Preparation Techniques of Luminal and Hard Tissues for Scanning Electron Microscopy

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1 Preparation Techniques of Luminal and Hard Tissues for Scanning Electron Microscopy Y. Ersoy Canillioglu and G. Erkanli Senturk Bahcesehir University, School of Medicine, Department of Histology and Embryology, Goztepe, Istanbul, Turkey The use of Scanning Electron Microscopy (SEM) in life sciences has increased dramatically in the last decade. This has given rise to develop new technical methods. This chapter will be provided a needed survey of these advances by using three dimensional details of SEM. Moreover, it includes outstanding array on techniques for sample preparation for a number of specimen types ranging from luminal organs to hard tissues. Although the presenting information is specific to organs used in the laboratories of researchers, the providing information and methods will benefit to all who use SEM in their research. Also, this chapter will be explained friendly used techniques and some trick information to take quick results in their laboratories for luminal and hard tissues. Keywords: SEM; techniques; luminal organs; hard tissues 1. Scanning Electron Microscopy (SEM) SEM is the powerful technique in the examination of materials. It is widely used in metallurgy, geology, biology and medicine. The user can obtain high magnification images, with a good depth of field, and can also analyze individual crystals or other features. A high-resolution SEM image can show detail down to 25 Angstroms, or better. A normal scanning electron microscope operates at a high vacuum. The basic principle is that a beam of electrons is generated by a suitable source, typically a tungsten filament or a field emission gun. The electron beam is accelerated through a high voltage (e.g.: 20 kv) and pass through a system of apertures and electromagnetic lenses to produce a thin beam of electrons., then the beam scans the surface of the specimen by means of scan coils (like the spot in a cathoderay tube "old-style" television). Electrons are emitted from the specimen by the action of the scanning beam and collected by a suitably-positioned detector. The microscope operator is watching the image on a screen. Imagine a spot on the screen scanning across the screen from left to right. At the end of the screen, it drops down a line and scans across again, the process being repeated down to the bottom of the screen. The key to how the scanning electron microscope works (and this is the clever bit) is that the beam scanning the specimen surface is exactly synchronized with the spot in the screen that the operator is watching. The electron detector controls the brightness of the spot on the screen - as the detector "sees" more electrons from a particular feature, the screen brightness is increased. When there are fewer electrons, the spot on the screen gets darker. These days, the screen is generally a digital monitor, but the principle is the same. The magnification of the image is the ratio of the size of the screen to the size of the area scanned on the specimen. If the screen is 300 mm across and the scanned area on the specimen is 3 mm across, the magnification is x100. To go to a higher magnification, the operator scans a smaller area; if the scanned area is 0.3 mm across, the magnification is x 1000, and so on. There are different types of electron image. The two most common are the secondary electron image and the backscattered electron image. The secondary electron image is used mainly to image fracture surfaces and gives a high resolution image. The backscattered electron image is used typically to image a polished section; the brightness of the backscattered electron image is dependent on the atomic number of the specimen (or, for compounds, the average atomic number). For example, lead will appear brighter than iron and calcium oxide will appear brighter than calcium carbonate. The backscattered electron image is, in essence, an atomic number map of the specimen surface. All SEM images are in black-and-white, although they may subsequently have false colors applied to them for aesthetic reasons or to aid interpretation. A development of the normal high-vacuum scanning electron microscope is the ESEM, or Environmental SEM. The ESEM can operate with air in the specimen chamber - the pressure is lower than atmospheric pressure but higher that the high-vacuum of a normal SEM. This has the advantage that wet specimens can be examined without them dehydrating and is especially useful for biological specimens and other specimens containing water, such as freshlymixed cement paste [1]. 741

2 2. Luminal Tissues Alimentary tract is the part of digestive system and composed of luminal organs. The alimentary canal constitutes four concentric layers. These are mucosa, submucosa, muscularis externa and serosa or advetitia. These layers are similar but in some places display regional modifications. The lumen of the alimentary canal is lined by epithelium, it supports by connective tissue layer as lamina propia beneath the epithelium, Surrounding this connective tissue coat is the muscularis mucosae, consists of smooth muscle. The epithelium, lamina propia and muscularis mucosae are collectively called mucosa [2]. If you want to examine internal mucosal surface of alimentary canal, SEM is the better technique to give effective results (Figure 1, 2). 3. Hard Tissues When mention about hard tissues, first you consider skeletal system and tooth. Skeletal system is important in protecting and supporting vital organs in body. Teeth are also responsible for mastication which is mechanical digestion of food under high pressure. Therefore its structure needs to be organized suitably for this function. Hardness of the tooth and bone is due to their inorganic structure. A lot of metabolic diseases effects hard tissues in our body. One of the most important diseases is diabetes mellitus in this century. Previous research shows that diabetes mellitus adversely effects skeletal system [3]. 3.1 Structure of the Bone Tissues Bone is one of the hardest and strongest substances in the body. Its hardness and strength are due to the association of hydroxyapatite crystals with collagen. Bone tissue is composed of cells (osteocytes, osteoblast, osteoclast) lying in an extracellular matrix that has become calcified. The calcified matrix is composed of fibers and ground substance. The fibers contain primarily type I collagen. Bone is the main component of the human skeleton where it plays different roles in supporting and protecting tissues and organs including the brain, spinal cord, lungs and heart. In addition, bone is an important reservoir for several minerals of the body and it is involved in mineral homeostasis. It stores about 99% of the body s calcium. Osteoblasts synthesize, secrete and deposit the collagenous matrix, which then gradually mineralizes. Bone has a complex hierarchical structure, which is essential for its remarkable biomechanical performance. The mineral content of the bone matrix is playing a significant role in tuning its hardness, strength [4]. Bone also host hemopoiesis, the production of blood cells by the bone marrow where central cavity, the marrow cavity [2]. During development, new bone is formed as reticular bone, which is defined as an immature bone type where the collagen fibers are arranged in irregular random arrays and contain smaller amounts of mineral substance and abundant osteocytes bone cells. In reticular bone, osteocytes are present in greater numbers. Reticular bone is a temporary tissue that is eventually converted into lamellar bone, the latter being so called because of the typical osteocytes arrangement in its matrix as ordered lamellae. In the adult there are two types of bone tissue: compact bone, also called cortical bone and trabecular bone, also called cancellous bone or spongy bone [2]. Compact bone is the outer layer of bone, and it represents approximately 80% of the skeletal system mass. Compact bone blends into trabecular bone, which accounts for 20% of the bone mass. Compact bone consists of closely packed osteons or Haversian systems. The osteon consists of a central canal called the osteonic (Haversian) canal, which is surrounded by concentric rings of lamellar bone. Between the rings, the osteocytes are located in spaces called lacunae. Small channels (canaliculi) radiate from the lacunae to the Haversian canal. These small channels provide a passageway for the diffusion of molecules and biochemical signals through the hard tissue. In compact bone, the Haversian systems are packed tightly together to form what appears to be a solid mass. Each osteonic canal hosts a blood vessel that is parallel to the long axis of the bone. Trabecular bone is lighter and less dense than compact bone; it consists of plates and bars of bone adjacent to small, irregular cavities that contain red bone marrow. Unlike compact bone where Haversian canal ensures the blood supply, in trabecular bone, the canaliculi connect to the adjacent bone marrow cavities. The organic matrix of bone is composed primarily of protein. The most abundant protein in bone is the collagen type I, which provides the tissue with flexibility; about 10% of adult bone mass is collagen. There are also a number of noncollagenous proteins that play important roles in the regulation of both cell activity and tissue biomineralization. The main mineral component of bone is hydroxyapatite, which is an insoluble ceramic made of calcium and phosphorus; about 65% of adult bone mass is hydroxyapatite. Bone also contains small amounts of magnesium, sodium, and bicarbonate. The unique combination of collagen fibers and hydroxyapatite confer the appropriate mechanical properties to the bone tissue. In fact, the collagen fibers of bone have great tensile strength while the mineral phase provides great compression strength. These combined properties are obtained through the chemical interactions between collagen fibers and the crystals, and provide a bony structure that has both extreme tensile and squeeze strength [5]. 742

3 3.2 Structure of the Tooth Tooth is another hard tissue in our body. The mineralized structures of tooth are enamel, dentin and cementum. Dentin surrounds the pulp chamber and root canal and is covered on the crown by enamel and on the root by cementum. Enamel and cementum meet each other at the cervix of the tooth [2]. Enamel is the hardest substance in nature after diamonds. Tooth enamel is the most mineralized tissue of human body. Its composition is 96% inorganic material and 4% organic material and water. In dentin, the inorganic material represents 70%. This inorganic material is mainly composed by a calcium phosphate related to the hexagonal hydroxyapatite, whose chemical formula is Ca 10 (PO 4 ) 6 2(OH). Enamel and dentin also indicated the presence in small quantities of other elements such as Na, Cl and Mg [6]. Dentin is primary layer of tooth with mesodermal origin bonelike structure % of its dry weight is composed of calcium salt, therefore dentin is harder than bone tissue. Human teeth are exposed to a different point-to-point pressure during mastication. 4. Fixatives for SEM Technique 4.1 Glutaraldehyde Glutaraldehyde is one of the most frequently used fixatives. It reacts rapidly with proteins and because it is a dialdehyde, it stabilizes structures by cross-linking before there is any opportunity for extraction by the buffer. Hence more ground substance of the cytoplasm (glycogen) and of the extracellular matrices is preserved. Glutaraldehyde alone is not an adequate fixative. Because certain cell components especially lipids, are not fixed and may be extracted during dehydration, therefore secondary fixation is necessary using osmium tetroxide [1]. 4.2 Osmium tetroxide It reacts with lipids and also acts as a stain. Osmium will penetrate most tissues at a rate of about 1mm / hour. Extended times will cause extraction of many proteins, therefore keep time of immersion to a minimum. 4.3 Formaldehyde Commercially available solutions are unsuitable for electron microscopy because of their content of methanol. Paraformaldehyde powder is used to prepare methanol-free formaldehyde. Main advantage of formaldehyde is that it has a higher rate of penetration than either glutaraldehyde or osmium tetroxide so that large blocks of tissue are well fixed. Will penetrate most tissues at a rate of about 10 mm / hour, but will take a longer time to stabilize the tissue. 4.4 Formaldehyde and Glutaraldehyde mixtures Fixatives containing both paraformaldehyde and glutaraldehyde provide a much better quality of fixation, than either aldehyde alone. Formaldehyde penetrates tissues rapidly and mildly stabilizes proteins etc. which are then permanently fixed by the glutaraldehyde Karnosky s fixative Karnovsky s fixative followed by a second fixing in osmium tetroxide increases the bulk conductivity of the sample [7]. Karnovsky s fixative includes 2 % paraformaldehyde; 2.5 % glutaraldehyde in 0.1 M phosphate buffer ph 7.4. [8] F:1G Fixative [9]. 4F:1G fixative contains 4% formaldehyde and 1% glutaraldehyde in monobasic phosphate buffer in ph: Uranyl acetate - (EN-BLOC stain) Used as 2% solution in distilled water, this takes 24 hours to dissolve. The solution will keep for 1 week if stored in dark at 4 o C. 4.6 Acrolein This is a highly reactive, volatile and flammable liquid, always handle in fume cupboard. Once opened must be stored in freezer at -15 o C. Any spills should be neutralized with 10% sodium or potassium bisulphite solution. Always prepare fixative immediately before use. 743

4 5. Decalcification of Hard Tissue Human dentition and bone has been the subject of intense histological investigations for many years. Dental pulp evaluation is often a part of research protocols followed in assessment of pulpal biological response to new restorative materials. Examination of demineralized sections of other dental tissues and bone is necessary to study various pathological and developmental processes. There are a number of options available when the histologist is required to produce sections from bone or other calcified specimens. Much more commonly calcified specimens like bone and teeth are decalcified following fixation and processed using a standard method to investigation both on light level and on electron microscopy level. Decalcification is a process of removal of calcium salt from mineralized tissues like bone and teeth and other calcified tissues using chemical solutions like acids and chelating agents following fixation. The decalcification procedure is relatively straightforward but some points are valuable of emphasis. Mineralized structures of bone are dissolved out during the process of decalcification, if performed correctly. Demineralization or decalcification of tissues is a routine process carried out in most laboratories by the use of various acids or chelating agents. Where the stronger acids provide the fastest result with the poorest quality of sections, chelating agents provide the slowest result and the best sections [10]. Scientists have tried to introduce new decalcifying substances or tried to modify known decalcification agents [11] in order to meet the criteria of a good decalcifying agent which Ensures complete removal of calcium; Causes minimal damage to cells and tissues; Causes non-impairment to subsequent staining and; Decalcifies at reasonable speed [12, 13] 5.1 Decalcifying agents Decalcification is done with different chemicals. Strong mineral acids Weaker organic acids Chelating agents Strong mineral acids Strong mineral acids such as hydrochloric or nitric acid at concentrations up to 10% are the most rapid in action. In this process time plays a significant role. 5%-10% Nitric Acid gives the quickest results, but the large bubbles that form during decalcification can severely disrupt cells. Decalcify until no bubbles are visible when the material is examined under a dissection microscope, but check thicker specimens thoroughly as a still calcified core may remain after all bubbles cease. After decalcification is complete, the tissues should be neutralized by sodium sulfate for 12 hours before dehydration by an ethanol. 10% HCl, this solution can be used when you need to make sections quickly. Decalcification time varies depending on size of specimens and it may require 2 to 6 hours or more at room temperature for complete decalcification. Another decalcifier with Perenyi s fluid (1882), which contains 10% nitric acid, 0.5% chromic acid and absolute alcohol, is a traditional decalcifier that decalcifies more slowly than aqueous nitric acid [14] Weak acids Weak acids such as formic acid are popular and are widely used for decalcification. Formic acid can be used as a simple 10% aqueous solution or combined with formalin or with a buffer. It is more economical than buffer mixtures and equally satisfactory and effective. It permits satisfactory nuclear and marrow cell staining when decalcification is performed at room temperature. Although it is slower than the strong acid agents it is much gentler in action. Another decalcifier with week acids is Morse s Solution which contains 10% sodium citrate, 20% formic acid Chelating agents Chelating agents such as ethylenediaminetetracetic acid (EDTA), work by capturing the calcium ions from the surface of the apatite crystal, slowly reducing its size. This is also the preferred solution for decalcifying bone material for transmission electron microscopy. The process is very slow but very gentle. Specimens can be decalcified in this solution over several days up to several weeks in a refrigerator at 4 C, depending on degree of mineralization and size of specimen. The fresh solution is changed several times or once a week. 744

5 6. Luminal Tissue Preparation Techniques for SEM There is friendly used preparation technique as below; Wash tissue to remove any mucus, blood or any other contaminant. The washing medium must be physiological. Fix tissue with 2.5% glutaraldehyde in 0.1M phosphate buffer ph for hours. Wash tissue with 0.1M phosphate buffer 4 X 15 minutes Rinse tissue with distilled water 3 X 5 minutes Dehydrate tissue with ethanol. NOTE: Times depends on specimen size and density 50% ethanol 20 minutes 70% ethanol 20 minutes - Can store tissue in 70% ethanol 80% ethanol 20 minutes 90% ethanol 20 minutes 95% ethanol 20 minutes 100% ethanol 3 X 20 minutes Samples then ready for Critical Point Drying Samples investigate by SEM (Figure 3). Fig. 1 SEM photography of stomach. Fig. 2 SEM photography of colon. 745

6 Fig. 3 SEM photography of urothelium. 7. Hard Tissue Preparation Techniques for SEM SEM provides much higher magnifications and better depth of field than optical microscopes. Therefore SEM plays an important role in morphological examination of hard tissues. There is a friendly used preparation technique as below; Fix tissue with Karnovsky solution containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M phosphate buffer (ph ) for hours at 4 C. Rinse tissue with distilled water Immerse in a NaOH solution for 3-5 days at room temperature to remove the adherent tissues. Rinse with distilled water for 12 h at 4 C Post-fix tissue in 1% osmium tetroxide for 2 h at 4 C. Dehydrate tissue with ascending graded ethanol (70%, 80%, 90%, 95%, 100% ethanol) Dry with CO2 liquid in a critical point dryer or in air Mount on metal stubs Coat with gold/palladium using an ion sputter. References [1] Dykstra MJ. Biological Electron Microscopy. Theory, Techniques, Troubleshooting. Plenum Press. New York and London [2] Gartner LP, Hiatt JL. Color Textbook of Histology. 3rd ed. Philadelphia: Elsevier; [3] Gogas Yavuz D, Parmaksız I, Cetınel S, Ahıskalı R, Ersoy Y, Tugtepe H, Yazıcı D., Sırıkcı O., Akalın S.Aminoguanidine prevents trabecular bone structure in streptozocin-induced diabetic rats. American diabetes association 68th Scientific Sessions; 06/2008. [4] Roschger P, Paschalis EP, Fratzl P, Klaushofer K. Bone. 2008; 42: , Bone mineralization density distribution in health and disease). [5] Merolli A and Leali PT, Hard tıssue structure and functıonalıty. Biomimetic, Bioresponsive, and Bioactive Materials: An Introduction to Integrating Materials with Tissues, First Edition. Edited by Matteo Santin and Gary Phillips. John Wiley & Sons, Inc. Published, [6] Gutiérrez-Salazara MP, Reyes-Gasga J. Microhardness and Chemical Composition of Human Tooth. Mat. Res. [online]. 2003; 6-3: [7] [8] Karnovsky MJ. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol. 1965; 27:137. [9] Mc Dowell and Trump. Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100:405, 1976] [10] Prasad P, Donoghue M. A comparative study of various decalcification techniques. Indian J of Dental Research. 2013; 24, 3: [11] Callis MG. Bone. In: Bancroft JD, Gamble M, editors. Theory and practice of histological techniques. 6th ed. Philadelphia: Churchill Livingstone; 2008; [12] Culling CF, Allison RT, Barr WT. 4th ed. London: Butterworths; Cellular pathology technique; pp [13] Sanjai K, Kumarswamv J, Patil A, Papaiah L, Jayaram S and Krishnan L. Evaluation and comparison of decalcification agents on the human teeth. J Oral Maxillofac Pathol. 2012; 16(2): [14] Clayden EC. Practical section cutting and staining. Edinburgh: Churchill Livingstone,

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