The inadequacy of microwave radiation as a means of fixation for electron microscopyкод для вставкиСкачать
INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Quiimipiac College THE INADEQUACY OP MICROWAVE RADIATION AS A MEANS OF FIXATION FOR ELECTRON MICROSCOPY By STEVEN ?. SCHMIDT B.S. Wheaton College, 1983 A THESIS Presented to the School of Allied Health and Natural Sciences and Quinnipiac College in partial fulfillment of the requirements for the degree of Master of Health Science June 1985 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number 1414127 UMI’ UMI Microform 1414127 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT THE INADEQUACY OF MICROWAVE RADIATION AS A MEANS OF FIXATION FOR ELECTRON MICROSCOPY Steven P. Schmidt Master of Health Science Quinnipiac College June 1935 The aim of this project was to evaluate the use of microwave radiation as a means of fixation for electron microscopy. Routine methods, though adequate, have failed to provide an all inclusive method of fixation without ser iously damaging the fragile cellular ultrastructure. Two variables (temperature and fixative mediums) were manipulated to determine the optimal temperature-medium combination that gave the best results. Temperatures ranged o o o from 50 - 80 C in 5 C increments and glutaraldehyde, formalin and saline were the mediums used. From this study it can be stated that microwave radia tion cannot be used as a fixative for electron microscopy. The effects of the high, rapid, random heating characteris tic of microwave radiation, destroyed the delicate ultra- QU1NNIPIAC COLLEGE U A M P kC M f* T LIBRARY n e rto Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16167 structure leaving the tissue inadequate for diagnostic interpretation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THE INADEQUACY OF MICROWAVE RADIATION AS A MEANS OF FIXATION FOR ELECTRON MICROSCOPY This thesis is approved as a creditable and independent investiga tion by a canidate for the degree of Master of Health Sciences, acceptable as meeting the thesis out implying that the conclusions requirements and is for this degree, but with reached by the canidate are necessar ily the conclusions o f the major department. r Quinnipiac College Thesis A dvisor 2al '< U Mary Jane Clarke, M.S., R.T. Assistant Professor, Director of Radiography Quinnipiac College Clinical Thesis Advisor David S. Papermaster, M.D. Director, EM Facility West Haven Veterans Administration Medical Center Associate Professor o f Pathology, Yale University Medical Director P athologists1 Assistant Training Prog Rosa E. Enriquez, M.D. Director of Pathologists' Assistant Training Program, West Haven Veterans Administration Medical Center ^tholoftyy, yQ\e Univei^ity /? Assistant Professor, Director, Graduate Studies_ Quinnipiac College Kent S. Marshall, Associate Professor ofyChemistry Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ac knowledgement s I would like to thank the following people for their support in this endeavor: Lillemor Wallmark whose technical expertise, assist ance and patience were integral to the completion of this proj e c t . Dr. Papermaster and Mary Jane Clark who were of im measurable assistance in the preparation of this work. My mother and father who not only made this education economically feasible, but who have also taught me the value of finishing each project that is begun. To my patient wife, Tamara, whose friendship, motivation and affection are an inspiration daily. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OP CONTENTS PAC-E INTRODUCTION Statement of the P r o j e c t ............................ 1 Literature Review .................................... 2 Microwave R a d i a t i o n ................ ...... . 5 Chemical Fixation Proc es s....................... ~ Physical F i xa tion ............................... Microwave F i x at io n............................. Ra tio nale ...............................................22 MATERIALS AMD MET HODS ....................................... 23 R E S U L T S ...................................................... 25 D I S C U S S I O N ................................................... 46 Data Analysis.......................................... 4& Overall Conclusions.................................. 51 RE FE R E N C E S .................................................. 52 A P P E N D I X E S .................................................. 55 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OP TABLES Page Table 1. Energies of Electromagnetic Radiati ons ........ 6 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF PLATES Page Plate 1. Rat kidney controls fixed in two stan dard fixatives and examined by light microscopy, a) formalin, b) glutaraldehyde Plate 2. Rat kidney fixed in saline with m i c r o wave radiation at various temperatures examined by light microscopy, a) 50 C. 1800x, b) 55 C, 1800x, c)60 C, 1800x, d) 65°C, 1800x. Plate 5. Rat kidney fixed in formalin with micro wave radiation at various temperaturesand examined bv light m i c r o s c o p y , a) 50 C, 1800x, b) 55 C, 1800x, c) 60 C , 1800x, d) 65 C , 1800x. Plate 4. Rat kidney fixed in glutaraldehyde with microwave radiation at various temperatures and examined bv light microscopy, a) 50 C, 1800x, b) 55 C, 1800x, c) 60 C , 1800x, d) 65°C, 1800x. 26 28 50 52 Plate 5. Rat kidney fixed in saline at two temperatures with microwave radiation and examined by light 55 microscopy, a) 70 C, 1800x, b)80 C, 1800x. Plate 6. Rat kidney fixed in formalin with m ic ro wave radiation at two temperatures and examined by light microscopy, a) 70 C, 1800x, b) 80 C, 1800x. Plate 7. Rat kidney fixed in glutaraldehvde with microwave radiation at two temperatures and examined light microsrcopv, a) 70 C, 1800x, b) 80°C, 1800x. Plate 8. Rat kidney control fixed in formalin and examined by electron microscopy. Plate 9. Rat kidney control fixed in glutaraldehyde and examined by electron microscopy. 54 55 ^8 J Plate 10. radiation Rat ^idney fixed in saline with microwave at 70 C and examined by electron microscopy. 40 Plate 11. radiation Rat £idney fixed in saline with microwave at 80 C and examined by electron microscopy. 41 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Page Plate 12. Rat kidney fixed in formalin with microwave radiation at 70 C and examined by electron microscopy. 42 Plate 13. Rat kidney fixgd in formalin with microwave radiation at 80 C and examined by electron microscopy. 43 Plate 14. Rat kidney fixed inoglutaraldehyde with microwave radiation at 70 C and examined by electron microscopy. 44 Plate 15. Rat kidney fixed inQglutaraldehyde with microwave radiation at 80 C and examined by electron microscopy. 45 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION Statement of the Project The aim of this project is to evaluate the use of microwave radiation as a means of fixation for electron microscopy. Routine methods, though adequate, have failed to provide an all inclusive method of fixation without ser iously damaging the fragile cellular ultrastructure. This damage is of great concern to those involved in the rapidly developing field of immunocytochemistry. Moreover, the time, money, and complexity of routine methods have signif icantly limited the use of electron microscopy in the diag nostic laboratory. These limitations have led to many areas of research in the goal of finding non-chemical methods of fixation. Supported by numerous reports of success at the light micro scope level, microwave radiation may be the key to the com plete utilization of the electron microscope. Therefore, the goal of this project is to determine the feasibility of microwave radiation as a means of fixation for the elec tron microscope. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2 Literature Review Since the days of Liewenhook (1662) scientists have continually strived for ways to look at the world around them. This drive to look into the microscopic world has led to the development of the most powerful instrument of inspection, the electron microscope. By using electrons as the source of energy, researchers have been able to m a g nify objects well over 40,000 times, allowing the individ ual the opportunity to examine a world unattainable by con ventional light microscopy. However, along with the advan tages, there comes the disadvantage of magnifying the det ri mental effects characteristic of tissue preparation. The result is that since the development of the electron micro scope in the 1940's, researchers have sought ways to limit the damage that fixation can incur in tissues. Fixation, by definition, is the method by which tissues are preserved in a state that holds its components "fixed" in situ so that they may be studied with the minimal amount of alteration from the living state. The main objectives of fixation are to preserve the structure of cells with the minimal amount of alteration from the living state with regards to volume, morphologic detail, and spatial rela tionships of organelles and macromolecules, minimum loss of tissue constituents and protection of specimens against the subsequent treatments including dehydration, embedding, staining, vacuum, and exposure to the electron beam (1). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Within recent years, attention has shifted from tradi tional chemical methods to the products of modern technology in search of methods that will better realize the goals of fixation while minimizing any side effects. Furthermore, with the advent of new immunohistochemical studies, the need to find a non-chemical fixative has been accentuated in hopes of minimizing chemical modifications of antigenic determin ants. One possible method is microwave radiation; which if proven reliable has the benefits of speed, ease, and low cost without using chemicals. Microwave Radiation A complete explanation of microwave radiation is far beyond the scope of this paper (see Collins 2). However, a basic understanding of the properties of microwaves and their effects are imperative for an understanding of how one might utilize microwaves in the fixation process. Microwave radiation is a form of electromagnetic radiation which falls within the frequency of 300 m e g a hertz to 300,000 megahertz (3). A hertz is one variation or cycle per second and a megahertz 1,000,000 cycles per second. Its characteristics are based upon the theory of electromagnetism and the understanding that microwave radi ation, like visible light moves in waves with frequency, wavelength and period being its fundamental characteristics. Microwave radiation exists naturally as a part of the spectrum of radiant energy released by the sun. It is now possible to conduct and control man-made microwave energy Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by c o n d uc ti ng it from a t r a n s m i t t i n g source antennae, re ce iv er antennae. to a The w av es tr an s fe r thr ou g h free space or a m e d i u m as o s c i l l a t i n g electric and magnet ic g the speed of light, 3 X 10 me te rs per second. fields at Not only does m i c r o w a v e r a d i a t i o n travel at the same speed as light, but tics of light. be reflected, it also has several other c h a r a c t e r i s Like light, mi c r o w a v e s carry the a b i l i t y to scattered, r e fr ac te d and a bs orb ed by a medium. It is by ab so rpt io n that m i c r o w a v e rad ia ti on can a c t iv el y in fl uen ce bio logical systems. However, a bs o r p t i o n dep en ds on the electromagnetic p ro p e r t i e s of the media. important variables are the d ia lec tri c c o n d u c t i v i t y of the tissues (3). The most constant and the It has been sh own that at best only 1 0 % of the ra d i a t i o n is a b so r be d by b io l o g i c a l tiss ue (4). It is im portant to rea liz e that w a te r compri ses 60-70% of a c e l l ’s volume. 10? is absorbed, Therefore, even t h o ug h only bec au se of wat er 's hig h conductivity, this small p r o p or ti on of a b so rb ed r a d i a t i o n can have a drastic effect on the mo l e c u l a r e n v i ro nm en t of water and the tiss ue molecules immersed in the water. Fr om the ele ct ro ma gn et ic r a d i a t i o n absorbe d by the cell the amount of ener gy r e l e a s e d per p h o t o n can be c a l c u l a t e d using E = hf (E=6.625 x 10 “ ^ Joules second). This e q u a t i o n is known as Pl ank's e q u a t i o n after its ori gi na t or , Max Plank. constant, The energy (E) equals the pr oduct of P l a n k ’s h, times the f re q u e n c y (f) o f the radiation. e n e r g y a bso rb ed is r e l e a s e d into the mo l e c u l e as kinetic Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The energy system. (thermal motions) that raises the temperature of the If the absorbed energy is above 10 eV it can cause ionization of the molecules. The energy per photon released by microwave radiation -3 _ is 1.24 x 10 eV which is far below the 1.24 x 10' and 2.48 x 10^ eV released by a photon of X or Gamma radiation respec tively. The importance of this is that microwave radiation, unlike X or Gamma radiation (and other ionizing radiations, see Table 1) is far below the energy level needed to cause ionization of molecules. Ionizing radiation may generate destructive genetic mutations (3). The energy released by the microwave radiation does have distinct effects on the cellular constituents, particu larly the proteins, nucleic acids and lipids. Its effect is indirect and mediated by its absorption by the water present in the tissue. The energy released by the microwave radia tion causes water molecules to vibrate at approximately 2,450 million times per second thereby rapidly raising the tissue temperature. Since microwaves travel at the speed of light, the rise in temperature is extremely rapid. Chemical Fixation Tissue fixation can be induced by both physical and chemical means, but chemical fixation has been studied the most since it causes the least amount of cellular damage at this time. Chemical fixation, unlike physical methods such as freezing, freeze drying or heating adequately pr e serves many cellular components. Because of these ad- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 6 Table 1 ENERGIES OP ELECTROMAGNETIC RADIATION O 1 Type of Radiation Wavelength Freauency Energy per Photon Tnm) (MHz) (eV) 3.0 X 10 21* 1.24 X 107 1.0 X X 5.0 X 10"1 6.0 X 1 0 23 2.48 X 106 Ultraviolet 1.5 X 1 0 1 Visible 3.9 X 1 0 2 2.0 X 1 0 17 8.27 X 101 0 7.7 X 1017 3.18 X 10 Infrared 7.8 X 1 0 2 3.8 X 1017 1.59 X 10° Microwave 1.0 X 1 0 6 3.0 X 106 1.24 X 10"3 Radio Frequency 1.0 X 1 0 8 3.0 X 102 1.24 X 10-7 H Gamma Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. vantages, chemical fixation (i.e. formaldehyde, glutar aldehyde, osmium tetroxide) is the most extensively used method of fixation. There are three common factors which affect all methods of fixation: 1) rate of penetration, 2) pH of solution and 3) osmolarity of the fixative. These common factors must be discussed before one attempts to discuss the individual che m ical reactions with cell components. The rate of penetration by the fixative upon five conditions: of fixative, 1) size of block, 3) fixative concentration, ation, and 5) temperature of fixative. is dependent 2) diffusability 4) duration of fix The size of the specimen has been reported to be the most common source of failure in achieving good results (1). proper fixation for electron microscopy, In order to achieve the tissue block must not be larger than 1 mm in thickness and ideally 0.5 mm. Moreover, it is of equal importance that the tissue blocks be uniform in size, otherwise irregular fixation will occur which results in a decrease in the quality and greater variability of preservation. Brunings and Preister (4) reported the difference between large and small blocks and demonstrated artifacts such as extrusions of midgut epithelium of insects in blocks which were thicker than 1 mm. The second condition affecting the rate of fixation is the rate of fixative diffusion. This area has been studied extensively and the results have shown a complex of variables all of which can be altered thereby increasing the diffusion. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Of these the best understood is the molecular weight of the fixative. However, recent studies by Dempster, et a l . (5) have shown that the rate of diffusion is dependent upon a coefficient of diffusability K where: K = __________time________ , (depth of pe ne t r a t i o n ) “ K, the diffusability constant, is a property of each chemical independent of molecular weight. Based on this equation and numerous experiments it has been suggested that the rate of diffusability for common fixatives in decreasing order is: (1) Formaldehyde— Glutaraldehyde— Osmium Tetroxide Fixation concentration is the third factor which pro foundly influences the rate of diffusion. Hyatt (1) re ports that low concentrations of fixative require longer duration of fixation than high concentrations. Longer fixation time may result in numerous detrimental side effects including tissue autolysis. It is important to realize that the concentration of fixative can easily be tailored for the needs and goals of thestudy. The final two factors affecting the rate of fixation is the reciprocal effect of temperature and duration of fixation. These factors are noteworthy for their flex ibility and relevancy to this study. By increasing the temperature one is able to increase the rate of penetra tion. This decreases the duration of fixation by increas ing the availability of fixative and at the same time in creasing the reactivity of the cellular chemicals with the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fixative. However, one must contend with an increased rate of autolytic destruction which is directly associ ated with increased temperatures. It has been shown that increasing the temperature with rapid fixatives (i.e. form aldehyde) results in better fixation because extremely rapid fixation is capable of compensating for the increase in autolytic activity a slower fixative (6). On the other hand, heating of (i.e. osmium tetroxide) results in ex cessive autolytic destruction (6). Little is known about the effects of temperature on the kinetics of the chemical reactions of fixation. There fore, unless an optimal temperature for rapid molecular fixation is determined the period between exposure to the fixative and completion of the fixation process will permit continued deterioration of tissue integrity (7). The regulation of the ti s s u e ’s pH must be taken into consideration for proper fixation. The need for an ex ogenous buffer is necessitated by the inability of the t i s s u e ’s buffering system to adequately handle the action of the fixative (1). In fact, it has been shown by Claude (8), that the application of unbuffered osmium tetroxide for 48 hours resulted in a pH drop from 6.7 to 4.4. The effects of this unchecked pH are serious, especially in light of the 7.0-7.4 physiologic range for animal tissues (9). Wrigglesworth and Packer (10) showed that the pro- tiens, which are the major cellular constituents, are de natured by low pH, resulting in abnormal protein struc Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 tures and subsequent deterioration of the structural pro teins. Moreover, proteolytic and lipolytic enzymes are activated at lower pH and are thereby increased in auto lysis of tissue. In order to compensate for this drop in pH exogenous buffers are required. physiologic range. These buffers keep the pH within It is the narrowness of this range that has limited the number of buffers in use. The buffer must be able to accommodate the hydroxyl and hydrogen ions generated by the reaction between fixative and macromole cules through acid-base mechanisms. Finally, osmolarity must be regulated for satisfac tory fixation. To n i c i t y ’s effect on easily understood, tissue integrity Is although regulation of tonicity is not nearly as easy to achieve. If tissues are subjected to a hypotonic solution, laws of diffusion dictate that water will move through the cell's semi-permeable membrane from a greater concentration of solute to a lesser concentra tion. This movement results in the abnormal and destruc tive swelling of tissues. The opposite movement is ex perienced in a hypertonic solution and the result is a destructively shrunken cell which cannot be studied. Therefore, it is crucial for the buffer and the fixative medium to be isotonic in relation to the tissues. In their comprehensive study, Bone and Ryan (11) report that both the buffer and the fixative affect the osmolarity of the solution. Two possible methods can be used to regu- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 late osmolarity of the buffer. These are the addition of electrolytes and non-electrolytes to the fixative solution. Common non-electrolytes used are polyvinylpyrrolidine (PVP) and sucrose. However, these non-electrolytes are large enough to decrease the penetration rate of the fixa tive and increase the extraction of cellular components (12, 13). Furthermore, some non-electrolytes such as PVP interfere with the enzymatic activity of the cellular en zymes (1). It is these deleterious effects that have limited the use of non-electrolytes in buffers. Electrolytes, such as sodium chloride and calcium dichloride and others, have been more successful in ade quately preventing osmolarity-evoked destruction. Unlike non-electrolytes, most electrolytes do not have the detri mental effects upon penetration rate and cellular extraction synonymous with the larger non-electrolyte buffers. With the three factors of rate of penetration, pH of solution, osmolarity of the fixative, in mind, it is now pos sible to turn the discussion towards the mechanism of fixation. In the 1950's an assortment of fixatives were used which proved to be inadequate. Sabitini (14), in 1964, demonstrated conclusively that glutaraldehyde was the most effective fixa tive for preserving cellular ultrastructure, while being flex ible enough to use for a broad spectrum of tissues. More over, glutaraldehyde was shown to have a lower detrimental effect on tissue proteins, both structural and enzymatic, and therefore could be used for some enzymatic studies Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (14). 12 The specific intermolecular action of glutaraldehyde is a complex and not completely understood. Glutaraldehyde is a straight chain five carbon dialdehyde. It has a molecular weight of 100.12, a low viscosity and reacts to an increased pH by polymerization (15). The success of glutaraldehyde is directly related to its ability to irreversibly crosslink proteins, thereby stabilising cellular structure and deactivating its enzymes. The reaction was first thought to be due to di ald ehydes ' ability to react with the free amino groups to form Schiff bases: 1) (16) protein - NH^+ + CHO - (CH2 )3 " CH0---------protein 2) -N = CH - (CHg) - protein - M = CH - (CH^)^ - CHO CHO + +NH^_ protein protein - N * CH - (CHp )3 - CH = N protein However, this proposed mechanism of crosslinking was not supported by experimental evidence that the glutaralde hyde fixed tissue can withstand acid treatment, which a Schiff-base formation cannot (18) and Hopwood (17). Moreover, both Richards (19) have shown that the kinetics of this reaction would not account for the fact that Schiff base formation is reversible while the protein-glutaraldehyde reaction is not (17). The recent theory of the reaction is complex. What is known is that the glutaraldehyde reacts primarily with the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13 epsilon amino groups of lysine (18). Jansen, et al.(20), using chymotrypsin and zymogen, detailed the loss of enzyme activity due to the reaction of epsilon amino groups with glutaraldehyde. It is now thought that alpha, beta unsatur ated aldehydes react with lysine to give a Michael type adduct. A Michael addition is a conjugate nucleophilic ad dition of enolate ions to alpha, beta- unsaturated carbonyl compounds. The characteristics of this reaction are consis tent with the known glutaraldehyde reaction In that they are irreversible, stable to acid and have the appropriate pK (17). Also, the resulting crosslink of amino groups with a five carbon chain between two nitrogen atoms is consistent with the known data detailing electron bridges observed at low resolution studies on crosslinked lysozyme (1 8 ). It is important to realize that these are theories of intermolecular reactions between the dialdehyde, glutaral dehyde, and proteins. They are not fact and are, therefore, still open to debate. No matter what the specific reaction is the result is still the same: the proteins are cross- linked and polymerised and as a result, the cell's structure is stabilized and its enzymes deactivated. The reaction of glutaraldehyde with nucleic acids is not as complex nor as Important to glutaraldehydes action as a fixative. Furthermore, the reaction has not been studied as extensively as it has with proteins. Hopwood (21), demon strated that the reaction of glutaraldehyde with RNA was o o slight until 45 C and absent with DNA until 64 C. The reason Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 14 for this non-reactivity Is related to the tertiary structure of nucleic acids which are held together by hydrogen bonds. It is only when the temperatures become high that these hydro gen bonds can be broken and the purine and pyrimidine bases can be made available for reaction with the aldehyde as the nucleic acids unwind. It is known that routine glutaraldehyde fixation pro cedures utilize low temperatures and therefore, it has been theorized that there has to be another mechanism responsible for the reaction of glutaraldehyde with nucleic acids. It has been suggested that the proteins associated with the nucleic acids are polymerized through their reaction with glutaraldehyde and form a complex meshwork which ensnares the nucleic acids thereby "fixing” the nucleic acids without specifically reacting with the nucleic acids. Lipids, the third major intracellular component, also react with glutaraldehyde In the fixation process, however, like the nucleic acids the reaction is not nearly as complete as with theproteins. There has been little examination of the reaction, but Roozemond (22) with rat thalamus, showed that buffered glutaraldehyde is thought to be due to the presence of free amino acids in the phospholipids which are then bound by the aldehyde in the same manner as are the pro teins. It Is important to realize that glutaraldehyde fixa tion of lipids is so incomplete that post-fixation with osmium tetroxide is required, otherwise it is impossible to adequately study structures made of lipids (23). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 Presently the two most common chemical fixatives are variations of pure glutaraldehyde: Karnovsky's solution and Miloneg's buffered glutaraldehyde. Karnovsky (24), in 1965, first reported a medium which used a mixture of for maldehyde and glutaraldehyde in an attempt z o broaden their individual spectra of positive effects while minimizing their limitations (24). The reason for the superiority of the mixture over using each fixative alone can be attributed to the symbiotic effect of the two fixatives. a monoaldehyde, Formaldehyde, is a small compound able to penetrate a tissue faster, thereby stabilizing the cellular constituents before autolysis can distort cellular architecture. Also, the lack of permanence and limited range associated with formaldehyde is alleviated by the presence of glutaraldehyde. This diaidehyde penetrates more slowly due to its larger size, however, its previously discussed ability to permanent ly crosslink proteins is the source of stability and per manence (1) . By determining the proper concentrations of each fixative the microscopist can take advantage of their respective posi tive characteristics while minimizing their drawbacks. The original mixture suggested by Karnovsky was a 52 glutaraldehyde buffer (pH 7.2) containing 0.05% calcium dichloride formulation, reports Hayat (24). This (1), is extremely hypertonic with an osmolarity of 2010 osmols, which results in severe cellular distortion. This has been corrected and now the most widely used solution is 1-3% glutaraldehyde and 0.5%-2% formaldehyde with a lower osmolarity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 The Miloneg's solution, on the other hand, utilizes a pure 2 % glutaraldehyde with a phosphate buffer (25). The use of this system is based primarily on the phosphate's abil ity to keep the tissue within the proper pH range without severely affecting the osmolarity. By keeping the solution within these restricted parameters one is able to optimize the effects of glutaraldehyde fixation. The result is that although chemical fixation is the most frequently used method of fixation for electron microscopy, it still has its drawbacks, primarily due to its dependence on chemicals. The detrimental effects of varying tissue pH and osmolarity have previously been discussed at length. But along with these, there are other causes of tissue destruction in chemically fixed tissue. One variable introduced by routine chemical fixation is the effect of the time delay upon structural preservation. Standard glutaraldehyde fixation takes two hours; during this time delay allows for a significant amount of autclytic activi ty especially if conditions such as pH are not optimal. Pre sently, there is no way to determine exactly the effects of pro longed fixation, but it is reasonable to assume they are signifi cant . In the discussion of fixation mechanisms it becomes obvious that although glutaraldehyde is the most effective chemical fix ative, it is also limited as to the cellular constituents it can fix effectively. fixing proteins, While extremely effective in crosslinking and glutaraldehyde is net nearly as effective in immobilizing carbohydrates and lipids. Lipid fixation is so Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 17 poor that post-fixation with osmium tetroxide is often neces sary. Therefore, the tissue is placed in a second medium which, like the first, must be adjusted with respect to osmolarity, pH, temperature and concentration. This multiplies the number of variables that will influence the quality of tissue preser vation . A final disadvantage of chemical fixation is the effect the chemicals have on various special techniques which are imperative for complete utilization ^f the electron microscope. Of the special techniques limited by glutaraldehyde fixed tissue are its deleterious effects on antigen, hormone and enzyme reactivity in such fields as immunchistochemistry and hormone receptors. It is thought that the glutaraldehyde changes the secondary and tertiary conformations of the pro teins (antigens, recepters and enzymes), thereby destroying their ability to react with the reagents of the specific stain ing techniques. In conclusion, it becomes obvious that chemical fixation, despite its position as the preeminent method of fixation, does not meet the requirements of the demanding, rapidly changing field of electron microscopy. Therefore, the goal of this study grows out of the need tc find a method of fixation which removes the negative effects introduced by chemicals. Physical Fixation Physical fixation methods, on the other hand, have so far proven to be seriously limited for electron microscopic use. The two primary methods of physical fixation are Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 heating and freezing. Freeze techniques, using special media and temperatures below -20°C are presently used In surgical pathology. This method introduces low temperatures and slow freezing resulting in the coagulation of tissue molecules, stabilizing the tissue and causing it to be hard enough for sectioning. However, along with the coagulation of molecules is the production of ice crystals which cause severe damage at the light microscope level. This mthod offers adequate fixation for light microscope examination, however it is com mon knowledge that these standard freeze techniques are inad equate for electron microscopy. Other methods of freeze fixation are presently in use in research electron microscopy. Tokuyasu (2b) has been successful using a combination of chemical and freeze fixa tion. The procedure consists of a I hour fixation in a less than 2 % concentration of glutaraldehyde fixative at 4JC. This step stabilizes the tissues, but does not cause anti genic damage or complete fixation. Thin sections are then o cut at 300-900 Angstroms at a temperature of -7 0 to -90 C. using a special cry oattachment. This step completes the fixation process by freeze methods thereby preserving the antigenic sites while causing complete fixation. A more recent development is a method known as freeze substitution. Here the tissues are fixed in liquid helium o at 4 Kelvin and then transferred through liquid nitrogen into liquid acetone at temperatures around 0°C. The acetone Infiltrates and stabilizes the tissues by replacing the water present in the tissues (27). This technique has Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 19 seen extensive use in the immunocytochemical research laboratories. Despite the tremendous utilization and results gathered by these techniques in research laboratories, they are not used in diagnostic laboratories. These methods are limited mainly by the tremendous cost for routine use. Special equipment such as cryoattachments, liquid helium o and nitrogen, and freezers capable of 4 K all are too ex pensive for the clinical laboratory. Moreover, the time consumption and difficulty of the technique make its routine usage unfeasible. The use of heat as a method of fixation, not met with great success. likewise has Theoretically it is possible because high heat coagulates and stabilizes proteins. But, the time lapse between routine heating and complete protein stabilization is long and allows enough time for severe destruction of the tissue by proteolytic enzymes and fat oxidation. Microwave Fixation As discussed previously, microwave radiation is cap able of vibrating molecules at speeds high enough to cause a rapid increase in kinetic energy. This property of al most instantaneously increasing temperature to a high level is the basis of* microwave radiation as a method of fixation. Proteins, the major cellular constituent, natured and coagulated, thereby fixed when the of the system reaches 50-60°C. can be de temperature However, traditional heating Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 methods take several minutes before these temperatures are reached. As Bell (28) vividly details, the enzymatic activity is increased significantly to the point of severely damaging the tissues when the temperature of the system is increased slowly. If one v/ere to limit the time lag during the rise in temperature to only a few seconds, (i.e. microwave radiation) it might then be possible to de nature and coagulate the proteins without subjecting the tissues to the prolonged autolytic activity of the un harnessed enzymes. Like proteins, nucleic acids react to high tempera tures in a similar manner. Temperatures above 50°C re sult in denaturation of the nucleic acids thereby fixing them. Unlike proteins, however, if the increase and sub sequent decrease in temperature are slow and gradual enough it is possible to renature the nucleic acids (28). In this case, the additional important characteristic of microwave radiation is it does not have the energy content capable of causing mutagenic ionization which could destroy the structure of the nucleic acids (28). Therefore, it is reasonable to assume that microwave radiation could serve as a possible method for fixation of nucleic acids. Lipids, likewise react to high temperatures in some way. However, the exact effect of the rapid increase in temperature on these lipids has not been determined. The use of microwave radiation for tissue fixation has already been shown to be effective at the light microscope level (29). Login (30), reports that tissue heated to 60°C Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 In a saline medium in the microwave oven were superior structurally to all other microwave and traditionally fixed tissue. The result is that by using microwave fix ation the time for fixation was reduced, cheaper solutions were used (saline vs. formaldehyde), toxic fumes were eliminated all without damaging the cellular architecture more than what is found in standard procedures. Other reports have appeared detailing the advantages of microwave fixation of fetal tissues tured cells (31), tissue cul (3 2 ), and even the elution of antibodies from sensitized red blood cells (33). However, only one study has been reported concerning the use of microwave radiation for electron microscope study (3*0. In their study, Chew, et al (3*0 examined the preservation of ultrastructure in microwave fixed rat tissue (kidney and liver). The result of their work is the proposal that the preservation of ultrastructure was equal to the standard fixation in 2.5^ glutaraldehyde for two hours while also causing no diffi culty in sectioning or staining. As stated previously, the feasibility of microwave radiation depends upon the speed at which it stabilizes the cellular constituents. If it does not achieve this, microwave radiation will be no better than the discarded methods of conventional heating. Another cause for con cern is the random, rapid heating of both structural and enzymatic proteins. Whereas denaturation of enzymatic proteins is beneficial to ultrastructural preservation, the denaturation of structural proteins without the benefit Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22 of crosslinking agents might cause severe structural damage. Nucleic acids also might suffer from denaturation without crosslinking resulting in destruction instead of preserva tion. Yet, despite these possible drawbacks previous re search has only been positive. It is now important to de termine the feasibility of fixing human tissues with micro wave radiation. Rationale The aim of this investigation is to evaluate the use fulness and reliability of microwave fixation of human tissues for electron microscope study. Theoretically and practically, microwave radiation is a successful method of fixation for light microscopy (30-32). However, very scanty evidence is available as to the effect of high in tensity microwave radiation on tissues examined at the ultrastructural level. This study will compare convention al formalin and modified Miloneg's glutaraldehyde fixatives to a series of trials where the fixation will be done by microwave radiation using chemical fixatives or saline. Furthermore, the temperature to which the tissue will be heated will also be systematically varied to determine the optimal temperature of fixation. Ultimately, the goal will be to determine the fastest and most efficient method of fixation which maintains the quality of preservation needed for diagnostic use. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MATERIALS AND METHODS Login (30) showed that the optimal temperature for high o quality fixation for electron microscopy ranged from 60-65 C. Using these temperatures as a base, a two stage project was proposed that first used 3 fixative mediums (saline, glutar aldehyde, formalin) at six separate temperatures (50, 55, 60, o 65, 70, 80 C) under optimal conditions in hope of isolating the proper temperature and mediums. The temperature-medium combinations that gave adequate quality under optimal con ditions would then be tested on surgical specimens. is defines as: Optimal amount of time between excision of tissue and complete fixation less than 15 minutes. The kidneys of a 125 gram albino rat were excised and placed immediately in 0.09? saline at room temperature. Tissue blocks at 1 mm^ were cut and randomly placed in either saline, formalin or glutaraldehyde and heated to temperatures _ o ranging from 50-80 C. Control tissues placed in 2% glutar aldehyde, and in formalin were fixed overnight. The samples were individually heated to the proscribed temperatures using a Kennore model 565 Microwave oven oper ating at 2450 MHz and 600 watts. The temperature of the medium was monitored by a heat sensor which was previously shown to be accurate before the experiment. The microwave fixed tissues and controls were processed by routine osmium tetroxide post-fixation, dehydration with 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ethanol and propylene oxide and epon embedding (Appendix 1). Thick sections were cut at 1 micron were stained with toluidine blue and basic fuschin (Appendix 2). ated by light microscopy. These were evalu Those tissues which exhibited ade quate fixation were thin sectioned at 8rnm. Sections were stained with lead citrate and uranyl acetate (Appendix 3) and were viewed with a Philips 300 electron microscope. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULT? The thick sections, seen in Plate 1, show excellent preservation of the renal anatomy using normal glutaralde hyde and formalin fixation methods. The glomeruli are well preserved with no evidence of cellular distortion or dis organization. The tubules, likewise, are preserved with good cellular integrity and continuity. The tissues fixed with the microwave oven using all three media (saline, formalin and glutaraldehyde) have a great variation in preservation fixed at 50°, 55 °t (see Plates 2-4). Those 60 ° 65° all had inadequate preservation for further study. The glomeruli were obliterated with little recognizable structure. The tubules are disorganized with severe nuclear and cytoplasmic clumping within the cells. The lumina are filled with displaced epithelial cells Overall, these temperatures did not produce adequate pre servation regardless of the medium used. When the temperature of fixation was raised to 70° and 80°C the preservation of structure in all media was im proved (see Plates 5-7). The glomeruli were well organized and the tubules were intact with prominent nuclear staining. These sections were equal to if not better than the control sections. From these results tissues fixed in all media at o n o 70 and 80 C were chosen for further processing and study by electron microscopy. 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 26 Plate 1. Rat kidnev controls fixed in two standard fixatives and examined by light microscopy, a) formalin, 1800x, b) glutaraldehyde, 1800x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 Plate 2. Rat kidney fixed in saline with microwave radiation at vagious temperatures and examined by light microscopy, a) 50 C, 1800x, b) 55°C, 1800x, c) 60°C, 1800x, d) 65 C, 1800x. I Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Plate 3. Rat kidney fixed in formalin with microwave radiation at various temperatures and examined by light microscopy, a) 50 C, 1800x, b) 55 C, 1800x, c) 60°C, 1800x, d) 65°C, 1800x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 31 Plate 4. Rat kidney fixed in glutaraldehyde with microwave va radiation at varigus temperatures and examined by light microscopy, a) 50 C, 1800x, b) 55°C, 1800x, c) 60°C, 1800x, microgcopy, d) 65 C , 1800x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Plate 5. Rat kidney fixed in saline at two temperatures with microwave radiation and examined by light microscopy, a) 70 C , 1800x, b) 80°C, ISOOx. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 34 Plate 6. Rat kidney fixed in formalin with microwave radi ation at two temperatures and examined bv light microscopy, a) 70 C , 1800x, b) 80°C, 1800x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 Plate 7. Rat kidney tixed in glutaraldehyde with microwave radiation at two temperatures and examined by light m icros copy, a) 70 C , 1800x, b) 80°C, 1800x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 The selected tissues were thin sectioned at The controls shown in plates 8 and 9 demonstrate good preservation of kidney ultrastructure. Both glomeruli and tubules show excellent organization and continuity with no damage to structural integrity. fy all cell types It is possible to identi (endothelial, epithelial and mesangial) and the nucleus and cytoplasm of each have excellent clari ty. ing. There is no appreciable disruption or chromatin clump The pedicles are seen as extensions of continuous epithelial cells and the basement membranes are clearly discernable. Tubules were comparably preserved (not shown). Plates 10 and 11 are representative of microwave fixed tissue at 7 0° and 80°C in saline. Glomeruli and tubules are distorted with obvious destruction of their anatomical continuity and integrity. The glomeruli are not preserved and it is difficult to understand the structure and identify its characteristic cells. The podocytes have lost their cellular attachment and are partially degraded. The base ment membrane has lost its integrity and continuity. The tubular epithelial cells contain altered mitochondria, dis torted borders with loss of intercellular adhesion and clumping of both the nuclear and cytoplasmic matrix. Overall the preservation is totally inadequate. The microwave-formalin fixed tissues at 70° and 80^C likewise show an unacceptable amount of ultrastructural damage with nearly the same negative features as seen In the tissues fixed in saline. As can be seen in Plates 12 and 13 the preservation though slightly better is still inadequate. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 Finally, Plates 14 and 15 illustrate that tissues microwave fixed in glutaraldehyde at 70^ and 80°C are also inadequately preserved despite its better preservation of glomerular podocytes. Once again, the overall continuity and integrity of both the glomeruli and tubules along with the intracellular damage, particularly the nuclear damage, is too great to use the tissues for diagnostic study. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. r 38 Plate 8 . Rat kidney control fixed in formalin and examined by electron m icroscopy,10,260x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 Plate 9. Rat kidney control fixed in glutaraldehyde and ex amined by electron microscopy, 10,260x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 PlateQ 10. Rat kidney fixed in saline with microwave radiation at 70 C and examined by electron microscopy, 4500x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 Plate ll.QRat kidney fixed in saline with microwave radia tion at80 C and examined by electron microscopy, 4500x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■I 42 Plate 12. Rat kidney fixed in formalin with microwave radiation at 70 C and examined by electron microscopy, 4500x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 43 Plate 13. Rat kidney fixed in formalin with microwave radiation at 80°C and examined by electron microscopy, 4500x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. f 44 Plate 14. Rat Jeidney fixed in glutaraldehyde with microwave radiation at 70 C and examined by electron microscopy, 4500x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 Plate 15. Rat jeidney fixed in glutaraldehyde with microwave radiation at 80 C and examined by electron microscopy,4500x. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION Data Analysis This study was carried out to investigate the feasibil ity of using microwave fixation for electron microscopy in the surgical pathology laboratory. duce prior claims of success Its design was chosen to repro (34) and to test the hypothetical advantages of non-chemical fixatives. The two stage program was used in order to eliminate the number of trials by elimin ating those trials that were inadequate under ideal conditions. The different temperatures were used in hopes of isolating the optimal temperature for fixation. The different media were used to determine if saline could replace formalin and/or glutaraldehyde in the hope that this would eliminate the need for expensive, damaging, toxic chemicals. The results illustrate that none of the temperaturemedium combinations resulted in an acceptable preservation of ultrastructure. Since these results were obtained with rat tissues obtained promptly, there was no justification for studying microwave fixation of surgical pathology spec imens which have varying amounts of delay prior to fixation. Exactly why microwave fixation resulted in such poor preservation in light of certain. Chew et a l . (34) results is un The light microscope study was consistent with other investigations (30- 3 2 ) and originally led to expecta tions of good results at the electron microscope level. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 However, it is obvious that the demands of electron micros copy are much more rigorous than light microscopy. As stated previously, the quality of fixation is dir ectly related to the fixative's ability to stabilize cellu lar proteins, particularly enzymatic proreins. Microwave heating does this by increasing the system's vibrational energy to the level where the heat produced unravels the complex protein structure. This process, ation, allows the proteins to crosslink, known as denaturleading to fixation. There are two ways in which the microwave heating mechanism might result hibited. in the structural alterations ex First, the time elapsed between introduction of the heat and when denaturation and fixation are complete might be long enough to allow oxidation of lipids and in creased activity of proteolytic enzymes to destroy the tissue. Since the heating is almost instantaneous and temperatures of 70° and 80°C are reached in the matter of seconds, it is unlikely that the increase in proteolytic ac tivity associated with the rise in temperature will have a prominent effect in this short period of time. Alternatively, the energy increase may be too random and severe resulting in the non-selective denaturation of both globular (enzymes) and fibrous (structural) proteins. On the one hand, denaturation of enzymatic proteins needs to be complete. The rapid rise in temperatures to levels of 70° and 80°C are excellent for deactivating the enzymatic proteins thereby inactivating their autolytic activity. If enzymes were the only proteins denatured by high tempera- Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 cures the results would have been excellent quality of fixation. However, enzymatic proteins, the heat increase affects not only the but also the structural proteins. Fibrous proteins, on the other hand, are proteins noted to have an axial secondary configuration which makes them ideal for their structural responsibilities. Located both intra and intercellularly, examples include collagen, alpha keratin and elastin. It is probable that the rapid heating of these proteins results in the denaturation of not only their quananery and tertiary structures, their secondary structures. but also Whereas the denaturation of enzymatic proteins is a positive factor in fixation, the denaturation of the structural proteins secondary struc tures results in an unravelling and disassociation of their basic chemical structure without the crosslinking action of chemical fixatives. As a result, the elastin and collagen fibers integral for intercellular adhesion and basement membrane integrity may be totally disrupted and destroyed. Moreover, the organelle destruction exhibited in the trials may be due to the disruption of the membrane proteins. Therefore, it can be theorized that the majority of ultra- structural damage seen in the 70° and 80°C trials can be attributed to the denaturation of the secondary structures of the fibrous proteins. However, it is unlikely that the tremendous destruc tion of tissue seen at 70° and 80°C can be attributed solely to the denaturation of fibrous proteins. The other cellular Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4S constituents, fats and nucleic acids, also showed evidence of heat destruction, though not as pronounced as the pro teins denaturation. The nuclear chromatin clumping and destruction results from the destruction of the nucleic acid structure by high Q temperatures. Temperatures above 35 C are enough to rup ture over 50% of the hydrogen bonds, which hold the double helical structure intact. In doing this, there is complete denaturation and destruction of nucleic acids. there is evidence of partial cleavage Below 35°C (less than 50 % ) of the hydrogen bonds and also denaturation of the hydrophic forces between stacked pyrimidine and purine bases. though the rupture of the structure is incomplete, Al there is enough energy to cause the clumping and destruction seen in the 70° and 80°C trials. At lower temperatures (50°, 55°, 60°, 65°C) the tis sue destruction can be attributed to non-deactivation of the enzymatic proteins. As previously discussed, temperatures over 65°C until reached, the levels of enzymatic activity actually increases proportionally to the tempera ture increase. Therefore, fix the proteins, the lower temperatures do not but actually increase their activity. Their unleashed activity can easily cause the tremendous destruction seen at light microscopy, through increased autolytic and biodegradative processes. The fact that 70°C fixed tissue, independent of medi um, resulted in the best fixation accentuates the diphasic pattern of destruction exhibited by these tissues. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. At this 50 temperature the enzymatic proteins had been best stabil ized with the minimal amount of structural protein dena turation, fat oxidation and nucleic acid destruction which are artifacts of higher temperatures. However, it is uno o likely that a temperature between 70 and 80 C would re sult in better preservation because the difference between the preservation in these two temperatures is very subtle. The result of using microwave radiation is poor pre servation for electron microscopy no matter what the tem perature-medium combination used. Though the damage may be minimized to the extent that the tissues can be studied with the light microscope, the demands of electron micros copy are far too rigorous to accept the poor preservation and severe distortion induced by microwave radiation. Therefore, unless there is devised a way to selectively heat the proteins, lipids, and nucleic acids so as to mini mize the side effects of microwave radiation, it will not be a successful tool for fixation of tissues for electron microscopy. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 Overall Conclusions Prom this study it can be stated that microwave radi ation cannot be used as a fixative for electron microscopy. The effects of the high, rapid, random heating, character istic of microwave radiation, destroyed the delicate ultra structure leaving thetissue inadequate for diagnostic inter pretation. This study can be used to remind those who wish to investigate other methods of heat fixation that the ef fects of high heat are still too severe for the delicate structures. Unless there is developed a technique to limit the negative effects inflicted by the heat while retaining its fixative capabilities, heat will never be useful as a fixative for electron microscopy. However, this does not exclude microwave radiation from the diagnostic laboratory. This study lends credence to prior results of good fixation for light microscopy. Al so, with a better understanding of how microwave radiation is used in light and electron microscopy, to use microwaves for other steps in the preparation of tis sues for electron microscopy; ising. it may be possible staining being the most prem Therefore, although microwave radiation is an inad equate means of fixation for electron microscopy, its useful ness might be realized in other areas of routine preparation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES Hayat M.A.: Fixation for Electron M i c r o s c o p y , Academic Press, Mew York, N .Y . , 1 9 8 1 . 2. Collins R.E.: Foundations for Microwave Engineering, McGraw Co., New York, N.Y., 1966. 3. Lin J.: Microwave Auditory Effects and Applic a t i o n s , Charles C~. Thomas C o . , Springfield II., 1978. 4. Metalsky I.: Non-Ionizing Radiation. In Industrial Hygiene Foundation , L.V. Cralley and G. D. Clayton, Eds., McGraw Co., Mew York, N.Y., 1968, pp. 140-179. 5. Brunings E.A. and dePriester W.: Effect of Mode Fixa tion on the Formation of Extrusions In the Midgut Epi thelium of Calliphora. Autobiology 4_, 487 (1971 ). 6. Depmster W.T.: Rates of Penetration of Fixing Fluids. AM. J. Anat. 107, 59 (I960). 7. Hayat M.A.: Principles and Techniques of Electron Microscopy: Biological A D D lications, University Press, Balti more^ ~MD . , 1981“ 8. Paris R.B., Kelley J., Drury S., Sauer K . : The Hill Re action of Chlcroplasts Isolated from Glutaraldehydefixed SDinach Leaves. Proc. Natl. Acad. Sci. 55, 1056 (1 9 6 6 ). — 9. Claude A.: Fixation of Nuclear Structure by Unbuffered Solutions of Osmium Tetroxide In Slightly Acid Distilled Water. Proc. Inter. Congr. Electr. Microsc. 5th ed. 2, 1-14 (1962). 10. Butler T.C., Waddell W.J., Poole D.T.: Intracellular pH based on the Distribution of Weak Electrodes. Fed. Proc. 26, 1327 (1967). 11 . Wrigglesworth J.M., Packer L.: pH Dependent Conforma tional Changes In Submitochondrial Particles. Arch. Biochem. B i c p h y s . 1 3 3 » 194 (196 8 ). 12. Bone Q., Ryan K.P.: Osmolarity of Osmium Tetroxide and Glutaraldehyde Fixatives. Histochem. J. 2, 1 (i9 6 0 ). 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13. Wood R.L., Luft J.H.: The Influence of the Buffer System on Fixation with Osmium Tetroxide. J. Cell Biol. 19, 83A (1974) . 14. Hagstrom L. Bahr G.F.: Penetration Rates of Osmium Tefcrox ide with Different; Fixation Vehicles. Histochemistry 2, (I960). 15. Sabitini D., Bebsch K., 3arnett R.: New Means of Fixation for Electron Mxcroscooy and Histochemistry. A n a t . Record 142, 274 (1 9 6 2 ). 16. Korn A.H., Feairheller S.H., Filaohione E.M.: Glutaraldehyde: Mature of the Reagent. J. Mol. Biol. £5, 525529 (1972). 17. Peters T., Richards F.: 46, 253 (1 9 7 2 ). 18. Richards F.M., Knowles J.R.: Glutaraldehyde as a Protein Crosslinking Reagent. J. Mol. Biol. _37 231-233 (1968). 19. Annual Review of Biochemistry. Hcpwood D.: Review of Glutaraldehyde as a Histochem. J. 4, 267-303 (1972). Fixative. 20. Jansen E. Tomimnastee Y., Olsen F . : Crosslinking of Alpha Chymotrypsin and Other Proteins by Reaction with Glutaraldehvde. Arch. Biochem. Bioohys. 144, 394-400 (1971). 21. Hopwood D.: The Reactions of Glutaraldehyde with Nucle ic Acids. Histochem. J. 7, 267-276 (1975). 22. Roozemond R.B.: The Effects of Fixation ’ w ith Formalde hyde and Glutaraldehyde on the Composition of Phospho lipids Extractable from Rat Thalamus. J. Histochem. Cytcchem. 5, 123-128 (1972). 23. Wood J.: The Effects of Glutaraldehyde and Osmium Tet.roxide on Froteins and Lioids of Myelin and Mitochondria. 3iochem. 5ioph.ys. Acta. 329, 118-127 (1973 ). 24. Karnovsky M . : A Formaldenyae-Glutaraldehyde Fixative of High Osmolarity for use In Electron Micro s c c o y . J. Cell Biel. 27, 137 (1965). 25. Miloneg G.: Advantages of a Phosphate Buffer for CsO^ Solutions in Fixation. J. Appl. Phys. 32_, 1637 (1961). 26. Tokuyasu K.T.: A Study of Positive Staining of ultrathin Frozen Sections. J. Ultrastr. Research 63, 287-307 (1978). 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Personal Communication. May 1 9 8 5 . 27. Papermaster D.: 28. Bell G. Davidson J., Ensile-Smith D.: Textbook of Physiology and B i o c h e m i s t r y . 8th e d . Williams and Wilkens Co., Baltimore, M D . , 1972. 29. Schneider D., Pelt B., Goldman H . : On the Use of Micro wave Radiation Energy for Brain Tissue Fixation. J. Neurochem. 38_, 749-752 (1 9 8 2 ). 30. Login G.: Microwave Fixation versus Formalin Fixation of Surgical and Autopsy Tissue. Am. J. Med. Technol. 44, 435-437 (1978). 31. Petere J., Scharden J.: Microwave Fixation of Fetal Specimens. Stain Technology 55_, 71-75 (1980). 33. _______ : Microwaves Elute Antibodies from Sensitized RBC's. Lab Management 6 (1984). 34. Chew E., Riches D., Lam T . , Hon Chan H . : A Fine Struc tural Study of Microwave Fixation of Tissues. Cell Biol. Intern. Reports 7, 135-139 (1 9 8 3 ). 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A P P E N D IX I ROUTINE FIXATION, POST FIXATION, DEHYDRATION AND EMBEDDING PROCESS EMPLOYED AT THE WEST HAVEN VA MEDICAL CENTER Fixation: 1. 2% Glutaraldehyde for 2 hrs. or overnight. 2. Rinse tissues in Q.05M sodium cacodylate for 5 minutes at 25°C. 3. Post Fix in 1% Osmium Tetroxide for 1 hour at 4°c. 4. Rinse in sodium cacodylate for 5 minutes at 25 C . Dehydrat i o n : 1 . 50$ ethanol for 5 minutes at room temperature. 2 . 7 0 $ ethanol for 5 minutes at room temperature. 3 . 9 5 % ethanol for 5 minutes at room temperature. 4. Two rinses, 15 minutes each at room temperature, 100$ ethanol. i 5. 100$ ethanolrpropylene oxide at room temperature. 6. Propylene Oxide for 15 minutes at room temperature 7. Propylene oxide:Epon 8. Epon until embedding. (1:1) for 15 minutes (1:1) 1 hour or longer. EMBEDDING: 1. Embed in epon for 48 hours at 5c°-60°C. 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A P P E N D IX II ROUTINE TOLUIDINE BLUE AND BASIC FUSCHIN STAINS USED AT THE WEST HAVE VA MEDICAL CENTER Toluidine Blue: 1. Place thick section on slide in a drop of* distilled water. 2. Dry on hot plate to permanently Tix section to slide 3. Place a Lev/ drops of toluidine blue stain so to cove the sections. Heat until steam is visible, but do not evaporate s tain. 5. Wash slide under running tap water, thenfinal rinse with distilled water. Basic Fuschin: 1. Make sure slide is completely clear of* toluidine blue by adequately rinsing with distilled water. 2. Drop a few drops of basic fuschin on slide so to cover the sections. 3. Heat for 15 seconds. 4. Wash slide under running tap water, rinse with distilled water. 5. Dry, then final coverslip and label. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX III URANYL ACETATE AND LEAD CITRATE STAINING PROCEDURES USED AT THE WEST HAVEN VA MEDICAL CENTER Uranyl Acetate: 1. Centrifuge the uranyl acetate In 25% ethanol m i n utes. 2. Place drop of uranyl acetate on a piece of parafil in a petri dish. 3. Put grids with sections down on top of uranyl ace tate drop. Stain for 15 minutes. 4. Wash by immersing the grids in 3 changes et h anol. 5. Dry on #50 of for 5 25% filter paper. Lead Citrate: 1. 2. 3. Centrifuge the lead citrate for 5 minutes before staining. Place drop in a Petri of lead citrate on a piece of parafilm dish containing a few pellets of NaOH. Place grids with sections down on top of lead cit rate drop. Stain for 4 minutes. Wash in a stream cf 0.02M NaOH and Immerse in 3 changes of distilled water. 5. Dry on #50 filter paper. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.