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Impregnation of soft biological specimens with thermosetting resins and elastomers.

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Impregnation of Soft Biological Specimens with
Thermosetting Resins and Elastomers
GUNTHER VON HAGENS
Anatomisches Znstitut der Uniuersitat Heidelberg, 0-6900Heidelberg,
Federal Republic of Germany
ABSTRACT
A new method for impregnation of biological specimens with
thermosetting resins and elastomers is described. The method has the advantage that the original relief of the surface is retained.
The impregnation is carried out by utilizing the difference between the high
vapor tension of the intermedium (e.g., methylene chloride) and the low vapor
tension of the solution to be polymerized. After impregnation, the specimen is
subject to polymerization conditions without surrounding embedding material.
The optical and mechanical properties can be selected by proper choice from
various kinds of resins and different procedures, for example, by complete or
incomplete impregnation. Acrylic resins, polyester resins, epoxy resins, polyurethanes and silicone rubber have been found suitable for the method.
Excellent results have been obtained using transparent silicone rubber since
after treatment the specimens are still flexible and resilient, and have retained
their natural appearance.
Preservation of soft biological specimens by
conventional method has major disadvantages. Preservation in fluids, for example,
alcohol, or embedding in blocks of plastic
(Bridgman and Humelbough, '63),makes i t
impossible to touch the specimen and to inspect it under magnification. Freeze-dried
specimens or specimens which are impregnated with paraffin (Hochstetter, '271, are not
resistant against mechanical stress and paraffin is not transparent.
The method described in the following enables one to choose optical and mechanical
properties of the specimen while its surface
and appearance are preserved, as record or
teaching purposes may require.
by its higher boiling ingredients. The pressure
is decreased gradually to 5-0 mm Hg and a cold
trap must be used to protect the oil vacuum
pump from the solvent vapors. After such
impregnating procedure the specimen is removed from the solution. The excess liquid is
drained off or may be removed by washing
with solvents or monomer substance. The
specimen is then subjected to polymerization
for curing.
The procedure may be carried out in two
modifications: (1)as complete impregnation to
obtain transparent as well as non-transparent
specimens and (2) as incomplete impregnation
which always results in nontransparent specimens. The latter method is preferable when
the inner and outer surfaces of the specimen
PRINCIPLES OF THE PROCEDURE
are to remain free of visible resin.
The specimen is fixed and dehydrated as
(1) Complete impregnation gives preparausual, for example, with formalin as fixative tions in which tissue fluid is fully replaced by
or alcoholfacetone for dehydration. It is then resin. Transparent silicone rubber yields nonimmersed in an intermedium of low boiling transparent specimens. Transparent acrylic
point, such as methylene chloride, b. p. 40°C, resins, polyester resins, epoxy resins and
which is miscible with the solution of resin to polyurethanes give - more or less - transbe used. The specimen is transferred from this parent specimens, for example, of large obintermedium into the unpolymerized solution jects where the internal structures (bone,
of the resin for full immersion. The volatile in- blood vessels or others) are visible; or of mactermedium is removed from the specimen by roscopic sections (e.g., from brain, lung, kidevaporation in vacuo. Thereby the intermeReceived July 20, '78. Accepted Oct. 31, '78.
' Patent pending.
dium is successively replaced by the resin and
ANAT. REC. (19791 194: 247-256.
247
248
GUNTHER VON HAGENS
ney) which can be examined favourably in
transmitted light.
(2) Incomplete impregnation gives porous,
non-transparent specimens. The tissue fluid is
partly replaced by resin and partly replaced by
air. The resin impregnates and stabilizes the
cell membranes, connective tissue fibers or
other structures. Since resin cannot be detected a t the surfaces, also nontransparent or
even coloured resins can be used. A fractured
surface gives a three-dimensional view of the
tissue.
Incomplete impregnation can be carried out
in two ways: (a) By incomplete extraction of
the last intermedium, but the extent of
impregnation is then difficult to control; (b)
by dilution of the unpolymerized solution of
resin with an organic solvent which is not
volatilized during removal of the volatile intermedium a t low pressures. This variation
enables to choose precisely the percentage of
resin in the cured specimen.
When curing incompletely impregnated
specimens, shrinkage must be avoided. This
can be accomplished by generating a high
vapor tension within the specimen a t elevated
temperature or by generation of gas, for example, CO, when polyurethanes are used. Such
conditions must be maintained until the specimen is sufficiently hardened.
The method has been successfully applied to
materials such as human arm with shoulder
girdle, human brain, larynx, liver, kidney,
testis and embryos; newborn pigs, rats, mice
and other complete organisims; and to sections of human brain, lung, kidney and
forearm. It was also applied to other materials
of biological interest, for example, plant
leaves, cactus and ferns. Many of the specimens, in particular from plants, retained their
natural colour when preserved.
EXPERIMENTAL PROCEDURES
Complete impregnation, non transparent,
of a human heart (fig. I )
andjejunum (fig. 2)
A human heart recovered three days after
death was fixed in a solution of 5% formalin.
Dehydration was carried out by sequential
immersion in increasing concentrations of
alcohol (70%, 90%, 96%, 100%) and acetone.
Methylene chloride was then used as intermedium. After draining excess methylene
chloride, the heart was placed in a container
with a solution of transparent silicone-rubber
(Sil-Gel@-604).The specimen was completely
submerged in this solution by use of a glass
plate. The container was placed then in a bell
jar a t - 5°C to prevent polymerization.
Evacuation was carried out gradually (!I by
means of an oil pump. The pressure was
lowered gradually to 1 mm Hg by closing a
bypass and the pump was protected by a trap
cooled with liquid nitrogen. Observation of the
progressing impregnation should be possible
during this procedure. The heart was removed
after 48 hours by which time not more than
a few bubbles of methylene chloride were
released.
Excess of silicone rubber was allowed to
drain off and the specimen cured by keeping it
at room temperature in a hanging position for
24 hours. Silicone rubber held between the
folds of the heart was removed during the curing process with the aid of methylene chloride
and a paint brush.
The human jejunum (fig. 2) has been fixed
by a solution of 5%formalin via the arteries.
Dehydration and impregnation has been carried out as described above for the human
heart.
The ready specimens are flexible and resilient. Exposure to 90°C for four months did
not change the mechanical or optical properties.
Complete impregnation, transparent, of
sections of a human lung (fig. 3)
A human lung was fixed and sectioned with
a meat slicer. The sections, 1-2mm thick, were
dehydrated as described. After soaking in
methylene chloride as intermedium, the sections were transfered in a solution of epoxy
resin (Araldite CY 221 100 parts of weight and
Harter HY 2954, 25 parts of weight). The
impregnation period was 60 minutes, during
which time the pressure was lowered to 50 mm
Hg. The sections were then placed on a foil of
polyester. Some additional resin was dropped
on the section which was finally covered by
another polyester foil without trapping air
bubbles. The samples were cured a t room temperature within two weeks. After peeling off
the polyester foils, the sections are transparent, flexible and resistant against mechanical stress.
Incomplete impregnation by dilution of the
resin, applied to a piece of
human lung (fig. 4)
A piece of human lung was fixed, dehydrated and immersed in methylene chloride.
IMPREGNATION OF SPECIMENS WITH POLYMERS
249
Fig. 1 Human heart, completely impregnated with silicone rubber. All fine details (valves, chordae tendineae) are
preserved and can be touched. Normal size.
Impregnation was carried out a t room temperature using a solution of polyurethane
(BaymidurB-VPKL 3-5002,43parts of weight
and castor oil 100 parts of weight). One
hundred fifty parts of xylene were added,
which is much less volatile than methylene
chloride. The piece of lung was impregnated
with the diluted solution of resin. Full impregnation with the diluted resin was reached
with final vacuum of about 80 mm Hg after 40
minutes. Curing and evaporation of the solvent xylene took several days. However sufficient curing to prevent shrinkage was reached
within a few hours.
The resulting specimen was broken into two
parts. With the aid of a stereomicroscope, the
alveoli, alveolar septs and bronchioli of the
lung are clearly visible on the fractured surface, giving a three-dimensional view.
Incomplete impregnation by incomplete
removal of last intermedium, applied
to a piece of rumen (figs. 5a,b),
rectum of cow (fig. 5c) and
human jejunum (fig. 5 d )
Pieces of rumen and a piece of rectum of a
cow were fixed by immersion in a solution of
formalin (5%), dehydrated and transferred
into methylene chloride. To achieve incomplete impregnation, not all of the intermedi-
250
GUNTHER VON HAGENS
Fig. 2 Human jejunum, completely impregnated with silicone rubber. The specimen is photographed twice to demonstrate its flexibility. One half of normal size. Surface details as demonstrated in figure 5d require incomplete
impregnation.
IMPREGNATION OF SPECIMENS WITH POLYMERS
Fig. 3 Section of a human lung, completely impregnated with epoxy resin. The section is bent (arrow), to
demonstrate its flexibility. Normal size.
Fig. 4 Fractured surface of a human lung, incompletely impregnated with polyurethane. Alveoli and
alveolar septs are clearly visible (arrows). For photography a PHOTOMAKROSKOP M 400, WILD-LEITZ
was used. x 80.
251
252
GUNTHER VON HAGENS
um was removed. Therefore, the impregnation
with a solution of silicone rubber (Sil-Gel@
604) took only five minutes a t room temperature down to a pressure of 150 mm Hg. Under
these conditions the gut was impregnated
with silicone rubber to about 65%.The samples were washed with benzene to remove excess of silicone rubber and cured a t 70°C for
four hours.
The specimens are flexible and appear
whitish because of the air within the specimen. Details of the surface are well seen under
a stereo microscope (figs. 5a-c). The human
jejunum (fig. 5d) was treated equally, except
that fixation was done by injection.
DISCUSSION
The impregnation of soft biological specimens in macroscopy with polymerizing thermosetting resins and elastomers involves the
difficulty of replacing low viscous fluids
(water, organic solvents) in soft tissue against
solutions of resins of usually higher viscosity
within a reasonable time, without major
shrinkage and without excessive consumption
of resin. These problems are minimized by
using an intermedium of high volatility and a
solution of resin of low volatility.
When impregnating large specimens, the intermedium has to be removed continuously by
evaporation in vacuo so that it is replaced by
the resin, as described in the examples above.
The larger the specimen, the lower the viscosity of the resin should be. When impregnating tissue sections up to about 1 mm thickness, the section saturated with a volatile intermedium (e.g., diethyl ether), may be placed
on a layer of resin which is poured on a film of
polyester. The intermedium evaporates at a t mospheric pressure and is replaced gradually
by the resin.
It is obvious that besides the type of resin,
the last volatile intermedium is of major
importance. The greater the difference between vapor tensions of intermedium and
resin, the less resin will evaporate. In a typical
example (heart, fig. 1) described in the foregoing, the difference is, expressed by boiling
points a t atmospheric pressure, 40°C for
methylene chloride versus 200-300°C for the
particular solution of silicone rubber. Methylene chloride seems to be the intermedium of
choice since it is not combustible, it is compatible with almost all resins and it is inexpensive. Acetone can substitute when the high
specific gravity of methylene chloride or
swelling of polymerized silicone rubber offers
difficulties with specific samples.
Direct impregnation by water soluble resin
(e.g., glycol methacyrlate) is not possible by
the method described. The tissue water cannot
be evaporated from the specimen through the
aqueous solution of the surrounding resin.
Heating would induce or accelerate polymerization and this would make the evaporation
of tissue water more difficult or even impossible. Moreover, excessive heating for an extended period of time would be detrimental to
the tissue.
It is possible to impregnate freeze-dried
specimens or specimens dried by the critical
point method. However, freeze-drying, in particular of large specimens, is time-consuming
and the polymerization of most resins seems to
be inhibited by various components of the
specimen, possibly lipids or amines (Lautenschlager, '76). Such inhibitors are washed out
from the tissue a t least to some extent by the
dehydrating organic solvents.
So far, silicone rubber, polyurethanes,
polyester, acrylic and epoxy resins have been
found useful in the procedure. The choice
depends on the material and the purpose for
which the specimens are meant. Solutions of
viscosities up to 5,000 mPa's are applicable.
Silicone rubber gives specimens which are
most natural in appearance. They are flexible
and resilient and the rate of curing can be
easily observed. The polymerization is only
slightly exothermic, which is of importance
when working with large specimens.
Polyurethanes are highly reactive even with
small amounts of water and may cause formation of solid foam. This type of resin should be
used for incomplete impregnation.
Polyester resins: The surfaces remain tacky
under the influence of oxygen. Advantages of
polyester resins are their broad spectrum of
desirable qualities like broad range of refractive index, low viscosity and flexibility. ThereFig. 5 a,b. Mucosa of a bovine rumen, tongue shaped
(a) and leaflike (b) villi. x 10. c. Surface view of a longitudinally sectioned anal channel of a cow; rectal mucosa
with zona columnaris (stippled) above, zona intermedia in
the middle, zona cutanea with single hairs, free of silicone
rubber, below. Normal size. d. Human jejunum, surface
view of the mucosal face, exhibiting intestinal villi. x 10.
Insert: higher magnification ( X 25) reveals the openings
of the crypts of Lieberkdhn between the villi (arrow). All
specimens are incompletely impregnated with silicone
rubber; they are flexible and resistant against mechanical
stress. From the macroscopic aspect the specimens are
similar to the jejunum in figure 2. For photography a
PHOTOMAKROSKOP M 400, WILD-LEITZ was used.
IMPREGNATION OF SPECIMENS WITH POLYMERS
Figure 5
253
254
GUNTHER VON HAGENS
fore, specific grades and formulations are to be
used.
Acrylic resins are crystal clear. The polymerization reaction is markedly exothermic and
therefore controlled curing is most critical in
such preparations.
Epoxy resins have the advantage to be most
resistant against mechanical stress.
In regard to optical properties, the degree of
transparency of the specimens is of major concern. Incomplete impregnation results in nontransparent specimens which appear whitish
due to the air within the specimen, even when
strongly dyed resins have been used.
When complete impregnation has been applied, the refractive index of the resin in reference to that of the tissue is important. The refractive index of tissue generally is around
n~2oOC= 1.54 (Spalteholz, '24). For transparency, the refractive index of the polymerized resin must be close to that value. Therefore, transparent silicone rubber, nD2ooC =
1.40, will not yield transparent tissue preparations. Transparent polyurethanes, polyester
resins and transparent epoxy resins have essentially the same refractive index as the
common tissue. These resins yield - more or
less - transparent products by complete impregnation. Acrylic resins (e.g., polymerized
polymethyl methacrylate n~2oOC = 1.49)
yield in slightly transparent specimens. The
finished specimen has to have a smooth surface. Large objects must be bleached, otherwise they will turn brown or even black, in
particular, when blood was not removed before
impregnation. Transparency is required when
inner structures of blood or lympatic vessels,
bones or other structures are to be shown for
demonstration purposes. Preservation of natural colour, for example in lung sections, or
staining of kidney sections with ferrum hematoxilin is desirable. In some specimens, the
color has been preserved by treating hemoglobin with sodium nitrite and ascorbic acid.
Transparency can be avoided by staining the
specimen strongly before impregnation. Obviously each material requires specific methods
and resin for optimal results.
Regarding the mechanical properties of the
specimens it should be pointed out that in general complete impregnation gives better resistance against mechanical stress than incomplete impregnation.
Incomplete impregnation is easy to carry
out in tissues which have extensive lumina
(e.g., blood vessels, glandular ducts). Such
preparations have been made from lung,
kidney, liver and gut.
Major shrinkage during dehydration and
impregnation can be avoided by proper sequence of dehydrating solutions and careful
adjustments of the gradient of vacuum applied. After incomplete impregnation, shrinkage can be kept minimal by balancing the solvents vapour tension and the mechanical
strength of the tissue against the forces causing shrinkage until sufficient hardening is
reached. The extent of shrinkage has been
roughly estimated by inspection; in specimens
with surface differentiations a stereo-microscope was helpful.
In several specimens the histological quality has been controlled. Standard 10-p HEsections revealed a quality of the tissue
fully comparable to that known from paraffin
sections.
Embedding of small pieces of tissue for light
and electron microscopy with acrylic, polyester or epoxy resins commonly is done by immersion, using increasing concentrations of
resins (Bennett et al., '76; Anker et al., '74).
This method is not practical for the impregnation of macroscopic specimens. It is not possible to exchange resins of higher viscosity
against water or organic solvents by immersion within a reasonable time. Besides, i t
would be very difficult to prevent polymerization during impregnation which may require
weeks, depending on the specimens size and
viscosity of the resin. In addition, the resin
would have to be renewed several times in
view of dilution by the intermedium and this
would involve considerable waste.
Only one solution of resin is needed for the
method described here. The resin becomes not
diluted since the last intermedium is removed
and the procedures can be carried out within a
reasonable time. When the resin solution can
be sufficiently stabilized, the surplus can be
re-used.
The disadvantage of the technique may be
that the unexperienced will not get optimal
results in the beginning. Some knowledge of
tissue dehydration and some overall experience in handling of thermosetting resins and
elastomers is desirable.
One may expect that many additional variations and applications of the method can be
developed. Many practical purposes can be
named: preservation of morphological struc-
IMPREGNATION OF SPECIMENS WITH POLYMERS
tures for demonstration; preservation of
plants; documentary evidence in forensic
medicine. Perhaps a major point in preparing
samples by the method will be the possibility
to correlate macroscopic and microscopic demonstration in teaching.
ACKNOWLEDGMENTS
I thank Professor H. Schlenk from the
Hormel Institute, University of Minnesota for
reviewing the text and Miss I. Ertel and Mr.
M. Liedtke for taking the photographs.
The study could not have been done without
continual advice and contribution of materials by Bakelite GmbH, Duisburg; BASF,
Ludwigshafen; Bayer AG, Leverkusen; Busing & Fasch KG, Oldenburg; CIBA-GEIGY,
Wehr/Baden; Kalle AG, Wiesbaden; PeroxidChemie GmbH, Hollriegelskreuth; RohmGmbH, Darmstadt; Alfons Schmidt, Speyer;
Wacker-Chemie GmbH, Munchen.
I wish to express my appreciation especially
to Professor W. Kriz for his patience and helpful efforts to improve the concept and clarity
of this paper.
LITERATURE CITED
Anker, G. Ch., K. Scheers-Dubheldam and C. Noorlander
255
1974 An epoxy resin embedding technique for large
objects. Stain Technol., 49: 183-188.
Bennett, H. S., A. D. Wyrick, S. W. Lee and J. H. McNeil
1976 Science and a r t in preparing tissues embedded in
plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain
Technol., 51: 71.97.
Bridgman, C. F., and F. A. Humelbough 1963 Plastic
embedded teaching specimens. Med. Biol. Illus., 13: 177185; 14: 265-272.
Hochstetter, F. 1927 Ueber ein Verfahren zur Herstellung von Trockenpraparten von Tieren und Pflanzen in
naturlicher Form und Farhe. Forschungen und Fortschritte, 3: 140-141.
Lautenschlager, E. 1976 Einhettungen in Kunstharz.
Wepf & Co., Verlag, Basel, pp. 55-58.
Spalteholz, W. 1924 Das “Durchsichtigmachen” als
biologische Arbeitsmethode. Handbuch der hiologischen
Arbeitsmethoden by Emil Abderhalden. Verlag Urban &
Schwarzenherg, Berlin, Wien. Aht. IX, Teil 1: 409-438.
APPENDIX
Products, which have been used for impregnation of specimens: Silicone rubber: Sil-Gel 8
604, Wacker-Chemie, D-8000 Munchen 2;
Epoxy resin: Araldit@ CY 221, Harter, HY
2954, CIBA-GEIGY GmbH D-7867 Wehr/
Baden; BayrnidurB VP KL 3-5002, Bayer-AG,
Sparte KL, D-5090 Leverkusen.
Further product information is given by the
author.
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