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From Willi Salvenmoser, Electron Microscopy of Flatworms:
Standard and Cryo-Preparation Methods.
In: Thomas Müller-Reichert, editor,
Methods in Cell Biology (Volume 96).
Academic Press, 2010, p. 307.
ISBN: 978-0-12-381007-6
© Copyright 2010, Elsevier Inc.
Academic Press.
Author's personal copy
CHAPTER 14
Electron Microscopy of Flatworms:
Standard and Cryo-Preparation Methods
Willi Salvenmoser*, Bernhard Egger*, Johannes G. Achatz*,
Peter Ladurner*, and Michael W. Hess†
*
Center for Molecular Biosciences, Institute of Zoology, University of Innsbruck, Innsbruck, Austria
†
Division of Histology and Embryology, Innsbruck Medical University, Innsbruck, Austria
Abstract
I. Introduction
A. Systematics, Phylogeny, and Morphology
B. Flatworms as Models
II. Rationale
III. Methods
A. Chemical Fixation—General Aspects
B. Chemical Fixation—Protocols
C. Cryo-Processing—General Aspects
D. Cryo-Processing—Protocols
IV. Materials
A. Animals
B. Chemical Fixation
C. Cryo-Processing
V. Results and Discussion
A. Chemical Fixation
B. Cryo-Processing
VI. Concluding Remarks
Acknowledgments
References
Dedicated to the memory of our mentor and friend Reinhard Rieger.
METHODS IN CELL BIOLOGY, VOL. 96
Copyright � 2010 Elsevier Inc. All rights reserved.
307
978-0-12-381007-6
DOI: 10.1016/S0091-679X(10)96014-7
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Willi Salvenmoser et al.
Abstract
Electron microscopy (EM) has long been indispensable for flatworm research, as
most of these worms are microscopic in dimension and provide only a handful of
characters recognizable by eye or light microscopy. Therefore, major progress in
understanding the histology, systematics, and evolution of this animal group relied
on methods capable of visualizing ultrastructure. The rise of molecular and cellular
biology renewed interest in such ultrastructural research. In the light of recent devel­
opments, we offer a best-practice guide for users of transmission EM and provide a
comparison of well-established chemical fixation protocols with cryo-processing
methods (high-pressure freezing/freeze-substitution, HPF/FS). The organisms used in
this study include the rhabditophorans Macrostomum lignano, Polycelis nigra and
Dugesia gonocephala, as well as the acoel species Isodiametra pulchra.
I. Introduction
Flatworms (= Platyhelminthes sensu lato) comprise a diverse group of organisms
ranging from free-living taxa to human and animal parasitic groups. For a long time,
they have been at the center of regeneration research, being called “almost immortal
under the edge of the knife” (Dalyell, 1814). Some flatworms are able to regenerate all
body parts from tiny tissue fragments—including the head (Egger et al., 2006;
Montgomery and Coward, 1974). While not all flatworms are able to regenerate
equally well, most species can restore at least the posterior part of the body behind
brain or pharynx (Egger et al., 2007).
Regeneration in flatworms is made possible by totipotent stem cells, called
neoblasts (Ladurner et al., 2008). These undifferentiated cells have a high nucleus­
to-cytoplasm ratio (Bode et al., 2006) and are the only proliferating cells in juvenile
and adult flatworms. For studying neoblasts during regeneration, growth, and tissue
maintenance, small and transparent flatworms in the “millimeter range”, such as
Macrostomum lignano and Isodiametra pulchra, have recently been introduced to
research in addition to the traditionally used large (centimeter range) and pigmented
triclads, often called “planarians” (De Mulder et al., 2009; Egger et al., 2007).
A. Systematics, Phylogeny, and Morphology
Molecular studies suggest that the Platyhelminthes sensu strictu, comprising the sister
taxa Catenulida and Rhabditophora, belong to the Lophotrochozoa, whereas the Acoe­
lomorpha represent a separate offshoot at the base of the Bilateria (Hejnol et al., 2009).
However, this issue is not entirely settled yet (Egger et al., 2009b). Because all these
organisms are processed similarly for EM, the acoels are also included in this chapter.
Within the Platyhelminthes the large taxon of the Rhabditophora is characterized by the
presence of rhabdite glands and is composed of the Macrostomorpha, Polycladida, and
the Neoophora, including among others the Tricladida and the parasitic Neodermata
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(Ehlers, 1985). The latter comprises the tapeworms, the flukes (e.g., the blood fluke
Schistosoma mansoni that causes bilharzia and the liver fluke Fasciola hepatica that
affects sheep, cattle, and human), and the ectoparasitic monogeneans that cause problems
in aquaculture.
Flatworms are acoelomate, bilaterally symmetric animals that primarily lack seg­
mentation, a circulatory system, respiratory organs, and an anus (Rieger et al., 1991).
The cephalized nervous system consists of an anteriorly positioned brain and poster­
iorly extending longitudinal nerve cords, interconnected by transverse commissures.
Flatworms possess an elaborate muscle system consisting of longitudinal, circular, and
oblique fibers. Small free-living species primarily use their epidermal cilia for locomo­
tion, assisted by muscular contractions in larger forms. The organization of the gut is
sometimes reflected in the name of a flatworm clade, as in the stunningly beautiful
marine polyclads, which possess a highly branched diverticular gut, the triclads, which
exhibit three gut branches, or the rhabdocoels with a rod-shaped gut. Many acoel
flatworms have no gut lumen at all but are characterized by a compact central digestive
syncytium. The tapeworms (Cestoda) have completely abolished their gut, taking up
nutrients through their epidermis. Unpaired (Catenulida) and paired (Rhabditophora)
protonephridia with “flame cells” are responsible for osmoregulation, but are absent in
the basal group of the Acoelomorpha. Most flatworms are sexually reproducing
hermaphrodites (one prominent exception is the diecious S. mansoni), while other
species are asexual and multiply by fission.
B. Flatworms as Models
Except for stable transgenic cell lines, the whole molecular “toolbox” is available for
the flatworm models. Additionally, small flatworms are exceptionally amenable to
whole-mount immuno-cytochemistry, i.e., for studying stem cells and other tissues,
such as musculature, nervous system, epidermis, gut, and various glands (Egger et al.,
2009a; Inoue et al., 2004; Ladurner et al., 2005).
Among acoel flatworms, three species have emerged as model systems, particularly
for developmental studies: the small (0.5–1 mm in length) Isodiametra pulchra (De
Mulder et al., 2009; Ladurner and Rieger, 2000) and the relatively large species of
Convolutriloba longifissura (up to 6 mm in length) (Åkesson et al., 2001; Gaerber et
al., 2007; Hejnol and Martindale, 2008) and Symsagittifera roscoffensis (4 mm in
length) (Moreno et al., 2009; Semmler et al., 2008), the latter two having symbiotic
green algae in the parenchyma.
Unfortunately, in the Catenulida, no model system has been established so far. In the
largest group of flatworms, the rhabditophorans, a couple of models have been used
extensively, such as Macrostomum lignano*, Schmidtea mediterranea*, Dugesia japo­
nica*, Girardia tigrina, Polycelis nigra, Echinococcus granulosus*, and Schistosoma
mansoni*. Whole genomes are being sequenced or already available for all species
marked with an asterisk.
In recent years, Schmidtea mediterranea (Salo et al., 2009) and Macrostomum
lignano (Ladurner et al., 2005) in particular have emerged as promising model systems
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Willi Salvenmoser et al.
for embryonic development (Morris et al., 2004; Sanchez Alvarado, 2003), regenera­
tion (Egger et al., 2006; Forsthoefel and Newmark, 2009; Sanchez Alvarado, 2006),
reproductive biology research (Janicke and Scharer, 2009; Newmark et al., 2008),
ageing (Mouton et al., 2009), and stem cell research (Ladurner et al., 2008; Palakodeti
et al., 2008).
II. Rationale
Here we discuss the most appropriate protocols for thin-section EM for ultra­
structure and immuno-cytochemistry studies. All three major systematic groups are
covered, including marine and freshwater flatworms. In particular, chemical fixation
as well as cryo-based sample processing (high-pressure freezing/freeze-substitution,
HPF/FS) is described for the triclads Polycelis nigra, Dugesia gonocephala, the
macrostomorph Macrostomum lignano, and the acoel species Isodiametra pulchra1.
III. Methods
A. Chemical Fixation—General Aspects
Simultaneous use of glutaraldehyde and osmium tetroxide (OsO4), even with
low concentrations of OsO4, gives best results for marine and freshwater species.
We recommend cacodylate buffer-based fixatives for marine organisms for
avoiding the so-called “fixation pepper” (i.e., artifactual precipitation of electrondense substances, see Mollenhauer, 1988). The concentration of buffers used is also
crucial. For freshwater organisms, buffer concentration should be low (e.g., 0.01–
0.05 M) to prevent shrinking artifacts (Gremigni and Falleni, 1991). Some authors,
however, recommend higher concentrations for proper buffering (Griffiths, 1993), espe­
cially if the primary fixative does not contain OsO4. For marine organisms, sucrose and/
or sodium chloride is added to the cacodylate buffer to increase molarity. Magnesium
chloride used for relaxation also provides protection of membranes in marine species
(Glauert and Lewis, 1998). However, traces of ions should be added in a millimolar
concentration to fixatives for freshwater species. As a complement to aforementioned
simultaneous fixation, the consecutive application of glutaraldehyde and OsO4 also gives
acceptable results except for stem cell morphology, and is convenient for “field work”. In
general, marine organisms are easier to fix than freshwater animals, as long as the
fixative’s molarity is equivalent to seawater. Another important parameter in addition
to the size and the salinity of the natural environment is the presence and amount of
water-filled spaces within the body (e.g., chordoid vacuoles in acoels), the latter making
1
This contribution is meant as a practical instruction for laboratory work. Thus, the literature list is not
exhaustive and we apologize to all whose work—though related to the topic—could not be included. For
ease of access to relevant literature we cited preferentially original papers and—when necessary—book
chapters.
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a proper fixation sometimes more difficult to accomplish. Due to their soft body,
relaxation of flatworms prior to fixation is mandatory to keep them in a life-like shape
and to assure proper orientation for sectioning. Therefore, different relaxation methods
are in use to avoid a contraction of the musculature at the beginning of fixation.
Magnesium chloride solution isotonic to seawater is used for marine organisms. For
freshwater animals, different chemicals have been tested so far (e.g., 2% urethane, 0.1%
1-phenoxy-2-propanol), as well as cooling on ice.
B. Chemical Fixation—Protocols2
1. Marine Organisms (e.g., Macrostomum lignano, Isodiametra pulchra)
a. Recommended Method Simultaneous fixation with glutaraldehyde and OsO4
(Eisenman and Alfert, 1982); see Figs. 1A, 2, 4A and 5A.
Buffer A: 0.2 M cacodylate buffer, 0.1 M NaCl; 0.35 M sucrose; pH 7.2
Buffer B: 0.2 M cacodylate buffer, 0.3 M NaCl; pH 7.2
Fixative A: 4% (v/v) glutaraldehyde in 0.2 M cacodylate buffer containing 0.1 M NaCl;
0.35 M sucrose; pH 7.2
Fixative B: 1% (w/v) OsO4 in 0.2 M cacodylate buffer containing 0.3 M NaCl;
pH 7.2
Cocktail: mix 9.5 ml fixative A and 0.5 ml fixative B immediately before use
Fixation procedure:
Fix relaxed animals in cocktail for about 5–10 min on ice. Specimens should
become brownish; solution should remain clear. Subsequently, replace cocktail with
fixative A. Fix in fixative A at þ4°C for 1 h. Wash three times with buffer A and once
with buffer B. Fix animals for 1 h with fixative B at þ4°C. Wash three times with
buffer B, once with buffer B mixed with distilled water (1:1), and once with pure
distilled water. Dehydrate specimens in a graded series of either ethanol or acetone
(50, 70, 90, and three times 100% at least for 15 min each) and embed in Epon or
Spurr’s resin.
b. Alternative Method Consecutive fixation with glutaraldehyde and OsO4.
Buffer A: 0.1 M cacodylate buffer, 9% (w/v) sucrose; pH 7.2–7.4
Buffer B: 0.05 M cacodylate buffer; pH 7.2–7.4
Fixative A: 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer containing 9% (w/v)
sucrose; pH 7.2–7.4
Fixative B: 1% (w/v) OsO4 in 0.05 M cacodylate buffer; pH 7.2–7.4
Fixation procedure:
Fix relaxed animals in fixative A for 1 h at þ4°C, wash three times with buffer A
(but without sucrose!) 15 min each, postfix in fixative B for 1 h at þ4°C, and wash
2
For ease of reading, we used neutral terms such as “fixative/buffer A,B…” instead of the original
nomenclature from literature.
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(A)
e
g
n
e
mu
a
mu
a
nb
n
mi
(B)
rh
e
mu
mu
mi
n
ecm
t
Fig. 1
Comparison of the body wall of Macrostomum lignano after chemical fixation and high-pressure
freezing (HPF) and freeze-substitution (FS). a, axon; e, epidermal cell; ecm, extracellular matrix; g, Golgi
stack; mi, mitochondrion; mu, muscle; n, nucleus; nb, neoblast; rh, rhabdite; t, testis. Scale bar = 2 µm.
(A) Chemical fixation (Eisenman and Alfert, 1982). Epidermis, main nerve cord and neoblast, the latter
showing the typical morphology with large nucleus and a thin rim of cytoplasm. (B) HPF and FS. Detail of
the body wall including proximal parts of the testis.
again three times with buffer B. Dehydrate in an ethanol or acetone series and embed in
Epon or Spurr’s resin. For “field work”, fixation should be carried out up to the last
ethanol or acetone dehydration step, followed by sample storage in a refrigerator until
resin embedding can be performed.
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14. Electron Microscopy of Flatworms: Standard and Cryo-Preparation Methods
mi
sp
n
mi
sp
n
Fig. 2 Early spermatids in the testis of Macrostomum lignano after chemical fixation (Eisenman and
Alfert, 1982). mi, mitochondrion; n, nucleus; sp, spermatids. Scale bar = 2 µm.
c. Fixation for Immuno-Cytochemistry of Marine Organisms
phate buffer containing 9% (w/v) sucrose; pH 7.2–7.4
Buffer: 0.1 M phos­
Fixative: 4% (w/v) formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M
phosphate buffer containing 9% (w/v) sucrose; pH 7.2–7.4
Fixation procedure:
Fix relaxed animals in formaldehyde fixative for 1 h at þ4°C, or on ice, wash three
times with phosphate buffer (but without sucrose), dehydrate in alcohol, and embed in
an acrylic resin (e.g., LR-White).
2. Freshwater Organisms (triclads, e.g., Schmidtea mediterranea, Dugesia gonocephala,
Polycelis nigra)
a. Recommended Method Simultaneous fixation with glutaraldehyde and OsO4
(Rombout et al., 1978); see Fig. 6.
Buffer: 0.1 M cacodylate buffer; pH 7.2
Fixative A: 2.0% (v/v) glutaraldehyde þ 1% (w/v) OsO4 in 0.1 M cacodylate
buffer; pH 7.2
Fixative B: 2% (v/v) glutaraldehyde þ 1% (w/v) OsO4 þ 1% (w/v) potassium
dichromate in 0.1 M cacodylate buffer; pH 7.2
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Willi Salvenmoser et al.
sp
sp
n
n
sp
sp
g
mi
er
Fig. 3 Testis of Macrostomum lignano processed with HPF and FS showing early and late spermatids. er,
endoplasmic reticulum; g, Golgi stack; mi, mitochondrion; n nucleus; sp, spermatid. Scale bar = 2 µm.
Fixation procedure:
Fix relaxed animals for 1 h in fixative A on ice. Replace solution without washing with
fixative B and fix for 1 h on ice. Wash three times in buffer, dehydrate specimens in an
ethanol or acetone series and embed in Epon or Spurr’s resin.
b. Alternative Method
bell, 1987).
Simultaneous fixation with glutaraldehyde and OsO4 (Camp­
Buffer: 0.001 M Tris-HCl, 1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM KCl, 1 mM
NaH2CO3; pH 7.8 (Hydra culture medium)
Fixative: 1% (v/v) glutaraldehyde þ 0.2% (w/v) OsO4 in buffer
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14. Electron Microscopy of Flatworms: Standard and Cryo-Preparation Methods
(A)
c
mv
mi
g
gl
n
(B)
c
c
mv
er
mi
mu
g
g
mu
Fig. 4 Body wall of Isodiametra pulchra after chemical fixation (A) according to Eisenman and Alfert (1982)
and after HPF/FS (B). c, cilium; er, endoplasmic reticulum; g, Golgi stack; gl, gland cell; mi, mitochondrion; mu,
muscle; mv, microvilli; n, nucleus. Scale bar = 2 µm. The apical part of the epidermis shows microvilli and basal
parts of cilia, the ciliary rootlet system (arrows), and mitochondria. Note in chemically fixed samples (A) the
shrinkage of membranes of mitochondria and poor preservation of the Golgi stack as well as artifactual
extraction of cytoplasm, as compared to excellent ultrastructure preservation achieved with HPF/FS (B).
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Willi Salvenmoser et al.
(A)
mi
a
(B)
mi
a
mu
a
Fig. 5 Detail of the brain of Isodiametra pulchra after chemical fixation (A) according to Eisenman
and Alfert (1982) and after HPF/FS (B). a, axon; mi, mitochondrion; mu, muscle. Scale bar = 1 µm.
Synapses (arrow heads) and clusters of vesicles in their vicinity called synaptic clouds are seen. Note in
HPF/FS samples (B) the distinct appearance of the vesicles and the absence of shrinkage of any
membranes.
Fixation procedure:
Fix relaxed animals on ice or at room temperature for at least 1 h. Fixation on ice
works slower and therefore it is preferred for smaller animals. Due to the low
concentration of OsO4 the solution remains clear even at room temperature. If solution
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317
e
mg
n
e
*
*
*
*
ecm
mu
n
mu
Fig. 6 Juvenile triclad, Polycelis nigra after chemical fixation (Rombout et al., 1978). e, epidermal cell;
ecm, extracellular matrix; mg, mucous gland; mu, muscle; n, nucleus. Scale bar = 2 µm. Detail of the body
wall with a prominent nucleus of an epidermal cell, the extracellular matrix, and a nucleus of a parenchymal
cell. Note that soluble components of the extracellular matrix became extracted (asterisks mark these almost
empty spaces).
becomes brownish during extended fixation times it should be exchanged. After
fixation, wash three times in buffer and dehydrate with either ethanol or acetone and
embed in Epon or Spurr’s resin.
c. Alternative Method Simultaneous fixation with glutaraldehyde and OsO4 mod­
ified after Shigenaka et al. (1971).
Buffer: 0.06 M phosphate buffer; pH 7.2
Fixative A: 6% (v/v) glutaraldehyde in <0.05 M phosphate buffer, 2 mM sucrose, 0.02 mM
magnesium sulfate; pH 7.2 (i.e., mix 2.4 ml 25% glutaraldehyde, 0.2 ml 0.1 M sucrose,
0.2 ml 1 mM magnesium sulfate with 7.2 ml 0.06 M phosphate buffer; pH 7.2)
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Willi Salvenmoser et al.
Fixative B: 2% (w/v) OsO4 in distilled water
Cocktail: mix equal volumes of fixative A and B immediately before use
Fixation procedure:
Always prepare fresh solutions. Fix relaxed animals on ice for 1 h. High concentra­
tions of glutaraldehyde and sucrose lead to the reduction of OsO4. Thus, the fixative
should be exchanged once becoming dark. After fixation do not wash with buffer but
start directly with dehydration in a standard graded series of acetone. Usually the
solutions with low concentration of acetone will start to get black during the dehydra­
tion process, but solutions with a high concentration of acetone (~100%) should
remain clear. Embed in Epon or Spurr’s resin.
d. Fixation for Immuno-Cytochemistry of Freshwater Organisms Buffer: 0.05 M
phosphate buffer; pH 7.2–7.4
Fixative: 4% (w/v) formaldehyde (freshly prepared from paraformaldehyde) in phos­
phate buffer
Fixation procedure:
Fix relaxed animals in formaldehyde fixative for 1 h at þ4°C, or on ice, wash three
times with phosphate buffer (but without sucrose), dehydrate in ethanol, and embed in
an acrylic resin.
C. Cryo-Processing—General Aspects
Rapid freezing undoubtedly immobilizes subcellular structures and dynamics much
quicker and more reliably than conventional chemical fixation at ambient temperatures
(Heuser et al., 1979; McIntosh, 2001; Muller et al., 1980; Sitte et al., 1987). The only
freezing method that is suitable for cryo-immobilization of bulky specimens thicker than
~10 µm is HPF (Moor and Riehle, 1968). HPF lowers the freezing point of water, and
both crystal growth and nucleation rates are markedly reduced (Müller and Moor, 1984).
The maximum thickness of metazoan samples that can be high-pressure frozen without
or with negligible amounts of disturbing ice crystal damage ranges from 0.1 to 0.2 mm.
This is primarily dependent on the physicochemical and physiological properties of the
samples, e.g., their contents of water and natural “cryo-protectants” within the cyto­
plasm. One of the most feasible ways to process cryo-immobilized samples for cellular
EM comprises FS followed by resin embedding at either room or low temperatures; for
recent reviews on various other cryo-methods, see Hess (2007) and Mobius (2009). FS
of the sample is accomplished by dehydration at very low temperatures through organic
solvents, such as ethanol or acetone. For chemical stabilization and staining of the
biological sample, fixatives are usually added to the organic solvent. Out of a wide
variety of published FS recipes, we consider FS with anhydrous acetone containing 1%
OsO4 (Van Harreveld and Crowell, 1964) plus 0.1–0.2% uranyl acetate (UA) as optimal
for morphological studies. This popular cocktail may be further supplemented with
1–4% water (Buser and Walther, 2008; Walther and Ziegler, 2002) for improving the
contrast of certain membranes and the cytoskeleton. FS with pure, anhydrous acetone,
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319
optionally supplemented with UA, followed by embedment in (meth)acrylate resins can
be used for immuno- and lectin-cytochemistry (this aspect is not discussed here;
for reviews on this topic, see Hess, 2007; Humbel and Müller, 1986; Schwarz
and Humbel, 2007). Typically, FS is performed for ~8 h (Humbel and Müller, 1986)
up to several days (Zhang et al., 1993) at 90 to 80°C with the aid of commercial or
homemade FS devices or by using dry ice–acetone mixtures (Steinbrecht and Müller,
1987). Likewise, warming-up usually takes ~12–30 h ending with thorough rinsing of
the specimens with pure solvent at temperatures between 30 to þ20°C (Humbel and
Müller, 1986). Finally, specimens are embedded in resin for ultramicrotomy and
transmission EM. According to our experiences and the published literature from
various cell types and organisms, short and extended protocols work equally well
(plants, fungi, mammalian cell cultures, e.g., Hess, 2003 and our unpublished data).
A highly similar quality in terms of ultrastructure preservation is obtained, provided the
specimens have been cryo-immobilized properly.
D. Cryo-Processing—Protocols
1. Marine and Freshwater Organisms
a. Recommended Method Rapid cryo-immobilization by means of HPF followed
by FS and epoxy resin embedding; see Figs. 1B, 3, 4B, 5B, 7 and 8.
Freeze-substitution media
Recipe I: Anhydrous acetone containing 1% (w/v)
OsO4 (Van Harreveld and Crowell, 1964) plus 0.1–0.2% (w/v) UA (diluted from a
10% (w/v) stock solution of UA in methanol) is optimal for morphological studies.
Recipe II: FS medium of recipe I, further supplemented with 4% (v/v) water
(Walther and Ziegler, 2002).
The fixatives (prechilled OsO4 crystals, UA stock) are carefully added to the acetone in
a beaker at room temperature followed by a few seconds of gentle mixing. Subsequently,
cryovials are quickly filled with 1.2–1.5 ml FS media, closed and immediately frozen in
upright position with LN2 to prevent premature degradation. We prefer 2 ml cryovials
with screw-caps, plus the appropriate cryoracks with locking bottoms that match the
cryovials since they permit one-hand operation (for detailed instructions how to make the
cocktail see Hess, 2007; McDonald and Muller-Reichert, 2002).
High-pressure freezing procedure Worms are pipetted with their natural medium (sea­
water, freshwater) into an embryo dish and subsequently kept on ice for 15–30 min to
slow down the animals’ activity. Next, the cup-shaped aluminum HPF specimen carriers
(cavity depth: 0.1–0.2 mm) are slightly overfilled with a droplet of freshwater or sea­
water containing worms3. A sapphire coverslip (diameter 3 mm, thickness 50 µm) is
3
Our experience and, thus, recommendations given here relate to “BAL-TEC HPM-010-type” HPF
machines but not to the “EM-PACT-type” instruments, which have slightly different and smaller specimen
carriers.
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Willi Salvenmoser et al.
(A)
n
e
*
*
*
ecm
*
*
mu
mu
pn
pn
pn
(B)
e
n
*
*
mu
*
*
mu
* *
*
Fig. 7 Body wall of triclads processed with HPF and FS. e, epidermal cell; ecm, extracellular matrix;
mu, muscle; n, nucleus; pn, protonephridium. Scale bar = 2 µm. Asterisks mark components of the
extracellular matrix, which are usually extracted by aqueous chemical fixation and/or dehydration at
ambient temperature (see Fig. 6) but remained well preserved by HPF/FS; note their characteristic pattern
at the base of the epidermal cells. (A) Polycelis nigra, juvenile. (B) Dugesia gonocephala.
immediately placed on top of the droplet and excess water is soaked with filter paper so
that the specimen carrier is sealed. This “mini-aquarium” is then transferred with
tweezers into the sample holder of the HPF apparatus. A wetted, 0.3-mm-deep aluminum
carrier is placed with its flat side down on top of the sapphire4. Finally, the sample holder
4
Clearly, the additional sapphire disk does increase the thickness of the sandwich, and thus slightly delays
rapid freezing. However, the transparent sapphire lid allows for full visual control of the whole loading
process so that the light-shunning animals cannot escape unobserved from the carrier. Moreover, acciden­
tally trapped air bubbles, that might impede proper freezing or damage the sample, are easy to detect and
loading can be repeated.
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c
e
c
e
bwm
n
e
gc
gc
+
sc
+
n
n
gc
sc
sp
+
sc
sp
sp
sp
t
sp
Fig. 8 Macrostomum lignano (HPF/FS). Gross anatomy of an adult animal showing epidermis,
musculature, gut, and testis in a low magnification electron photomicrograph. The testis is ventral and the
gut dorsal to the extracellular matrix that is marked by crosses (+). Scale bar = 10 µm. bwm, body wall
musculature; c, cilia; e, epidermis; gc, gut cell; n, nucleus; sc, spermatocyte; sp, spermatid; t, testis lumen.
is closed and the “sandwich” is immediately cryo-immobilized by HPF. Frozen sand­
wiches are subjected to FS or stored in LN2 for later use. (Larger animals, e.g., fresh­
water triclads must be cut with a sharp scalpel blade into appropriate pieces to fit into the
HPF carriers.)
Freeze-substitution procedure and embedding The cryo-immobilized samples are
transferred under LN2 with precooled tweezers into the cryovials containing the
frozen FS cocktails. Subsequently, the lids are loosely screwed onto the vials to
permit safe evaporation of excess N2 gas. Finally, the vials are placed into the
precooled FS container and after about 1 h the lids are tightened and FS is started.
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A feasible example for FS (to be performed over the weekend) reads as follows: FS
for 36 h at 90°C (or at 80°C, if a dry ice–acetone mixture is used instead of an
FS device (Steinbrecht and Müller, 1987)), warming up to 55°C at a rate of 5°C
per hour, subsequently postfixation and staining at 55°C for 6 h, followed by
warming up to 30°C at a rate of 5°C per hour where samples are left for
additional ~8–12 h. The step-wise warming-up should allow fixatives/stains
included in the FS media to stabilize the specimens at low temperatures (for details
on the reactivity of selected fixatives at low temperatures see the following
reviews: Hess, 2007; Humbel, 2009). Finally, the specimens are allowed to reach
room temperature, rinsed three times with pure solvent (10 min each), and
embedded in any epoxy resin. We avoid embedding in Spurr’s mixture (Spurr,
1969) as this may solubilize and extract subcellular constitutents of FS specimens
(Hess, 1990). Ultrathin sections of FS samples were viewed without poststaining,
whereas chemically fixed samples were poststained with UA and lead.
IV. Materials
A. Animals
The rhabditophoran flatworm Macrostomum lignano, originally sampled in the
Mediterranean (Ladurner et al., 2005), and the acoel flatworm Isodiametra pulchra
from the Atlantic coast of Maine (Smith and Bush, 1991) are kept in permanent
laboratory cultures at the University of Innsbruck since 1995 and 2003, respectively,
and are being fed with the diatom Nitzschia curvilineata.
The freshwater tricladid rhabditophorans Polycelis nigra and Dugesia gonocephala
were sampled in creeks and ponds in Lower Austria in May 2009 and in a creek near
the University of Innsbruck in spring 2009, respectively, and have since been kept in
laboratory cultures on veal liver.
B. Chemical Fixation
1. Chemicals (be aware that most of the reagents are more or less toxic and/or hazardous to
health; for their safe use and disposal consult the relevant Material Safety Data Sheets)
0.05–0.2 M cacodylate buffer (contains arsenic compounds!), optionally supplemen­
ted with varying concentrations of sucrose.
0.05–0.1 M phosphate buffer, optionally supplemented with varying concentrations
of sucrose and/or magnesium sulfate.
Hydra culture medium: 0.001 M Tris-HCl, 1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM
KCl, 1 mM NaH2CO3; pH 7.8 (all Sigma-Aldrich or Merck).
Urethane and 1-phenoxy-2-propanol (Sigma-Aldrich).
Paraformaldehyde, potassium dichromate, as well as glutaraldehyde, OsO4 crystals,
epoxy resins (Epon, Spurr’s) from Sigma-Aldrich, Agar (Stansted, U.K.), EMS (Hat­
field, PA, U.S.A.), Polysciences (Warrington, PE, U.S.A.), or Ted Pella (Redding, CA,
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U.S.A.), LR-White acrylic resin from Sigma-Aldrich or London Resin Co (Woking,
Surrey, U.K.).
2. Tools
For specimen handling, embryo dishes (preferably black, if used for small, quasitransparent species) or crystallization dishes (e.g., from Agar) and fine glass or small
plastic pipettes were used.
C. Cryo-Processing
1. Chemicals
OsO4 crystals, uranyl acetate, epoxy resin (Epon) from Sigma-Aldrich, Agar, EMS,
Polysciences, or Ted Pella.
2. Tools
Embryo dishes or crystallization dishes and fine glass pipettes were used for animal
handling. Sapphire coverslips (diameter 2 mm; usually used for HPF of adherent 2D cell
cultures, see Hess et al., 2010, Chapter 27, this volume) were from Engineering Office
Martin Wohlwend (CH-9466 Sennwald, Switzerland; [email protected]) and
are also available from Leica Microsystems (Vienna, Austria).
Several types of aluminum carriers for HPF were from Martin Wohlwend Engineer­
ing or Leica.
For FS, we recommend 2 ml cryovials (e.g., Nalgene, from Nalge Nunc Interna­
tional, Rochester, NY; note that these vials are not explicitly LN2-certified!).
Tweezers, scalpels, slot, or mesh grids (copper) coated with formvar and carbon
were all from Agar, EMS, Polysciences, or Ted Pella.
3. Instruments
Further EM instruments used in this study: dissecting microscope (i.e., Leica EM
workstation), HPF apparatus HPM-010 (from BAL-TEC, Balzers, Liechtenstein;
note that this instrument is currently sold by ABRA-Fluid AG, Widnau, Switzer­
land), automated FS device AFS (Leica), ultra-microtome Ultracut S, diamond
knives for ultra-microtomy (from DIATOME, Biel, Switzerland), transmission EM
Libra 120 EFTEM (from Zeiss, Oberkochen, Germany), or Philips CM120 TEM
(from F.E.I., Eindhoven, The Netherlands).
V. Results and Discussion
A. Chemical Fixation
In our hands, simultaneous fixation with glutaraldehyde and OsO4 gives the best
results, especially for stem cell research. The generally accepted criteria for a good
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fixation for EM (Hayat, 2000) are: clearly contrasted membranes, including the nuclear
envelope and mitochondrial membranes, and a so-called “full cytoplasm” (e.g.,
Figs. 1A and 2). Inadequate fixation causes membrane undulations or interruptions,
shrinkage or swelling of mitochondria or endomembrane compartments, such as the
Golgi apparatus (e.g., Fig. 4A vs. Fig. 4B), as well as the (partial) loss of the matrix of
organelles such as mitochondria or peroxisomes (not shown). Extraction of easily
soluble compounds of the cytoplasm often leads to the appearance of an “empty
cytoplasm” (Fig. 4A).
A proper chemical fixation allows the study of the gross anatomy of both marine and
freshwater flatworms at the ultrastructural level, including more voluminous species
(Figs. 1A, 2, and 6). Research on stem cells and regeneration as well as studies on
changes of subcellular structures after gene silencing by RNAi (RNA interference) are
routinely possible (De Mulder et al., 2009). However, the preservation of certain
structures still remains unsatisfactory especially in comparison to cryo-processed
samples. Cellular structures with high concentrations of chemically bound water,
such as extracellular matrix, show alterations and shrinkage (Fig. 6 vs. Fig. 7). The
amorphous “ground substance” only is affected, but not the fibers. Depicting the
structures underlying certain physiological processes, such as liquid transport by
vesicles, is not always possible. In many cases, these processes are too fast to be
properly immobilized by chemical fixation. The preservation of the nervous system is
also frequently suboptimal. The ultrastructure of the different vesicle types, like dense
core and lucent vesicles but also synaptic sites, are adequately preserved thus allowing
distinction and functional analysis of the different axons types. However, the shape of
axons and the composition of the microtubular and neuronal fiber network differ in
comparison to cryo-processed specimens. As an example, axons in chemically fixed
samples do not appear round (Figs. 1A and 5A vs. Fig. 5B) and usually microtubules
and neuronal fibers are not equally distributed.
Out of the various immuno-cytochemical methods available, immuno-gold labeling
of resin sections has already been successfully applied to flatworm research (Bode
et al., 2006; Pascolini et al., 1992). Depending on the antigen and the quality of the
antibody, even simultaneous or consecutive fixation with glutaraldehyde and OsO4 is
occasionally possible (e.g., the immuno-labeling of BrdU (Bode et al., 2006)). Usually,
formaldehyde fixation and embedding in an acrylic resin gives satisfactory results. For
optimal tissue preservation and antigen localization, cryo-processing should be
considered.
B. Cryo-Processing
“Representatives” from all three major systematic groups of nonparasitic flatworms
were processed by means of HPF and FS. In part, excellent or at least adequate results
were obtained (Figs. 1B, 3, 4B, 5B and 8, and Fig. 7, respectively). The ultrastructural
patterns observed were fully consistent with those reported in the literature from cryo­
processed nematodes, e.g., C. elegans (McDonald, 2007; Muller-Reichert et al., 2003;
O’Toole et al., 2003), as well as other metazoa including mammalian systems. Briefly,
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the outlines of any cell type under investigation appeared smooth and not undulated,
and so were the membranes of the generally turgescent organelles. In case of sub­
optimal freezing, however, membranes showed somewhat slightly bent or “zigzag”
patterns, characteristic for minor ice crystal damage. Whether an artifact is acceptable
or not depends on its intensity and on the scientific question under investigation.
Matrix constituents (i.e., extracellular matrix, mitochondrial matrix, contents of
Golgi cistern/vesicles, secretory vesicles of various glands) were generally much better
preserved than after conventional chemical fixation, thus, stained more intensively
(e.g., Figs. 4B and 7 vs. Figs. 4A and 6). The cytoskeleton, too, proved well preserved
and clearly contrasted in our preparations. No major differences between anhydrous
and “watery” FS media were apparent, except that the triclad P. nigra showed very low
specimen contrast after anhydrous FS (not shown). Adequate staining of membranes
and cytoskeletons was achieved here by adding water to the FS media (e.g., Fig. 7A).
Taken together, we consider the preservation quality and the yield of usable specimens
obtained with small-sized animals, such as M. lignano and I. pulchra as very good
(Figs. 1B, 3, 4B, 5B and 8).
Concerning, however, the preparation of more voluminous flatworms, the following
steps need further optimization. Most importantly, the freezing quality achieved with
thicker animals, such as triclads, is not perfect yet (e.g., Fig. 7). The greatly reduced
cooling rate in the center of bulky objects is an inherent problem of specimens thicker
than ~200 µm (Studer et al., 2008). Nevertheless, improvements should be possible by
reducing the volume of the whole sandwich since the mass of aluminum specimen
carriers counts as well. For the “mini-aquarium” described here, for example, one
could use a suitable spacer ring instead of the solid lid on top of the sapphire disk, in
analogy to the two-sapphire sandwich described elsewhere (Hawes et al., 2007). An
alternative could be the use of custom-made specimen carriers (Craig et al., 1987).
Standard carriers for the BAL-TEC HPM010 (and similar instruments) have a 200-µm­
thick bottom. Carriers with a thinner bottom, so-called “membrane carriers”, were
developed for the EM PACT2 (McDonald, 2007) and quite thin steel foils may be
suitable as well (Sawaguchi et al., 2008). Furthermore, it remains to be tested whether
it is possible to enclose the animals within the sandwich in physiologically appropriate
media other than water. Usually, liquids with a lower freezing point and/or better heat
conductivity than water or buffer serve as so-called space “fillers” to improve the rapid
freezing process (Studer et al., 1989). Hexadecene (a paraffin oil) is widely used,
especially for plants and fungi (Hess, 2007; Studer et al., 1989) but is definitely not
suitable for flatworms. Hexadecene quickly disintegrates the flatworms’ epidermis and
parenchyma (our unpublished data) similar to its negative effects on nematodes
(Hohenberg et al., 1994). Concentrated bovine serum albumine solutions (~20% w/
v), for example (McDonald et al., 2007), has proven as to be apt for various cell types/
organisms, including marine sponges (McDonald et al., 2007) and ctenophores
(T. Müller-Reichert, pers. comm.). However, negative effects of this reagent were
also observed, in particular with certain triclads (T. Müller-Reichert, pers. comm.).
Thus, a suitable filler remains to be found by systematic testing the various reagents
employed for other organisms. Finally, it has to be mentioned that large-sized
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flatworms frequently stick firmly within the cavity of the specimen carrier. This leads
to accidental freeze-fracturing (especially at the level of the epidermis) when the
sandwich is removed from the sample holder or subsequently opened. Likewise, it is
almost impossible to peel stuck worms off the carrier’s cavity after FS or resin
infiltration without damaging the sample. Polymerizing the sample still contained
within the aluminum carrier solves this problem. After polymerization, one must
only free the carrier’s rim from excess resin by thorough, careful trimming with a
razor blade, followed by immersion in liquid nitrogen. Sometimes it takes several
freeze–thaw cycles until the resin block with the sample falls off the carrier. Finally, the
sample is re-embedded (see also Sawaguchi et al., 2003 in this context). Alternatively,
coating the carriers and the sapphire with lecithin (Craig et al., 1987) is worth a try so
that the sample is released already during FS.
VI. Concluding Remarks
The major advantages of chemical fixation are its low costs and ease of handling. It
is the method of choice for routine work, “fieldwork”, and for the fixation of large
specimens. The most serious disadvantage of chemical fixation is the well-known
problem of fixation artifacts (Bowers and Maser, 1988; Strangeways and Canti, 1927).
The degree of damage depends on the type and physiological condition of the cells/
tissue/organism under investigation.
Cryo-preparation techniques, namely, HPF and FS, were only recently introduced to
flatworm ultrastructure research (Egger et al., 2009a). Thus, we are at the very
beginning of exploiting their potential for this group of organisms. A well-established,
straightforward approach for (immuno-)cytochemistry that should be applied to flat­
worms is FS followed by embedding in acrylic resin, as described for various organ­
isms (Humbel and Müller, 1986; Schwarz and Humbel, 2007). Another promising
method serving the same purpose is HPF/FS followed by sample rehydration and
Tokuyasu cryosection immuno-labeling (van Donselaar et al., 2007); this somewhat
more laborious method has meanwhile been optimized and also employed for flies and
nematodes (Ripper et al., 2008; Stierhof et al., 2009). Finally, it is conceivable that
SDS-digested freeze-fracture replica labeling (Fujimoto, 1995; Kaufmann et al., 2009)
is suitable for flatworm research as well, as made likely by classical freeze-fracture
studies (Rieger et al., 1991).
Acknowledgments
We thank Karin Gutleben for the excellent technical assistance and Thomas Müller-Reichert for
communicating unpublished observations. P. Ladurner is supported by grant P-18099 from FWF Austrian
Science Funds; B. Egger is supported by a Sparkling Science grant (funded by the Austrian Ministry of
Science and Research); and M.W. Hess is supported by grants from FWF Austrian Science Funds (P­
19486-B12), Österreichische Nationalbank-Jubiläumsfonds (P-11050), and Tiroler Wissenschaftsfonds (P­
UNI-0404/100).
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