plant cell wall; only some host cytoplasm is arranged around the
loss of the diffusible compounds is regarded as the main haustorium. The haustorium (H) consists of the haustorial body (HB)
goal of all these techniques (Steinbrecht and Muller, with a bipolar arrangement of finger-like haustorial lobes (HF). The
1987). This cannot be achieved by conventional fixa- entire haustorium is surrounded by a sheath (s) composed of the
sheath membrane, a n extrahaustorial membrane (em) and the extra-
tion and dehydration procedures since most elements of haustorial matrix (ema). Bar = 3 km.
biological interest are quantitatively extracted from
the tissue under these conditions. Generally, fixatives Fig. 20. Enlarged view on haustorial fingers exhibiting some cy-
rapidly permeabilize the plasma membrane leading to toplasmic details: mitochondria (m), a sharply resolved plasmalemma
(pl), ribosomes, and multivesicular bodies (arrowheads).The cell wall
a leakage of cellular ions a t room temperature during (cw) of the haustorium is characterized by an outer electron-dense
dehydration. In particular, lipids are dissolved in all layer more or less loosely structured. Bar = 1 p m .
current organic dehydration liquids and monomeric
resins a t room temperature unless they are fixed with powderyFig. 21. Interface of the infection site between the haustorium of
mildew and the plant epidermal cell: the sheath (s) contains
OsO, (Weibull et al., 1983). Aldehyde treatment does fibrillar extrahaustorial matrix (ema) surrounded by the extrahaus-
not prevent solubilization of lipids in organic solvents. torial sheath membrane (em). Host cytoplasm (hcy) is visible closely
However, when the temperature is lowered below attached to the epidermal cell wall and in the vicinity of the hausto-
about 200 K, most lipids become insoluble and are de- rium (arrowhead). Bar = 1 Fm.
tectable on the ultrastructural level (Weibull and Fig. 22. Haustorial finger containing aggregations of glycogen (g)
Christiansson, 1986). On the other hand, the visualiza- particles arranged in the periphery Qf the cytoplasm. Bar = 2 pm.
tion of lipids is improved either by adding uranyl ace-
Fig. 23. Immunocytochemical localization of threonine-rich exten-
tate to methanol, by using complex substitution media, sin-like epitopes (THRGP; ex) in the extrahaustorial matrix (ema) of
o r by combining freeze substitution with low tempera- haustoria (H) of Puccinia graminis f.sp. tritici grown on compatible
ture embedding in Lowicryl HM20 (Humbel et al., wheat tissue. The haustorial cell wall (cw) is of low contrast. The well
1983; Muller et al., 1980; Verkleij et al., 1985). resolved extrahaustorial membrane (em) separates host and fungal
In this context, one example is presented. Figure 5 interface. In the host cytoplasm (hcy) a small section of a chloroplast
(c) is visible. Immunogold labeling by using a polyclonal rabbit anti-
shows that the accumulation of the sterol biosynthesis THRGP antibody; method according to Hippe et al. (1989) and Hippe-
inhibiting fungicide [3HlTriadimenol in lipid droplets Sanwald et al. (1991b). Bar = 0.25 IJ-m.
IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL EM 415

Figs. 19-23.
416 S. HIPPE-SANWALD
(Edelmann 1989, 1991; Harvey, 1982; Ross et al.,
1983; Wroblewski and Wroblewski, 1984; Wroblewski,
1989). Microanalytical results obtained from freeze-
substituted tissue are compared with X-ray investiga-
tions based on freeze-dried, Lowicryl-embedded or fro-
zen-hydrated material. In general, ultrarapid freezing
followed by cryosectioning a t low temperatures, as well
as freeze drying and Lowicryl embedding under vac-
uum conditions are recommended as methods of choice
for the retention of mobile ions in their in vivo state
(Wroblewski, 1989).
Nevertheless, promising data are available from lit-
erature on the retention of water-soluble components
by a modified composition of the substitution fluid, and
subsequent embedding under low temperature condi-
tions (Edelmann, 1989, 1991; Harvey, 1982; Ornberg
and Reese, 1981; Wroblewski and Wroblewski, 1984;
Van Zyl et al., 1976). In one of these experiments, the
cellular ion content has been shown to be better re-
tained in freeze-substituted or freeze-dried tissue
which was embedded in Lowicryl HM20 than in sam-
ples embedded in conventional epoxy resins (Wrob-
lewski and Wroblewski, 1984). Recent results pub-
lished by Edelmann (1989, 1991) indicate that freeze
substitution of biological material in pure acetone fol-
lowed by embedding in Lowicryl K l l M or HM23,
yields suitable preparations for microanalytical studies
on changes of the ultrastructure, the redistribution of
cellular water and intracellular movements of mobile
ions during muscle contraction. Cellular ions are re-
tained a t the same subcellular places as in frozen-hy-
drated and freeze-dried preparations (Edelmann,
1991). Obviously, the retention of diffusible cellular
ions in frog sartorius muscle is dependent on their in-
teractions with cellular macromolecules. The ions are
not homogeneously distributed in the cellular water,
but probably adsorbed to carboxyl groups of proteins.
By optimizing the freeze substitution and embedding
conditions, these features can be clearly defined (Edel-
mann, 1991).
Retention of Protein Antigenicity. In modern cell
biology, there is great interest in the subcellular local-
ization of unbound and bound proteins with structural
and/or enzymatic function by immunocytochemistry.
The most important prerequisite for protein detection
is a n adequate processing of the biological material. In
order to obtain reliable results, some important criteria
have to be considered: maintenance of protein antige-
nicity, immobilization of the antigen, preservation of
the cellular fine structure, accessibility of the antigens
and fixation of fast processes (Voorhout et al., 1989).
Dehydration a t progressively lower temperature af-
ter mild, conventional chemical fixation (the PLT tech-
nique) is mostly used in combination with immunogold
labeling (Bendayan et al., 1987; Roth et al., 1981).
However, aldehyde and OsO, fixation may destroy an-
tigenic sites, although in some cases the sensitivity of
epitopes appears not to seriously affect immunoreactiv-
ity (Acker and Kammerer, 1990; Bendayan and
Zollinger, 1983; Ichikawa et al., 1989; Roth et al., 1981;
Voorhout et al., 1989). This means that generally, the
conditions for optimum labeling must be worked out for
each class of binding site, since any manipulation of
the biological tissue during processing can result in
decreasing the maintenance of antigenicity (Bendayan
et al., 1987).
Cryofixation followed by freeze substitution and LT
embedding in Lowicryl appears particularly suited to
fulfil the aforementioned requirements for optimum
preparation. This procedure yields superior preserva-
tion of the general fine structure, with simultaneous
improvements in resolution, intensity, and specificity
of the immunogold staining. In particular, LT prepa-
ration prevents major conformational changes of pro-
teins, leading to better retention of protein antigenicity
(Bendayan, 1984; Kellenberger et al., 1987; Ichikawa
et al., 1989; Monaghan and Robertson, 1990). Presum-
ably, the hydration shells of proteins are better pre-
served a t low temperatures which enhance the acces-
sibility of antigenic sites (Kellenberger, 1987;
Kellenberger et al., 1987). Immobilization of diffusible
antigens and fixation of fast metabolic processes is
achieved by the preparation under LT conditions. How-
ever, for some specific problems, cryoultramicrotomy or
freeze drying in combination with ultrarapid freezing
and LT embedding is favored as the method of choice
(Jorgensen and McGuffee, 1987; Voorhout et al., 1989).
In particular, cryo-ultramicrotomy is sometimes pre-
ferred since it is less time-consuming and the ultra-
structural preservation is sufficient (Voorhout et al.,
1989).
Meanwhile, many promising results are available on
Figs. 24-27. Haustorial plant-pathogen interface in stem rust
(Puccinia graminis f.sp. tritici) infected mesophyll cells of susceptible
wheat processed by high pressure freezing, freeze substitution and
subsequent Lowicryl HM20 embedding according to section entitled
Technical Process; Protocols.
Fig. 24. Infection site containing a young haustorium (H) being
connected with the haustorial mother cell (HMC) located outside the
mesophyll cell. At the haustorial neck a n electron-dense neck band
(nb) is visible. The haustorium is enclosed by smooth host cytoplasm
(hcy). Bar = 1 pm.
Fig. 25. Haustorium of Puccinia graminis f.sp. tritici on compati-
ble wheat cultivar Little Club showing the host-parasite interface at
higher magnification. The host cytoplasm (hcy) containing a chloro-
plast (c) is separated from the extrahaustorial matrix (ema) by the
distinctly resolved extrahaustorial membrane (em). Small vesicles
are frequently seen at the extrahaustorial membrane. The extrahaus-
torial area is characterized by a homogeneously structured matrix
(ema) closely attached to the outer haustorial cell wall layer (cw).The
haustorial cell wall is composed of two layers, a n inner electron trans-
lucent layer and a n outer electron dense layer (cw). Bar = 0.25 pm.
Fig. 26. Evidence for the localization of elicitor-active glycopro-
teins in the outer haustorial (HI cell wall layer (cw; arrowheads) of
Puccinia graminis f.sp. tritici in a compatible wheat stem rust inter-
action after freeze substitution. There is also evidence for the release
of the elicitor glycoprotein into the extrahaustorial matrix (arrow-
heads). Immunogold labeling according to Hippe et al. (1989) and
Marticke et al. (1992) by using monoclonal mouse anti-elicitor anti-
bodies and the colloidal gold technique. Bar = 0.25 pm,
Fig. 27. Unlike Figure 24, less labelling (arrowheads) is shown in
the haustorial (H)cell wall (cw) after the use of the “progressive
lowering temperature” (PLT)method (compare Bendayan et al., 1987)
and embedding in Lowicryl HM20. To demonstrate the decreased
number of particles, no poststaining of the sections has been done. Bar
= 0.5 pm.
IMPACT OF FREEZE SUBSTITUTION ON BIOLOGICAL E M 417

Figs. 24-27
418 S. HIPPE-SANWALD
various biological materials: bacteria and fungi, a s
well as plant and animal tissue. Only a few selected
examples of the wide range of applications are dis-
cussed in the following section with particular regard
to the progress made by freeze substitution in biologi-
cal immunoelectron microscopy.
Carlemalm et al. (1986) clearly demonstrated a n in-
creased labeling of cytoplasmic proteins in Escherichia
coli cells after freeze substitution as compared to PLT
preparation. Enterobacterial cell surface glycolipid
precursors have been proven to be real constituents of
the cytoplasm also occurring in the inner and outer
cytoplasmic membrane of Escherichia coli (Acker and
Kammerer, 1990). Since soluble compounds are immo-
bilized by the LT method, displacement of the cytoplas-
mic antigen has been excluded. Using the PLT tech-
nique, translocations of diffusible cellular antigens
cannot be prevented because of the slowness of fixation
(Acker and Kammerer, 1990). In a similar approach,
improved retention of the antigenicity of labile lu-
ciferase epitopes of the bioluminescent bacterium
Vibrio harveyi has been revealed after freeze substitu-
tion (Nicolas et al., 1989).
In the field of mycology and plant pathology, a n in-
creasing number of investigations are dealing with the
immunocytochemical characterization of fungal anti-
genic sites. Applying antiserum specific to freeze-sub-
stituted hyphae of the coniferous, endophytic fungus
Lophodermium piceae, this combined method can be
used for the in situ identification of the fungus in ap-
parently healthy host tissue of Norway spruce needles
(Suske and Acker, 1989).
Several types of extramural substance(s) associated
with the intercellular hyphae of various rust fungi
have been localized using freeze substitution of HP-
frozen material (Harder et al. 1989). Considerably less
material has been discerned on the hyphal surface af-
ter conventional preparation. A similar fixation sensi-
tivity has been found in studies on the localization of
a n elicitor-active glycoprotein in the haustorial cell
wall and the extrahaustorial matrix of the stem rust
Puccinia graminis fsp. tritici infecting wheat leaves.
As shown in Figure 27, considerably fewer antigenic
sites have been discerned in material processed by the
PLT technique in comparison to LT prepared tissue
(Fig. 26). In the latter case, even some distinct labeling
of the extrahaustorial matrix is visible, indicating dis-
location of elicitor molecules from the fungal cell wall
to the plant-pathogen interface. This is the first report
on successful application of specific monoclonal anti-
bodies for the localization of elicitor-active glycopro-
teins in the haustorial cell walls of the wheat stem rust
Puccinia graminis fsp. tritici growing in compatible
wheat leaf tissue after H P freezing, freeze substitution
and Lowicryl HM20 embedding. Even after these pre-
liminary results, it becomes evident that molecular
components of the extrahaustorial matrix originate
from the haustorial cell wall of the rust fungus. Pre-
sumably, these constituents play a n important role in
the plant pathogen interaction by triggering the plant
resistance response in a still unknown way.
Powdery mildew, Erysiphe graminis f s p . hordei on
cereal is another important plant pathogen system be-
ing extensively investigated. Host cell wall extensin
enhancement (hydroxyproline-rich glycoprotein;
HRGP) has been demonstrated biochemically in com-
patible wheat powdery mildew infections (Clarke et al.,
1981). According to cross-reactivity of anti-THRGP-an-
tibodies against barley infected by Erysiphe graminis
f.sp. hordei, the immunocytochemical localization of
THRGP-like epitopes has been recently performed on
the basis of LT preparation (Hippe et al., 1992b;
Kieliszewski et al., 1990). The superior maintenance of
antigenicity is exhibited over a n electron-dense outer
haustorial cell wall layer and the extrahaustorial ma-
trix (compare also Fig. 23). Further interpretations of
the results are given by Hippe et al. (1991b).
In plant molecular biology, some current investiga-
tions concerning the localization and the transport of
bound and unbound cellular components make use of
the newly developed LT preparation techniques in com-
bination with immunoelectron microscopy. Due to the
high amount of cellular water, cryofixation by HP
freezing prior to freeze substitution of the plant mate-
rial is particularly important for the immunodetection
of labile cytoskeletal elements (Lancelle and Hepler,
1989; Lichtscheidl et al., 1990) as well as for the iden-
tification of soluble compounds in the intercellular
space (Hippe et al., 1989). Evidence exists that novel
information about processing and localization of chi-
meric proteins in transgenic plant tissue can only be
obtained by a combined molecular and cell biological
approach. The introduction of LT methods in conjunc-
tion with immunogold labeling has proven to be suc-
cessful for the in situ localization of the foreign protein
T4 lysozyme fused to a plant signal peptide in trans-
genic tobacco stem tissue (Hippe et al., 1989). By this
method, superior preservation of ultrastructural de-
tails of the plant cells (Figs. 14-16) and excellent re-
tention and antigenicity of the soluble polypeptides is
possible. In Figure 16, fibrillar, network-like struc-
tures are shown densely immunostained. In a similar
study, the detection of the synthesis and assembly of
mammalian immunoglobulin light and heavy chains in
the cytoplasm of transgenic tobacco tissue using spe-
cific secondary antibodies has been also achieved by
the aforementioned techniques (During et al., 1990).
Animal tissue appears to be particularly well suited
for demonstrating the advantages of freeze substitu-
tion for immunostaining of intra- and extracellular
proteins (Humbel et al., 1983; Hunziker and Herr-
mann, 1987; Ichikawa et al., 1989; Menco, 1989;
Monaghan and Robertson, 1990). In a n early study,
Humbel et al. (1983) succeeded in localizing M-line pro-
tein in muscle cells. The precise localization together
with successful immobilization of extracellular proteo-
glycan within r a t cartilage matrix is another example
of the efficiency of this method (Hunziker and Herr-
mann, 1987). In Figure 18, the high immunosensitiv-
ity of gold-labeled proteoglycan using monoclonal an-
tibodies against chondroitin sulfate glycosaminoglycan
is presented. Positive immunostaining of fixation-
labile antigens has been obtained in freeze-substituted
human kidney, human breast tumour cell lines, and
rat olfactory epithelia in the complete absence of chem-
ical fixatives during freeze substitution (Menco, 1989;
 IMPACT OF FREEZE SUBSTITUTION O N BIOLOGICAL EM
Monaghan and Robertson, 1990). As demonstrated by
Ichikawa et al. (1989) amylase labeling density over
the secretory granules of the cryofixed gerbil parotid
gland examined is about 1.5 times or more as high as in
those processed by conventional chemical fixation.
CONCLUSION AND PERSPECTIVES
Low temperature preparation techniques including
ultrarapid freezing, freeze substitution, and LT embed-
ding can be regarded as important tools in biological
electron microscopy. This combined method offers a
deeper insight into the architecture and the molecular
substructure of a biological cell. In this context, freeze
substitution is one important step during the transfer
of a frozen-hydrated specimen into the resin-embedded
state. This means that the quality of the sample ana-
lyzed under the electron microscope is not only deter-
mined by the feasibility of adequate ultrarapid freezing
techniques, but is particularly influenced by the freeze
substitution process itself. The high sensitivity of im-
munocytochemical labeling techniques and the possi-
bility of performing localization studies of proteins and
other cell compounds at the molecular level using
freeze-substituted objects offer new perspectives for a
combined cell and molecular biology. As an example,
the pathway and fate of a protein in the cell as well as
the spatial identification of compounds being possibly
involved in the cellular signal transduction during a
host-parasite interaction can be analyzed on the sub-
cellular level. Furthermore, the potential of this
method with regard to the in situ characterization of
genes and the localization of even transient gene prod-
ucts, including the study of foreign proteins in trans-
genic biological systems, is becoming increasingly sig-
nificant. On the basis of these new methodological
developments, a deeper understanding of the structure-
function relationship in biology can be achieved.
ACKNOWLEDGMENTS
Financial support of the Dr. Christian Drager Stif-
tung (Lubeck, Germany) is gratefully acknowledged.
The author gratefully acknowledges Dr. E.B. Hunziker
(University of Bern, Switzerland) for supplying one mi-
crograph used in this review. The valuable technical
assistance of Petra Sedlag, Monika Hermanns, Karl-
Heinz Marticke, Andreas Bentz, and Volker Heeschen
are greatly appreciated. Thanks are due to Dr. E. San-
wald for reading the manuscript.
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