Sample preparation for nuclear microscopy : cryotechniques S P

SUPPORT OF PUBLIC AND INDUSTRIAL RESEARCH
USING ION BEAM TECHNOLOGY
Integrated Activity
EU Project No. 227012
WP7 - Milestone M7.4 : Sample cryo-preparation protocol available
Institute :
CNRS (CENBG)
Sample preparation for nuclear microscopy : cryotechniques
1. Introduction
Sample preparation is fundamental to the field of biomedicine and can fully explain the difficulty
encountered by microanalysts in this domain. It is usually expected that the results of microanalysis
reflect a state quite close to that of the living. The achievement of a perfectly preserved ultrastructure
is of course impossible. However during the three last decades, due to the improvement of preparation
techniques, particularly cryotechniques, the preservation of morphology and ion distributions reached
a level that allows the investigation of most cellular mechanisms.
The processing of biological samples must take into consideration the possible degradation of tissues
when cells are removed from their natural environment. Two crucial steps are involved: the sampling
which results in the disruption of the blood supply and in the sudden change of the ionic osmolarity of
surrounding fluids. This step, if carried out under inadequate experimental conditions or prolonged in
time, may result in serious modifications in the distribution of inorganic diffusible ions such as Na+,
Cl-, K+ and Ca++. Hopefully, unlike these electrolytes, other trace elements, such as essential metals,
are usually firmly bound to macromolecules (nucleic acids, proteins, enzymes) and structures
(membranes). An efficient fixation of the tissue must thus be operated upon immediately. The second
crucial step is the introduction of the specimen in the analysis chamber under vacuum. In order to
avoid a destructive boiling, tissues must be dehydrated first.
The simplest and most efficient technique to obtain a sample suitable for microanalysis is based on
cryofixation/freeze-drying. By doing so, tissues are kept in a virginal state, thereby eliminating the
risks of element displacement and contamination generally associated with the diffusion of
endogenous substances in the tissue.
2. Cryofixation
2.1 Advantages of cryotechniques
The fixation step is crucial because (i) the ability of the specimen to undergo subsequent processes
without too much degradation will depend on its quality; (ii) according to the type of fixation used, it
will determine the whole preparation pathway. Conventional processing is generally based on fixation
with aldehydes, staining with heavy metal salt, dehydration by alcohol and embedding in plastic.
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During this invasive process, besides the problem of an eventual contamination, it seems evident that
diffusible substances may be re-distributed or washed out.
Freezing is the sole alternative to chemical fixation and must be the method per preference. Freezing,
when rapidly carried out, may not only preserve the original composition of a specimen but may also
arrest physiological processes with high time resolution. The last point is particularly interesting when
dynamic mechanisms such as electrolyte diffusion through cell membranes are investigated.
After cryofixation, it is possible to envisage different preparation schemes: (i) cryomicrotomy of
frozen samples with either subsequent freeze-drying or direct transfer of slices in a cold stage into the
analysis chamber for direct analysis in the frozen hydrated state. The analysis of hydrated specimens
has been routinely used in electron microscopy. However this procedure is still rather scarce for the
nuclear microanalysis. (ii) dehydration of the specimen by freeze-drying or freeze-substitution
followed by resin embedding and conventional microtomy.
SPECIMEN SAMPLING
CRYOFIXATION
FREEZE-DRYING
FREEZE-SUBSTITUTION
CRYO-MICROTOMY
RESIN EMBEDDING
MICROTOMY
FREEZE-DRYING
CRYOTRANSFER
NUCLEAR MICROPROBE ANALYSIS
Fig. 1. Sample preparation schemes for nuclear microanalysis based
on cryotechniques
Before describing cryotechniques in detail, it must be emphasised that the final result will depend on
the tissue sampling procedure. Severe artefacts may be produced during sampling. Disconnecting a
specimen from its blood supply or the disruption of its innervation could severely disturb its
physiological state and consequently the ion distribution and compartmentalisation. In particular,
intracellular increase of sodium and chlorine accompanied by a loss of potassium have often been
reported, for instance, during liver specimen sampling.
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The cryofixation must occur immediately after sampling. All attempts to reduce the time interval
between sampling and fixation will result in an improvement of the preservation. To solve this
problem, an ingenious system was developed with the aim of carrying out sampling and cryofixation
simultaneously. This device allows in situ freezing by using the so called “cryopunching” technique.
This simple apparatus, derived from an impact freezing device, was described in great details by
Zierold (1993).
2.2 Mechanism of freezing
As mentioned by Robards and Sleytr (1985) in their extensive review of all aspects concerning the
cryofixation of biological media, freezing is not a simple phase transition that transforms a liquid into
a solid state. Rather, it is a complex succession of events sensitive to such physical parameters as
cooling rate or pressure.
When pure water is cooled at a reasonable cooling rate, at a temperature below the melting point (i.e.
the equilibrium temperature at which the two phases, water and ice, can coexist) (273 K), it does not
freeze immediately but remains in a metastable super-cooled state. The duration of the so-called
super-cooling phenomenon is limited and the fluid can retrieve a more stable two-phase (water and
ice) equilibrium state by spontaneous crystallisation. When this happens, the latent heat of fusion is
released and warms the water until it reaches its melting point again. As cooling continues, the ice
nucleation process leads to the growth of ice crystals that act as the seeds for progressive
crystallisation of the whole water volume.
270
0
-20
250
S
230
S
Temperature
210
(K)
-40
-60
R
190
-80
170
-100
150
-120
130
R
Temperature
(°C)
-140
110
-160
90
-180
Fig 2. Freezing behaviour for pure water (left) and cells (right). Pure water melts at 273 K () but may be supercooled to about 235 K (S). The critical crystallisation range is displayed by the solid line. Recrystallisation of ice
may take place down to the recrystallisation point (R) (130 K for water). In living cells, the recrystallisation point
is raised and the critical interval is thus reduced. (Adapted and redrawn from Robards and Sleytr, 1985).
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In such freezing, the rate of growth of ice crystals depends on the temperature, with a maximum of
about 260 K. Below this maximum, the rate falls with a quasi-linear behaviour as the temperature
decreases. The vitrification temperature (or recrystallisation point), i.e. the temperature above which
water would transform from an amorphous solid state into a crystalline state, has been estimated at
127±4 K . This means that for pure water, crystallisation may occur if its temperature is in the range
127-273 K. But if this range is traversed quickly enough during the cooling procedure, so that the
heat of fusion is removed faster than it is produced, crystallisation can be totally avoided. In order to
achieve this, the cooling rate must be very high, a goal considerably complicated by the fact that the
heat capacity of super-cooled water is greatly enhanced (105 J.mole-1.K-1) compared to ice’s (32
J.mole-1.K-1) at the same temperature (238 K).
The behaviour of pure liquid is not necessarily reflected in living cells and tissues because of the
presence of solutes or lipid phases. The critical cooling velocity, above which vitrification occurs,
varies from sample to sample and thus cannot be precisely predetermined. In such media, the
recrystallisation point is raised as a consequence of a reduction in the volume of water available for
freezing. In biological compartments containing more than 80% water, the recrystallisation
temperature is raised by about 50 K, hence reducing the critical temperature interval through which
rapid cooling is necessary. In addition, 15% of cellular water is bound to macromolecules and does
not freeze.
2.3 Ice crystallisation artefacts
Ice crystallisation is a damaging process which alters the state of specimens from their in vivo
condition. It may cause the mechanical rupture of cell membranes leading to an irreversible loss of the
compartmentalisation. The extracellular crystallisation may also cause dehydration and shrinkage,
thereby disturbing the osmotic balance across cell membranes. Proteins may be denature. The
phenomenon of ice crystallisation usually leads to phase separation in the crystal growth, giving rise
to an increase in the concentration of solutes in the remaining liquid water. This process can be a
source of analytical artefacts and can only be avoided by the vitrification of the specimen (formation
of an amorphous state without crystal formation). A redistribution of mobile substances, such as
elctrolytes, may then occur during crystallisation. The solutes are swept into the intercrystalline space
by the growing ice-crystal dendrites. The ion displacement is therefore given by the size of the
segregation compartment because ions probably do not move further than one diameter of a
segregation hole (Zierold, 1993).
Ice crystal segregation often appears in water-rich compartments (e.g. extracellular space and
nucleus). A strong correlation between ice-crystal size and cooling rate is commonly accepted. In
aqueous suspensions, at cooling rates of approximately 100 K.s-1, ice crystals of 4 to 5 µm were
observed while at rates of several thousand K.s-1 crystals of 1 to 2 µm appeared. These values were
achieved in simple aqueous systems but they could represent a relevant order of magnitude for a real
specimen subjected to inadequate (i.e. low cooling rate) freezing.
2.4 Cryofixation with liquid cryogens
The physical problem of cryofixation is the extraction of heat from a sample as rapidly as possible.
But due to the poor thermal conductivity of ice, the cooling rate required to avoid the generation of
ice crystal artefacts can only be achieved within border regions of the specimen. It is generally
believed that at normal pressure, the thickness of the well-preserved zone, fit for use in high
resolution electron microscopy, does not exceed 50 µm. Hopefully, for the spatial resolution of
nuclear microanalysis, the requirements would not be so drastic.
Rapid freezing by immersion in a cold liquid is, without doubt, the simplest method. One can
intuitively tell that the intimate contact with the specimen surface, whatever its topography, is easy to
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establish. The main difficulty, however, appears when the high temperature specimen surface comes
into contact with the liquid cryogen. A stable vapour film forms at the interface, insulating the object
and thus reducing the rate of heat withdrawal. This is known as the “Leidenfrost” phenomenon.
The choice of the liquid cryogen is of primary importance in improving the rapidity of cooling. It
must be selected on the basis that it has a low melting point and high boiling point so that the
phenomenon of film boiling is minimised. The coolant must be liquid at a suitably low temperature
(low melting point at normal pressure). It must have good heat conduction, high specific heat and
density.
Liquefied gases with low boiling points (LHe, LN2), can easily cause the Leidenfrost phenomenon.
They are therefore only suitable for use as primary cryogens, i.e. to cool secondary cryogens which
come into contact with the specimen. From this point of view, LN2, when undercooled to 63 K and
partly solidified by vacuum (N2 slush), would provide the best results. Various compounds which fall
into the class of suitable cryogens are listed in Table 1. For instance, dichlorodifluoromethane is a
typical cryogen with a melting point of 118 K and a boiling point of about 243 K. It is classified under
the generic name “halocarbon” usually used for halogenated methane compounds (CCl2F2–
halocarbon 12-freon 12). Propane and ethane have been reported to provide optimum results for
routine preparation. They have a sufficient cooling power as well as the reputation of being easy and
cheap to acquire. They present no danger if small quantities are used and the appropriate safety
regulations are carefully observed. Isopentane, widely used in the past, gives inferior results but has
the advantage of being a liquid at ambient temperature and atmospheric pressure.
Liquid
Melting
point
°C(K)
Boiling
point
°C(K)
Specific
heat
(J.g-1.K-1)
Thermal
conductivity
(mJ.m-1.s-1.K-1)
Isobutane
CH3CH(CH3)2
- 159.2
(114)
-11.2
(261)
1.68
180
Isopentane
(CH3)2C3H6
-159.9
(113)
27.8
(301)
1.72
182
Propane
C3H8
-189.6
(84)
-42.1
(231)
1.92
219
Ethane
CCH3CH3
-183.5
(90)
-88.8
(184)
2.27
240
Halocarbon 12
CCl2F2
-158.0
(115)
-29.8
(243)
0.85
138
Halocarbon 22
CHCl2F2
-160.0
(113)
-40.8
(232)
1.08
152
Liquid nitrogen
N2
-210.0
(63)
-195.8
(77)
2.0
153
Table 1. Thermodynamics of liquid cryogens: some characteristic parameters
Several practical aspects must also be taken into account. Plunging must only be applied to tiny
specimens (up to approx. 1 mm in diameter). The heat capacity of the specimen carrier must be very
low in order to avoid the boiling of the cryogen and the recrystallisation phenomenon which may
sometimes take place due to the inflow of heat from the frame to the sample. Another critical
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parameter is the speed with which the object enters into and moves within the liquid. The path must
be chosen in such a way that by the time the specimen comes to a stop, it is completely frozen at a
temperature that excludes the recrystallisation process. In order to reach this cooling rate, high
velocity can be achieved by using mechanical injector systems. Optimal injection velocity of
approximately one m.s-1 has been proposed. Finally, layers of liquids which could remain on the
specimen surface from the last rinsing procedure, may drastically reduce the depth of the preserved
zone: the cryogen has to absorb the heat through this layer.
2.5 Alternative Techniques
Various techniques of cryofixation have been successfully applied to the problem at hand. Cryogen
jet, hammer freezing, metal mirror, slamming, spray freezing, and impact cryofixation are the various
cryomethods which the analyst has to choose from. Numerous apparatus, though not always
commercially available, have been designed; a fact which does not help the situation. Here we will
highlight some of their principles and main features. For a detailed description, the reader is referred
to the excellent review by Robards and Sleytr (1985) and Sitte et al. (1987).
High pressure freezing is a technique which permits one to freeze specimens using a liquid coolant at
a very high pressure of approximately 2000 bars. The water is easily sub-cooled so that the critical
cooling range in which crystallisation can occur (see section 2.2) is greatly reduced. Consequently,
cooling rates of only 102 K.s-1 are required for adequate freezing. This could be the only method
available for the preservation of large specimens (> 1 mm). Nevertheless, most biological cells are
rapidly damaged through exposure under such high pressure. It is therefore necessary to apply the
pressure for only a millisecond before the cooling process begins. This technique is thus expensive
because of the use of a complex experimental device.
Jet freezing (or cryogen jet) uses a jet of liquid cryogen accelerated to an extremely high velocity and
is then directed onto one or two faces of the specimen usually sandwiched between thin protective
metal plates. Theoretically, it does not make any difference whether the object is moved at high
velocity in resting cryogen, or is stationary and cooled with a moving jet of cryogen (Sitte et al.,
1987). This last technique is particularly suitable for subsequent freeze-fracturing of the sandwich
specimen by simply removing the sandwich supports.
Impact cryofixation (also called “metal mirror cryofixation”, “slamming” or “cold-block freezing”)
takes advantage of the large heat capacity and good thermal conductivity of some solids, a priori
better than any liquid coolant. Freezing is obtained by impacting the fresh sample onto the polished
surface of a metal block at very low temperature. It generates very high heat transfer rates and yields
excellent freezing in thin (10-20µm) surface regions. Experimental devices can be found in Sitte et al.
(1987).
3. Dehydration
3.1 Cryotechniques
Before being placed in vacuo in the specimen chamber, soft tissues must be dehydrated. Four
techniques could be used in theory:
(i) Air-drying at atmospheric pressure and ambient temperature
(ii) Freeze-drying of hydrated specimen (lyophilisation).
(iii) Freeze-substitution with a non-aqueous volatile solvent.
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(iv) Critical point drying after the replacement of water with an organic
solvent.
The first one concerns only samples which were previously chemically fixed. Since drastic structural
damages can be induced during this process, they must be avoided absolutely, be it for microanalysis
or morphological examination. The remaining three methods are employed according to the type of
tissue and particularly the experiment to be performed.
Temperature
(°C)
Temperature
(K)
400
650
Vapour
200
450
3
1
0
Liquid
250
2
Solid
-200
50
10-3
101
105
Pressure (bar)
Fig. 3. Pressure-temperature phase diagram of water. The three different ways of transforming from the liquid
phase to the vapour phase are: (1) air-drying, (2) freeze-drying, (3) critical point drying. (Redrawn from
Robards and Sleytr, 1985).
From the thermodynamical point of view, the three main pathways along which water is transformed
into vapour susceptible to diffusing out of the specimen are represented in Fig 3. On this p-T diagram,
the regions corresponding to the three phases of water, solid, liquid and vapour are clearly visible.
Air-drying is represented by the arrow number one crossing the vaporisation curve at a temperature
above that of the triple point. In the freeze-drying pathway (route number two), the liquid is at first
transformed into the solid state, crossing the melting curve. It is then sublimed at a temperature and a
pressure below those of the triple point (Patm and T = 0 °C). The third way is critical point drying. It
involves going round the critical point to move from the solid to vapour region. Under such
thermodynamical conditions, the water becomes the so-called super critical fluid. The critical point of
water is nevertheless not easily accessible (647K and 218 atm). It does not make sense to bring a
specimen to such a state. Water is thus usually replaced by a solvent such as CO2 whose critical point
lies within a less damaging range.
Freeze-drying is certainly the simplest method and is carried out directly on fresh thin cryosections of
unfixed material. It can be performed immediately after cryosectioning for an analysis of unembedded
sections. Freeze-substitution is necessary when specimens have to be embedded in resin subsequently.
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3.2 Freeze-drying
Freeze-drying is a dehydration technique in which ice is gently removed from the sample by
sublimation. If high level preservation is needed, the sample must be kept during the process at a very
low temperature below the recrystallisation point. It is thus necessary to decrease the pressure within
the drying chamber for sublimation to occur. A reasonable sublimation rate may be achieved when the
partial pressure of vapour is kept very low as compared to the saturation vapour pressure of ice. The
water vapour released from the specimen must therefore be continuously removed from the
atmosphere of the system. This can be effected through adsorption onto dessicants or by condensation
onto a surface kept at a temperature considerably lower than that of the specimen; for instance on a
cold trap chilled to 77 K with liquid nitrogen. Mechanical dessicants such as zeolite or synthetic
zeolites (also known as “molecular sieves”) can also be employed.
A simple vacuum system for freeze-drying can be constructed using a simple rotary vacuum pump.
The specimen is placed in a pre-cooled temperature-controlled stage in the chamber. When the
vacuum is established, water vapour is either trapped in a condenser kept at very low temperature or
on a dessicant positioned between the specimen and the vacuum pump. Special care needs to be taken
to avoid contamination by vacuum pump oils by using an adsorption trap on the pumping pipe.
When microtomy is performed in a conventional cryochamber at a higher temperature (–40°C), drying
at atmospheric pressure can be done directly in the microtome cryochamber. The risks of temperature
raising, a critical point during cryotransfer, are thus minimised. Even though these unfixed freezedried sections are unsuitable for high resolution morphological examination under electron
microscopy, the distribution of diffusible elements is generally preserved. Sections (5-10 µm in
thickness) obtained with such a procedure are at least suitable for low resolution microanalysis using
nuclear probes.
3.3 Freeze-substitution
The principle of freeze-substitution is to dissolve the ice of the cryofixed sample using an organic
solvent. The specimen is generally processed at a very low temperature to avoid recrystallisation. In
order to ensure the diffusion of the solvent through the tissues, it must be kept in a liquid state. The
temperature must then be maintained above the melting point of the solvent used. After the
completion of the substitution, different pathways are possible. For example, the temperature may be
raised without any risk of morphological damage to embed the specimen in a resin at room
temperature. The sample can also be kept at a low temperature and embedded under such conditions.
The dehydration rate depends on the temperature and the solvent used. The fastest substitute seems to
be methanol while acetone is considerably lower and diethylether is the slowest. Usually, complete
drying takes a very long time. Depending on the solvent, this process can take between several days
and several weeks, and must be followed by rewarming slowly the sample (1°C per hour). This
thawing procedure can nowadays be carried out in the best reproducible manner by using
commercially available freeze-substitution units. The frozen specimens are dehydrated automatically
under controlled conditions using any desired warming schedule. These systems are usually based on
LN2 cooling.
The choice of the substitution medium is a critical point. Numerous comparative studies have been
carried out in the past with the goal of achieving the best morphological preservation. In those
preparation schemes, freeze-substitution was often associated with simultaneous chemical fixation by
means of OsO4 or glutaraldehyde. The action of the fixing additive was found to be mostly efficient at
subzero temperature during the thawing procedure, another argument in favour of very slow
rewarming. Acetone is widely used nowadays and is often made water-free by adding molecular
sieves. On the other hand, methanol can substitute specimens more rapidly than acetone even with the
presence of large amounts of water. Diethyl ether has the drawback of acting very slowly but was
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reported to maintain the distribution of water-soluble substances. It is especially useful for the
microanalysis of electrolytes.
An excellent comparative study of freeze-drying procedures for X-ray microanalysis of biological
specimens was published recently (Pålsgård et al., 1994). In this study, tetrahydrofuran (THF) was
compared against the most widely used solvents (acetone, methanol and diethyl ether) using the
preservation of elemental ratios of diffusible intracellular electrolytes as criteria. The ratios were
determined by X-ray microanalysis of the pancreas sections of mice. They were dried using the
different solvents and then resin embedded at room temperature and sectioned.
A well-preserved elemental distribution is characterised by high K/Na and low Cl/K intracellular
ratios due to the high potassium and low sodium cellular contents in the living state. Pålsgård et al.
demonstrated that those methods which preserve a high K/Na ratio maintain a low Cl/K
simultaneously, thus showing unambiguously that major monovalent ions had been preserved.
According to these criteria, methanol was found to be a poor substitution fluid while THF and diethyl
ether gave the best results. When compared against direct cryosectioning of frozen tissues, these
results were confirmed and proved that freeze-substitution followed by resin embedding could be a
suitable preparation technique for the microanalysis of diffusible elements. Finally, acetone
substitution gave a relatively intermediate preservation of the distributions.
Zierold (1987) dealt with the problem of local ion’s redistribution during freeze-drying and freezesubstitution in the following terms: most of the diffusible elements of interest (from sodium to
calcium) are not volatile, even under high vacuum. When water is entirely replaced by vacuum or
substituted with a solvent, these ions are prone to bind to the closest macro-molecule. This process is
enhanced by the increase of electrostatic forces applied to such ions due to the drop in the dielectric
constant from 80 for water to 1 for a vacuum. Zierold concluded that in cells containing enough
organic matrix, the redistribution was expected to take place within small volumes of several nm. In
cell compartments rich in water, such as vacuoles or intercellular spaces, the spatial extent of this
phenomenon could be considerably increased. Taking this electrostatic force into consideration, he
explained that the reliable results obtained using the technique of freeze-substitution with diethyl
ether in the preservation of ionic distributions could be attributed to a very low dielectric constant
(4.3) when compared with that of ethanol, methanol or acetone (25.1, 33.5, 21.4 respectively).
4. The plastic embedding issue
The plastic embedding method is believed to ensure very good morphological preservation as well a
technically simple sectioning procedure at room temperature. This is a technique particularly suited
for the preparation of specimens intended for subsequent ultramicrotomy. Plastic embedding follows
the dehydration of the sample and can be carried out regardless of what the drying method may have
been: freeze-drying or freeze-substitution. The infiltration of plastic can be performed either at room
temperature after rewarming the tissue, or directly at a very low temperature immediately after the
drying is completed. Araldite is a resin widely used for routine treatment of specimens at high
temperature (293 K). But for a better result, low viscosity embedding media such as Spurr’s resin is
preferred. These media are expected to lower the structural damages which sometimes occur during
the plastic infiltration and this may be attributed to surface tension forces created by the advancing
plastic (Robards and Sleytr, 1985).
Roos and Barnard (1985) discovered a remarkably low ion compartmentalisation in specimens
embedded in Spurr’s resin compared to freeze-dried cryosections. They measured a higher K/P ratio
in cryosections, thus demonstrating that ion displacements had occurred during the diffusion of plastic
within the tissues. On the other hand, Pålsgård et al. (1994), after a careful freeze-substitution of
pancreas specimens compared such tissues embedded in araldite at room temperature to tissues issued
from a cryosectioning procedure. After X-ray analysis, they did not find any significant difference in
K/Na and Cl/K ratios.
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It is nevertheless believed that cryoembedding techniques could provide better retention of diffusible
elements. The interest in such techniques has increased since 1985 when it was proposed that new
methacrylate resins (lowicryl K11M and HM23) are able to infiltrate a tissue at temperatures below
213 K and 193 K respectively. The fact that the polymerisation of those resins can be operated at low
temperature under UV light is certainly beneficial. Several techniques using this kind of resin were
developed, including the impregnation in vacuo at –80°C (Wróblewski, 1989). For an example of a
versatile experimental device dedicated to freeze-substitution, freeze-drying and low temperature
embedding, see Sitte et al. (1994).
The elemental redistribution during the diffusion of polymer through the tissues is not the only
drawback associated with plastic or resin embedding. The artificial enhancement of the sample
organic matrix will decrease dramatically the peak to background ratio of X-ray analysis. The
emission of bremsstrahlung radiation is indeed proportional to the areal mass of the target. The
analytical sensitivity can thus be significantly reduced. In addition, the normalisation procedure based
on the determination of the organic sample matrix cannot be operated.
5. Microtomy and cryomicrotomy
If one excludes the analysis of isolated blood cells or cultured cells, biological tissues must be
sectioned prior to microanalysis. Three basic different procedures can be distinguished:
(i) sectioning of embedded specimens at room temperature
(ii) cryosectioning of hydrated frozen specimens (conventional
cryomicrotomy)
(iii) ultra-cryomicrotomy.
The third method refers to the sectioning at very low temperature (< –100°C) of tissues for high
resolution examination using an electron microscope. The thickness of cryosections obtained under
such conditions usually ranges from several tens to a hundred nanometers.
Very thin sections are necessary for an analysis using fine electron probes in order to maintain the
beam resolution through the whole specimen thickness. Electrons are by far more sensitive than
protons to straggling: protons generally undergo minor deflection in depth within the sample, whereas
electrons are extensively scattered, giving a “pear-shape” excitation volume which alters
progressively the resolution as it penetrates the sample. In addition, the path of protons is longer than
that of electrons. The optimum X-ray emission yield can therefore only be achieved in sections with a
thickness above 5 µm. The lateral resolution of nuclear microprobes does not necessitate sections
with a thickness below 5 µm. The latter must nevertheless not exceed 15 µm because of the problem
of unresolved structures due to overlapping cells.
Usually, a compromise has to be found between the sectioning temperature and the thickness of
sections, the two parameters being not really independent. At very low temperatures, it is not possible
to obtain semi-thin or thick sections because the tissues become too brittle. The use of ordinary
cryostats at higher temperatures was often reported by authors working with nuclear probes. The
technique of conventional microtomy generally fulfils all requirements of the nuclear microprobe
analysis. Many commercially available equipment can be used in the temperature range (–30°C/–
40°C). At this temperature, recrystallisation cannot be avoided but the spatial extent of induced
artefacts can be kept below the resolution of the probe. Another problem which can be addressed
concerns the eventual displacements of ions along the surface of the cryosections as is the case with
superficial melting.
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To carry out conventional cryosectioning, a fine thermal equilibrium has to be found between the
atmosphere of the cryostat, that of the knife and also the specimen itself. The knife must be slightly
colder than both the sample (several degree) and the cryochamber. Particular attention has to be paid
to the temperature of the polymer film which is used to pick up the section directly on the knife edge
to avoid thawing. The practical aspects of this technique have been briefly but carefully reviewed in a
paper by Kirby and Legge (1991).
6. Specimen mounting
For an analysis under ion beam, the tissue sections must be mounted on thin supporting foils. These
foils must be as thin as possible so as not to increase the X-ray background due to bremstrahlung
radiation. It must also be mentioned that the thinner the backing foil is the lower the energy deposited
by the particles in the sample. The resultant rise in temperature is a well-known cause for specimen
damage. Finally, it is important to keep the areal mass of this film at a value considerably lower than
that of the specimen when the mass of the latter has to be determined.
Polymer films with a thickness ranging from 0.1 to 0.4 µm can be easily obtained free from
contaminants. These foils are usually prepared from formvar, collodion or nylon dissolved in the
appropriate solvent and cast on fresh distilled water. In this way, the film is directly picked up on the
water surface and stretched over the sample holder or on electron microscope grids. For this purpose,
carbon grids which do not introduce interfering line on X-ray spectra are commercially available. It
must nevertheless be remarked that although the use of those grids as support improves the
mechanical rigidity, it also prevents the possibility of large scans during an analysis. The reader can
find the preparation procedures for formvar films in Echlin and Moreton (1979) and for collodion
films in Lamvik (1989).
When cryosections are picked up directly from the surface of the microtome knife, the supporting film
should have already been cooled at a temperature slightly above that of the knife. This is to ensure
specimen adhesion. Sometimes the section has to be sandwiched with another film to prevent
detachment.
Electron microanalysts use to coat the sample surface with thin conductive carbon layers in order to
improve the electrical conductivity. As a matter of fact, it is necessary to drain off charges brought by
the high current beam (several nA) on the specimen surface. The reader must be aware that this does
present a serious source of potential contamination where trace element analysis is concerned. The
problem of electrical conductivity of such supporting foil, even at low temperature, has been
addressed by several authors (Lamvik, 1989).
7. Preparation procedure for cultured cells
Cell culture has become the standard experimental method for conducting fine studies of cell
metabolism. The fundamental difference with other preparation schemes is that here the experimental
conditions of culture must fulfil, from the beginning, all analytical requirements. The extreme
physiological and mechanical stress which is inflicted on cultured cells when they are separated from
their culture support is suspected to modify their metabolism deeply. It is thus absolutely necessary to
culture cells directly on a supporting film compatible with the X-ray analysis. By doing so, cells can
grow as a monolayer. The microanalysis of individual cells is thus possible, even in a heterogeneous
population.
Models of cultured cells are usually employed for in vitro assays, in the framework of pharmacology
or toxicology studies. In most cases, an incubation is performed in a medium supplemented with the
substance under investigation. After a rapid rinsing procedure, the cells intended for individual
microanalysis are immediately cryofixed. Where the analysis of whole single cells is concerned, the
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goal is to determine the intracellular elemental composition and maybe the spatial distributions
without the need for sectioning. The medium surrounding cells (culture medium or experimental
buffer) must then be removed in order not to interfere with the analysis. Rinsing, using an appropriate
solution, must be performed very rapidly to prevent the loss of intracellular elements. The inorganic
cations are indeed very sensitive to changes in osmotic gradients. Volatile buffers such as ammonium
acetate can be employed (Wróblewski and Wróblewski, 1993). They have the advantage of being free
from minerals. Ice-cold distilled water can also be used for a short rinsing (5 sec). A detailed study on
the eventual effects of rinsing on cell electrolytes has been published in (Borgmann et al., 1994).
Thin film support
Grid bars
Cells
Sample holder
Fig. 4. Cells grown on a thin film with (down) and without (top) a supporting grid. When stretched over a large
hole (2–3 mm in diameter) without grid, the film is more fragile but ensures an easier analysis. (see text for
details) (Redrawn from Wróblewski and Wróblewski, 1993).
After the excess rinsing fluid has been absorbed using a filter paper, the cells can be cryofixed by
immersing them in liquid cryogen (see section 2.4). The thickness of the cells (< 10 µm) is low
enough to ensure cryofixation without any risk of crystallisation. The subsequent preparation will
depend on whether the cells need to be plastic embedded. Some authors carried out cryosubstitution
with diethyl ether and THF followed by plastic embedding. Ortega et al. (1996) employed the more
conventional method of freeze-drying and a microanalysis of unembedded individual cells. For
extensive reviews on preparation schemes and methodological aspects, the reader is referred to
Warley (1994).
The main difficulty in this procedure is finding a supporting film that is compatible with the
conditions of cell culture and the ion beam analysis. It must be inert to the cells and must not contain
contaminants. It is of extreme importance that this film should be thin enough so as not to enhance the
X-ray background and be transparent enough for a light microscopy examination of cells during
culture. The cells can be seeded on thin polymer foils stretched over a hole (2 or 3 mm in diameter)
drilled in the sample frame. The latter must be made from biocompatible material (carbon, nylon, pure
aluminium or titanium). This is especially important for metallic supports which are prone to corrode
in the culture medium and may therefore release cytotoxic high doses of metal such as copper, an
element present in most alloys.
All components, supports and films must be, of course, sterilised. It must be emphasised here that
thick growth supports, such as carbon plates which have been used in the past for low accelerating
voltage (1–5 kV) electron microscopy must be absolutely avoided. For such voltages, the electron
path is so short that the analysis of cells is possible without having the electrons impinging on the
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support. As mentioned earlier, MeV protons are more penetrating particles. Bremsstrahlung radiation
emitted along the path of the incident ions in the support would decrease dramatically the
signal/background ratio.
Different polymer films can serve as growth support. Formvar and Pioloform are excellent transparent
thin films (0.2–0.4 µm in thickness) (Wróblewski and Wróblewski, 1993). They have sufficient
mechanical rigidity to cover holes that are of 2–3 mm in diameter without needing additional support
grids. However, full success is not warranted, since some films break during cell culture. A more
acute problem is cell adhesion to the growth support. Some lines are perfectly adapted to a direct
culture on plastic supports that have already been made hydrophilic through chemical or physical
treatments. Classical plastic culture dishes may be treated, for instance, by either high voltage
discharges, u.v. light or electron bombardment. For the culture on thin films, extracellular matrix
compounds must be employed to pre-coat surfaces. However, no general rule can be given here since
cell growth is dependant on the line under investigation. Specific adhesion factors such as collagen or
gelatine are commercially available free from contaminants. Their use as a form of coating layer has
been described by different authors (Ortega et al. 1996)
References
Borgmann, S., Granitzer, M., Crabbé, J., Beck, F.X., Nagel, W. & Dörge, A. (1994) Electron
microprobe analysis of electrolytes in whole cultured epithelial cells. Scanning Microscopy
Supplement, 8, 139-148
Echlin, P. & Moreton, R.B. (1979) The preparation of biological materials for X-ray microanalysis. In
Microprobe analysis as applied to cells and tissues, ed. T. Hall, P. Echlin & R. Kaufmann, p 159.
London: Academic Press
Kirby, B.J. & Legge, G.J.F. (1993) The preparation of biological tissue for a trace element analysis on
the proton microprobe. Nucl. Instr. and Meth., B77, 268-274
Lamvik, M.K., Davilla, S.D. & Tuttle, J. (1989) Properties of substrates for low temperature
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Ortega, R., Moretto, Ph., Fajac, A., Bénard, J., Llabador, Y. & Simonoff, M. (1996) Quantitative
mapping of platinum and essential trace metals in cisplatin resistant and sensitive human ovarian
adenocarcinoma cells. Cellular and Molecular Biology, 42(1), 77-88
Pålsgård, E., Lindh, U. & Roomans, G.M. (1994) Comparative study of freeze-substitution techniques
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