Impregnation of soft biological specimens with thermosetting resins and elastomers.код для вставкиСкачать
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.