1. 1. Maria Mirasol L. Tuao, MD, DPPS Pediatric Consultant
2. 2. HISTOLOGY = is the study of the tissues of the body & how these tissues are arranged to constitute organs. The Greek root histo = can be translated as either tissue or web and both translations are appropriate because MOST of the tissues are webs of interwoven filaments & fibers, both cellular & noncellular, with membranous linings. It involves all aspects of tissue biology, with the focus on how cells structure & arrangement optimize functions specific to each organ.
3. 3. Tissues are made of 2 interacting components: Cells & Extracellular matrix
4. 4. EXTRACELLULAR MATRIX: Consists of many kinds of molecules, MOST of w/c are highly organized & form complex structures, such as collagen fibrils & basement membranes. The main function once attributed to the extracellular matrix were: to furnish mechanical support for the cells, To transport nutrients to the cells, & to carry away catabolites & secretory products.
5. 5. We now know that, although the cells produce the extracellular matrix, they are also influenced & sometimes controlled by molecules of the matrix. There is, thus, an intense interaction between cells & matrix, w/ many components of the matrix recognized by & attaching to receptors present on cell surfaces.
6. 6. Most of these receptors are molecules that cross the cell membranes & connect to structural components of the intracellular cytoplasm. Thus, Cells & Extracellular matrix form a continuum that functions together & reacts to stimuli & inhibitors together.
7. 7. Each of the fundamental tissues is formed by several types of cells & typically by specific associations of cells & extracellular matrix. These characteristic associations facilitate the recognition of the many subtypes of tissues by students.
8. 8. Most organs are formed by an orderly combination of several tissues, except the Central Nervous System, w/c is formed almost solely by nervous tissue. The precise combination of these tissues allows the functioning of each organ & of the organism as a whole.
9. 9. The small size of cells & matrix components makes histology dependent on the use of microscopes. Advances in Chemistry, Molecular Biology, Physiology, Immunology, & Pathology and the interactions among these fields are essential for a better knowledge of tissue biology.
10. 10. Familiarity w/ the tools & methods of any branch of science is essential for a proper understanding of the subject. These chapters reviews several of the more common methods used to study cells & tissues & the principles involved in these method
11. 11. Preparation of histological sections or tissue slices - the MOST common procedure used in the study of tissues that can be studied w/ the aid of the light microscope. Under the Light Microscope, tissues are examined via a light beam that is transmitted through the tissue.
12. 12. Because tissues & organs are usually too thick for light to pass through them, they must be sectioned to obtain thin, translucent sections, & then attached to glass slides before they can be examined.
13. 13. The ideal microscope tissue preparation should be preserved so that the tissue on the slide has the same structure & molecular composition as it had in the body.
14. 14. However, as a practical matter this is seldom feasible & artifacts, distortions, & loss of components due to the preparation process are almost always present.
15. 15. The Basic steps used in tissue preparation for Histology are shown in Figure 1-1.
16. 16. Sectioning fixed & Embedded Tissue. Most tissues studied histologically are prepared as shown. (A) Small pieces of fresh tissue are placed in Fixative solutions w/c generally cross-link proteins, inactivating degradative enzymes & preserving cell structures. The fixed pieces then undergo Dehydration by being transferred through a series of increasingly more concentrated alcohol solutions, ending in 100% w/c effectively removes all water from the tissue. The alcohol is then removed in a Clearing solution miscible in both alcohol & melted paraffin.
17. 17. Sectioning fixed & Embedded Tissue. When the tissue is then placed in melted paraffin at 58C it becomes completely infiltrated w/ this substance. All steps to this point are commonly done today by robotic devices in active histology or pathology laboratories. After Infiltration the tissue is placed in a small mold containing melted paraffin, w/c is then allowed to harden. The resulting paraffin block is trimmed to expose the tissue for sectioning (Slicing).
18. 18. Sectioning fixed & Embedded Tissue. Similar steps are used in preparing tissue for transmission electron microscope, except that smaller tissue samples are fixed in special fixatives & dehydration solutions are used that are appropriate for Embedding in epoxy resins w/c become much harder than paraffin to allow very thin sectioning.
19. 19. The Basic steps used in tissue preparation for Histology are shown in Figure 1-1.
20. 20. It is used for sectioning paraffin-embedded tissues for light microscopy. After mounting a trimmed block w/ the tissue specimen, rotating the drive wheel moves the tissue-block holder up & down. Each turn of the drive wheel advances the specimen holder a controlled distance, generally between 1 & 10 m, & after each forward move the tissue block passes over the steel knife edge, w/c cuts the sections at a thickness equal to the distance the block advanced. A Microtome
21. 21. Paraffin sections are then adhered to glass slides, deparaffinized, & stained for microscopic examination. For transmission electron microscopy sections < 1 m thick are prepared from resin- embedded cells using an ultramicrotome w/ a glass or diamond knife. A Microtome
22. 22. If a permanent section is desired, tissues must be fixed. To avoid tissue digestion by enzymes present w/in the cells (Autolysis) or by bacteria & to preserve the structure & molecular composition, pieces of organs should be promptly & adequately treated before, or as soon as possible after, removal from the animals body.
23. 23. This treatment Fixation-can be done by chemical or, les frequently physical methods. In CHEMICAL FIXATION: The tissues are usually immersed in solutions of stabilizing or cross-linking agents called FIXATIVES.
24. 24. In CHEMICAL FIXATION: Because the fixatives needs some time to fully diffuse into the tissues, the tissues are usually cut into small fragments before fixation to facilitate the penetration of the fixative & to guarantee preservation of the tissue. Intravascular perfusion of fixatives can be used.
25. 25. In CHEMICAL FIXATION: Because the fixative in this case rapidly reaches the tissues through the blood vessels, fixation is greatly improved.
26. 26. FORMALIN one of the best fixatives for routine light microscopy. A buffered isotonic solution of 37% formaldehyde. The chemistry of the process involved in fixation is complex & NOT always understood
27. 27. FORMALDEHYDE & GLUTARALDEHYDE : Another widely used fixative Are known to react w/ the amine groups (NH2) of tissue proteins. In the case of Glutaraldehyde, the fixing action is reinforced by virtue of its being a dialdehyde, w/c can cross-link proteins.
28. 28. In view of the high resolution afforded by the electron microscope, greater care in fixation is necessary to preserve ultrastructural detail. Toward that end, a double fixation procedure, using a buffered glutaraldehyde solution followed by a second fixation in buffered osmium tetroxide, is a standard procedure in preparations for fine structural studies.
29. 29. The effect of osmium tetroxide is to preserve & stain lipids & proteins.
30. 30. Tissue are usually embedded in a solid medium to facilitate sectioning. To obtain thin sections w/ the microtome, tissues must be infiltrated after fixation w/ embedding substances that impart a rigid consistency to the tissue.
31. 31. Embedding materials include: Paraffin - used routinely for light microscopy Plastic resins used for both light & electron microscopy
32. 32. The process of Embedding, or tissue impregnation = is ordinarily preceded by 2 main steps: Dehydration & Clearing
33. 33. DEHYDRATION : The WATER is first extracted from the fragments to be embedded by bathing them successively in a graded series of mixtures of ETHANOL & Water, usually from 70% to 100% Ethanol (DEHYDRATION)
34. 34. CLEARING: The Ethanol is then replaced w/ a solvent miscible w/ both Alcohol & the embedding medium. As the tissues are infiltrated w/ this solvent, they generally become transparent (CLEARING).
35. 35. Once the tissue is impregnated w/ the solvent, it is placed in melted paraffin in an oven, typically at 52-60C. The heat causes the solvent to evaporate, & the spaces within the tissues become filled w/ paraffin. The tissue together w/ its impregnating paraffin hardens after removal from the oven.
36. 36. Tissues to be embedded w/ Plastic resin are also dehydrated in Ethanol & depending on the kind of Resin used subsequently infiltrated w/ plastic solvents. The Ethanol or the solvents are later replaced by plastic solutions that are hardened by means of cross-linking polymerizers.
37. 37. Plastic embedding prevents the shrinking effect of the high temperature needed for paraffin embedding & gives little or no distortion to the cells. The hard blocks containing the tissues are then placed in an instrument called a MICROTOME & are sliced by the microtomes steel or glass blade into sections 1 to 10 m thick.
38. 38. Remember that one micrometer (1 m) = 1/1,000 of a millimeter (mm) = 10-6 m. Other units of distance commonly used in histology are the: nanometer (1nm = 0.001m = 10-6 mm = 10-9 m) & angstrom (1 = 0.1 nm or 10-4 m ).
39. 39. The sections are floated on water & then transferred to glass to be stained. An alternate way to prepare tissue sections is to submit the tissues to rapid freezing. In this process, the tissues are fixed by freezing ( physical, NOT chemical fixation) & at the same time become hard & thus ready to be sectioned.
40. 40. A freezing microtome the CRYOSTAT is then used to section the frozen block w/ tissue. Because this method allows the rapid preparation of sections w/o going through the long embedding procedure described above, it is routinely used in hospitals to study specimens during surgical procedures.
41. 41. Freezing of tissues is also effective in the histochemical study of very sensitive enzymes or small molecules, since freezing, unlike fixation, does NOT inactivate most enzymes. Finally, because immersion in solvents such as XYLENE dissolves cell lipids in fixed tissues, frozen sections are also useful when structures containing lipids are to be studied.
42. 42. To be studied microscopically sections must typically be stained or dyed because most tissues are colorless. Methods of staining tissues have therefore been devised that NOT only make the various tissue components conspicuous but also permit distinctions to be made between them.
43. 43. The dyes stain tissue components more or less selectively. Most of these dyes behave like acidic or basic compounds & have a tendency to form electrostatic (SALT) linkages w/ ionizable radicals of the tissues. Tissue components w/ a net negative charge (ANIONIC) stain more readily w/ BASIC dye & are termed BASOPHILIC;
44. 44. Example of Basic dyes are: Toluidine blue Alcian blue & Methylene blue Hematoxylin behaves like a basic dye, that is, it stains the basophilic tissue component.
45. 45. The main tissue component that ionize & react w/ basic dyes do so because of acids in their composition: Nucleic acids Glycosaminoglycans & acid glycoproteins
46. 46. Cationic components, such as proteins w/ many ionized amino groups, have affinity for ACIDIC DYES & are termed ACIDOPHILIC. Examples of Acid dyes: Orange G Eosin & Acid fuchsin
47. 47. Cationic components, such as proteins w/ many ionized amino groups, have affinity for ACIDIC DYES & are termed ACIDOPHILIC. Examples of Acid dyes: stain the acidophilic component of tissues such as : Mitochondria Secretory granules & Collagen
48. 48. The simple combination of HEMATOXYLIN & EOSIN (H&E) = is used MOST commonly. Hematoxylin = stains DNA of the cell nucleus & other acidic structures such as RNA-rich portions of the cytoplasm & the matrix of cartilage blue. Eosin = in contrast, stains other cytoplasmic components & collagen pink.
49. 49. Figure 1-2: (a) Micrograph stained w/ Hematoxylin & Eosin With H&E, basophilic cell nuclei are stained purple while cytoplasm stains pink. Micrographs of the columnar epithelium lining the small intestine
50. 50. Many other dyes, such as the TRICHOMES = are used in different histologic procedures. Examples of trichomes: Mallory stain Masson stain The trichomes, besides showing the nuclei & cytoplasm very well, help to distinguish extracellular tissue components better than H&E.
51. 51. A good technique for differentiating Collagen is the use of PICROSIRIUS, especially when associated w/ polarized light.
52. 52. The chemical basis of other staining procedure is more complicated than the electrostatic interactions underlying basophilia & acidophilia. DNA can be specifically identified & quantified in nuclei using the FEULGEN REACTION, in w/c deoxyribose sugars are hydrolyzed by mild hydrochloric acid, followed by treatment w/ PERIODIC ACID & SCHIFF REAGENT (PAS).
53. 53. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) Is based on the transformation of 1,2- glycol groups present in the sugars into aldehyde residues, w/c then react w/ Schiff reagent to produce a purple or magenta color. Polysaccharides constitute an extremely heterogenous group in tissues & occur either in a free state or combined w/ proteins & lipids.
54. 54. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : Because of their hexose sugar content, many polysaccharides can also be demonstrated by the PAS in liver, striated muscle, & other tissues where it accumulates.
55. 55. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : Short branched chains of sugars (oligosaccharides) are attached to specific amino acids of Glycoproteins, making most glycoproteins PAS-positive. Figure 1-2b shows an example of cells stained by the PAS reaction.
56. 56. Figure 1-2: (b) Micrograph stained by Periodic acid Schiff (PAS) reaction for glycoproteins. With PAS, staining is most intense at the cell surface, where projecting microvilli have a prominent layer of glycoproteins (arrow head) & in the mucin-rich secretory granules of goblet cells. Cell surface glycoproteins & mucin are PAS-positive due to their high content of oligosaccharides & polysaccharides. The PAS-stained tissue was counterstained w/ hematoxylin to show the cell nuclei. Micrographs of the columnar epithelium lining the small intestine
57. 57. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : Glycosaminoglycans (GAGs) are anionic, unbranched long-chain polysaccharides containing aminated sugars. Many glycosaminoglycans are synthesized while attached to a core protein & constitute a class of macromolecules called Proteoglycans, w/c upon secretion make up important parts of the extracellular matrix (ECM).
58. 58. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : Unlike a glycoprotein, a proteoglycans carbohydrate chains are great in weight & volume than the protein core of the molecule. GAGs & many acidic glycoproteins do NOT undergo the PAS reaction, but because of their high content of anionic carboxyl & sulphate groups show a strong electrostatic interaction w/ alcian blue & other basic stain.
59. 59. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : Basophilic or PAS-positive material can be further identified by enzyme digestion pretreatment of a tissue section w/ an enzyme that specifically digests one substrate, leaving other adjacent sections untreated.
60. 60. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : For example, pretreatment w/ ribonuclease will greatly reduce cytoplasmic basophilia w/ little effect on chromosomes, indicating the importance of RNA for the cytoplasmic staining.
61. 61. The PAS Technique (PERIODIC ACID & SCHIFF REAGENT) : Similarly, free polysaccharides are digested by amylase, w/c can therefore be used to distinguish glycogen from glycoproteins in PAS-positive material.
62. 62. In many staining procedures certain structures such as nuclei become labelled, but other parts of cells are often not visible. In this case a Counterstain is used to give additional information. A Counterstain is usually a single stain that is applied to a section by another method to allow better recognition of nuclei or other structures.
63. 63. Lipid-rich structures are best revealed w/ LIPID-SOLUBLE dyes to avoid the steps of slide preparation that remove lipids such as treatment w/ heat, xylene, or paraffin. Typically frozen sections are stained in alcohol solutions saturated w/ a lipophilic dye such as Sudan black.
64. 64. The stain dissolves in cellular lipid droplets & other lipid-rich structures, w/c became stained in black. Specialized methods for the localization of cholesterol, Phospholipids, & glycolipids are useful in diagnosis of Metabolic diseases in w/c there are intracellular accumulations of different kinds of lipids.
65. 65. In addition to tissue staining w/ dyes, Metal impregnation techniques usually silver salts are a common method of visualizing certain ECM fibers & specific cellular elements in nervous tissue.
66. 66. The whole procedure, from Fixation to observing a tissue in a light microscope, may take from 12 hours to 2 days, depending on the size of the tissue, the fixative, the embedding medium, & the method of staining. The final step before observation is Mounting a protective glass coverslip on the slide w/ adhesive mounting media.
67. 67. Conventional bright-field microscopy, as well as Fluorescence, a phase-contrast, differential interference, confocal, & polarizing microscopy are all based on the interaction of light & tissue components & can be used to reveal & study tissue features.
68. 68. With the Bright-field microscope, widely used by students, stained preparations are examined by means of light that passes through the specimen. The microscope is composed of mechanical & optical parts (figure 1-3). The optical components consist of 3 systems of lenses.
69. 69. Figure 1-3: Bright-Field Microscope Components & Light path of a bright-field microscope: Photograph of a bright- field light microscope showing its components & the pathway of light from the substage lamp to the eye of the observer.
70. 70. Figure 1-3: Bright-Field Microscope The optical system has 3 sets of lenses: a condenser, a set of objectives, & either one or 2 eyepieces. Condenser collects & focuses light, producing a cone of light that illuminates the tissue slide on the stage.
71. 71. Figure 1-3: Bright-Field Microscope Objective lenses enlarge & project the illuminated image of the object in the direction of the eyepiece. For routine histological studies objectives having 3 different magnifications are generally used : X4 for low magnification observations of a large area (field) of the tissue. X10 for medium magnification of a smaller field. X40 for high magnification of more detailed areas.
72. 72. Figure 1-3: Bright-Field Microscope Eyepiece or ocular lens further magnifies this image another X10 & projects it onto the viewers retina, yielding a total magnification of X40, X100, or X400 (with permission, from Nikon Instruments) , photographic film, or (to obtain a digital image) a detector such as a charge- coupled device (CCD) camera.
73. 73. Figure 1-3: Bright-Field Microscope The total magnification is obtained by multiplying the magnifying power of the objective & ocular lenses.
74. 74. The critical factor in obtaining a crisp, detailed image w/ a light microscope is its Resolving power. Resolving power = defined as the smallest distance between 2 particles at w/c they can be seen as separate objects. The maximal RP of the light microscope is approximately 0.2 m; this permits good images magnified 1000-1500 times.
75. 75. Objects smaller or thinner than 0.2m (such as a ribosome, a membrane, or a filament of actin) cannot be distinguished w/ this instrument. Likewise, 2 objects such as Mitochondria will be seen as only one object if they are separated by less than 0.2 m.
76. 76. The quality of the image-its clarity & richness of detail depends on the microscopes resolving power. The magnification is of value only when accompanied by high resolution. The resolving power of a microscope depends mainly on the quality of its objective lens.
77. 77. The eyepiece lens enlarges only the image obtained by the objective; it does not improve resolution. For this reason, when comparing objectives of different magnifications, those that provide higher resolving power .
78. 78. Fluorescence = When certain substances are irradiated by light of a proper wavelength, they emit light w/ a longer wavelength. In Fluorescence microscopy: Tissue sections are usually irradiated w/ ultraviolet (UV) light & the emission is in the visible portion of the spectrum. The fluorescent substances appear brilliant on a dark background.
79. 79. In Fluorescence microscopy: For this method, the microscope has a strong UV light source & special filters that select rays of different wavelengths emitted by the substances.
80. 80. Figure 1-4a. Fluorescent compounds w/ affinity for specific cell macromolecules may be used as fluorescent stains. Example: Acridine orange, w/c binds both DNA & RNA When observed in the fluorescence microscope, these nucleic acids emit slightly different fluorescence, allowing them to be localized separately in cells.
81. 81. Figure 1-4a. Components of cells in culture are often stained w/compounds visible by fluorescence microscopy. (a): Kidney cells stained w/ acridine orange, w/c binds nucleic acid. Under a fluorescence microscope, nuclear DNA emits yellow light & the RNA-rich cytoplasm appears reddish or orange.
82. 82. Figure 1-4b. Other compounds such as Hoechst stain & DAPI specifically bind DNA & are used to stain cell nuclei, emitting a characteristic blue fluorescence under UV.
83. 83. Figure 1-4b. Another important application of fluorescence microscopy is achieved by coupling fluorescent compounds to molecules that will specifically bind to certain cellular components & thus allow the identification of these structures under the microscope.
84. 84. Figure 1-4b. Antibodies labeled w/ fluorescent compounds are extremely important in Immunohistological staining.
85. 85. Figure 1-4b. Components of cells in culture are often stained w/compounds visible by fluorescence microscopy. (b): The less dense culture of kidney cells stained w/ DAPI (4,6- diamino-2- phenylindole) w/c binds DNA, & w/ phalloidin, w/c binds actin filaments.
86. 86. Figure 1-4b. (b): The less dense culture of kidney cells Nuclei of these cells show a blue fluorescence & actin filaments appear green. Important information such as the greater density of microfilaments at the cell periphery is readily apparent. (Figure 14b, with permission, from Drs. Claire E. Walczak and Rania Risk, Indiana University School of Medicine, Bloomington.)
87. 87. Some optical arrangements allow the observation of unstained cells & tissue sections. Unstained biological specimens are usually transparent & difficult to view in detail, because all parts of the specimen have almost the same optical density.
88. 88. Figure 1-5 Phase- contrast microscopy, however, uses a lens system that produces visible images from transparent objects.
89. 89. Unstained cells appearance in 3 types of Light microscopy. Neural crest cells growing as a single layer in culture appear differently w/ various techniques of light microscopy.
90. 90. These cells are unstained & the same field of cells, including 2 differentiating pigment cells, is shown in each photo. (a) Bright-field microscopy (b) Phase-contrast microscopy (c) Differential interference microscopy
91. 91. Figure 1-5a (a): Bright-field microscopy: w/o fixation & staining, only the 2 pigment cells can be seen
92. 92. Figure 1-5b (b): Phase-contrast microscopy: Cell boundaries, nuclei, & cytoplasmic structures w/ different refractive indices affect in-phase light differently & produce an image of these features in all the cells.
93. 93. Figure 1-5b (b): Phase-contrast microscopy: With or w/o differential interference, is widely used to observe live cells grown in tissue culture. All x200. (With permission, from Sherry Rogers, Department of Cell Biology and Physiology, University of New Mexico.)
94. 94. Figure 1-5c (c): Differential interference microscopy: Cellular details are highlighted in a different manner using Nomarski optics.
95. 95. Is based on the principle that light changes its speed when passing through cellular & extracellular structures w/ different refractive indices. These changes are used by the phase- contrast system to cause the structures to appear lighter or darker in relation to each other.
96. 96. Because it does not require Fixation or staining, Phase-contrast microscopy allows observation of living cells & tissue cultures, & such microscopes are prominent tools in all cells culture labs.
97. 97. A related method of observing unstained cells or tissue sections is the Nomarski differential microscopy, w/c produces an image w/ a more apparent three- dimensional aspect than in routine phase- contrast microscopy.
98. 98. With a regular bright-field microscope the beam of light is relatively large & fills the specimen. Stray light reduces contrast within the image & compromises the resolving power of the objective lens.
99. 99. It avoids stray light & achieves greater resolution by using : (1) a small point of high-intensity light provided by a laser & (2) a plate w/ a pinhole aperture in front of the image detector.
100. 100. The point of light source, the focal point of the lens, & the detectors pinpoint aperture are all optically conjugated or aligned to each other in the focal plane (Confocal) & unfocused light does not pass through the pinhole.
101. 101. This greatly improves resolution of the object in focus & allows the localization of specimen components w/ much greater precision than w/ the bright-field microscope.
102. 102. Most Confocal microscopes include a Computer-driven mirror system (the Beam splitter) to move the point of illumination across the specimen automatically & rapidly. Digital images captured at many individual spots in a very thin plane-of-focus are used to produce an Optical section of that plane.
103. 103. Moreover, creating optical sections at a series of focal planes through the specimen allows them to be digitally reconstructed into a three-dimensional image.
104. 104. Figure 1-6.
105. 105. Although a very small spot of light originating from one plane of the section crosses the pinhole & reaches the detector, rays originating from other planes are blocked by the blind. Thus, only one very thin plane of the specimen is focused at a time.
106. 106. The diagram shows the practical arrangement of a confocal microscope. Light from a laser source hits the specimen & is reflected. A beam splitter directs the reflected light to a pinhole & a detector.
107. 107. Light from components of the specimen that are above or below the focused plane is blocked by the blind. The laser scans the specimen so that a larger area of the specimen can be observed.
108. 108. Allows the recognition of structures made of highly organized molecules. When normal light passes through a Polarizing filter (such as Polaroid), it exits vibrating in only one direction. If a second filter is placed in the microscope above the first one, w/ its main axis perpendicular to the first filter, No light passes through.
109. 109. Figure 1-7. If, however, tissue structures containing oriented macromolecules are located between the 2 polarizing filters, their repetitive structure rotates the axis of the light emerging from the polarizer & they appear as bright structures against a dark background (Fig 1-7)
110. 110. Figure 1-7. Birefringence = the ability to rotate the direction of vibration of polarized light & is a feature of crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules, & microfilaments.
111. 111. Polarizing Light Microscopy: Produces an image only of material having repetitive, periodic macromolecular structure; features w/o such structure are Not seen.
112. 112. Polarizing Light Microscopy: Shown here is a piece of thin mesentery that was stained w/ red picrosirius, orcein, & hematoxylin, & was then placed directly on a slide & observed by bright-field & polarizing microscopy.
113. 113. Figure 1-7a A piece of thin mesentery (a): Under routine Bright-field microscopy Collagen fibers appear red, along w/ thin dark elastic fibers & cell nuclei.
114. 114. Figure 1-7b A piece of thin mesentery (b): Under Polarizing light microscopy Only Collagen fibers are visible & these exhibit intense birefringence & appear bright red or yellow; elastic fibers & nuclei lack oriented macromolecular structure & are Not visible.
115. 115. Transmission & scanning electron microscopes are based on the interaction of electrons & tissue components. The wavelength in the electron beam is much shorter than of light, allowing a thousand-fold increase in resolution.
116. 116. Figure 1-8a (a) The TEM is an imaging system that permits resolution around 3 mm. This high resolution allows magnification of up to 400,000 times to be viewed w/ details.
117. 117. Figure 1-8a (a) The TEM : Unfortunately, this level of magnification applies only to isolated molecules or particles. Very thin tissue sections can be observed w/ details at magnifications of up to about 120,000 times.
118. 118. Figure 1-8b (b): SEM : Permits pseudo-three- dimensional views of the surfaces of cells, tissues, & organs. Like the TEM this microscope produces & focuses a very narrow beam of electrons, but in this instrument the beam does Not pass through the specimen.
119. 119. Figure 1-8b (b): SEM : Instead the surface of the specimen is first dried & coated w/ a very thin layer of metal atoms through w/c electrons do Not pass readily.
120. 120. Figure 1-8b (b): SEM : When the beam is scanned from point to point across the specimen it interacts w/ the metal atoms & produces reflected electrons or secondary electrons emitted from the metal.
121. 121. Figure 1-8b (b): SEM : These are captured by a detector & the resulting signal is processed to produce a black-and-white image on a monitor.
122. 122. Figure 1-8b (b): SEM : SEM images are usually easily understood, because they present a view that appears to be illuminated from above, just our ordinary macroscopic world is filled w/ highlights & shadows caused by illumination from above.
123. 123. Is a method of localizing newly synthesized macromolecules (DNA, RNA, proteins, glycoproteins, & polysaccharides) in cells or tissue sections. Radioactively labeled metabolites (nucleotides, amino acids) incorporated into the macromolecules emit weak radiation that is restricted to the cellular regions where the molecules are located.
124. 124. Radiolabeled cells or mounted tissue sections are coated in a darkroom w/ photographic emulsion containing silver bromide crystals, w/c act as microdetectors of this radiation in the same way that they respond to light in common photographic film.
125. 125. After an adequate exposure time in lightproof boxes the slides are developed photographically. The silver bromide crystals reduced by the radiation are reduced to small black grains of metallic silver, indicating locations of radiolabeled macromolecules in the tissue.
126. 126. Figure 1-9 This general procedure can be used in preparations for both Light microscopy & TEM.
127. 127. Figure 1-9 Autoradiographs are tissue preparations in w/c particles called Silver grains indicate the regions of cells in w/c specific macromolecules were synthesized just prior to Fixation.
128. 128. Figure 1-9 Precursors such as nucleotides, amino acids, or sugars w/isotopes substituted for specific atoms are provided to the tissues & after a period of incorporation, tissues are fixed, sectioned, & mounted on slide or TEM grids as usual.
129. 129. Figure 1-9 This processing removes all radiolabeled precursors, leaving only the isotope in the fixed macromolecules. In a darkroom the slides are coated w/ a thin layer of chemicals like those in the photographic film & dried.
130. 130. Figure 1-9 In a black box the isotope in newly synthesized macromolecules emits radiation exposing the layer of photographic chemicals immediately adjacent to the isotopes location.
131. 131. Figure 1-9 The minute regions of exposed chemicals in the photographic layer are revealed as silver grains by developing the preparation as if it were film, followed by microscopic examination .
132. 132. Figure 1-9 Shown here are autographs from the salivary gland of a mouse injected w/ H- fucose 8 hr before tissue fixation. Fucose is incorporated into oligosaccharides & the results reveal location of newly synthesized glycoproteins containing such sugars.
133. 133. Figure 1-9a (a): Black silver grains are visible over regions w/ secretory granules & the duct indicating glycoprotein locations. X1500.
134. 134. Figure 1-9b (b): The same tissue prepared for TEM autoradiography shows silver grains w/ a coiled or amorphous appearance against localized mainly over the granules (G) & in the gland lumen (L). X7500. (Figure 19b, with permission, from Ticiano G. Lima and A. Antonio Haddad, School of Medicine, Ribeiro Preto, Brazil.)
135. 135. Live cells & tissues can be maintained & studied outside the body. In a complex organism, tissues & organs are formed by several kinds of cells. These cells are bathed in fluid derived from blood plasma, w/c contains many different molecules required for growth.
136. 136. Cell culture has been very helpful in isolating the effects of single molecules on specific type of cells. It also allows the direct observation of the behavior of living cells under a phase contrast microscope. Many experiments that cannot be performed in the living animal can be accomplished in vitro.
137. 137. The cells & tissues are grown in complex solutions of known composition(salts, amino acids, vitamins) to w/c serum components or specific growth factors are added. In preparing cultures from a tissue or organ, cells must be initially dispersed mechanically or enzymatically.
138. 138. Figure 1-5 Once isolated, the cells can be cultivated in a clear dish to w/c they adhere, usually as a single layer of cells (Figure 1-5).
139. 139. Culture of cells that are isolated in this way are called Primary cell cultures. Many cell types once isolated from normal or pathologic tissue have been maintained in vitro ever since because they have been immortalized & now constitute a permanent cell line.
140. 140. Most cells obtained from normal tissues have a finite, genetically programmed life span. Certain changes, however (some related to oncogenes), can promote cell immortality, a process called Transformation, w/c are similar to the initial changes in a normal cells becoming a cancer cell.
141. 141. Because of improvements in culture technology, most cell types can now be maintained in the laboratory. All procedures w/ living cells & tissues must be performed in a sterile area, using solutions & equipment, to avoid contamination w/ microorganism.
142. 142. As shown in the next chapter, Incubation of living cells in vitro w/ a variety of new fluorescent compounds that are sequestered & metabolized in specific compartments of the cell provides a new approach to understanding these compartments both structurally & physiologically.
143. 143. Other histological techniques applied to cultured cells have been particularly important for understanding the locations & functions of microtubules, microfilaments, & other components of the cytoskeleton.
144. 144. Cell culture has been widely used for the study of the metabolism of normal & cancerous cells & for the development of new drugs. This technique is also useful in the study of parasites that grow only w/in cells, such as Viruses, Mycoplasma, & some Protozoa.
145. 145. In Cytogenetic research, determination of human karyotypes (the number & morphology of an individuals chromosomes) is accumulated by short- term cultivation of blood cells or fibroblasts & by examining the chromosome during Mitotic division. In addition, cell culture is central to contemporary techniques of molecular Biology & recombinant DNA technology.
146. 146. Indicates methods for localizing cellular structures in tissue sections using the unique enzymatic activity present in those structures. To preserve these enzymes histochemical procedures are usually applied to unfixed or mildly fixed tissue, often sectioned on a Cryostat to avoid adverse effects of Heat & Paraffin on enzymatic activity.
147. 147. Enzyme histochemistry usually works in the following way : (1) Tissue sections are immersed in a solution that contains the substrate of the enzyme to be localized; (2) The enzyme is allowed to act on its substrate; (3) At this stage or later, the section is put in contact w/ a marker compound;
148. 148. Enzyme histochemistry usually works in the following way : (4) This compound reacts w/ a molecule produced by enzymatic action on the substrate;
149. 149. Enzyme histochemistry usually works in the following way : (5) The final reaction product, w/c must be insoluble & w/c is visible by Light or Electron microscopy only if it is colored or electron-dense, precipitates over the site that contains the enzymes. When examining such a section in the microscope, one can see the Cell regions (or Organelles) covered w/ a colored or electron-dense material.
150. 150. Examples of enzymes that can be detected histochemically include the following: Phosphatases split the bond between a phosphate group & an alcohol residue of phosphorylated molecules. The visible, insoluble reaction product of phosphatases is usually Lead phosphate or Lead sulfide.
151. 151. Figure 1-10 Phosphatases Both alkaline phosphatases w/c have their maximum activity at an alkaline pH & acid phosphatases can be detected.
152. 152. Examples of enzymes that can be detected histochemically include the following: Dehydrogenases remove hydrogen from one substrate & transfer it to another. Like phosphatases, Dehydrogenases play an important role in several hydrogen & precipitates as an insoluble colored compound.
153. 153. Examples of enzymes that can be detected histochemically include the following: Dehydrogenases Mitochondria can be specifically identified by this method, since dehydrogenases are key enzymes in the Citric acid (Krebs) Cycle of this organelle.
154. 154. Examples of enzymes that can be detected histochemically include the following: Peroxidase, w/c is present in several types of cells, promotes the oxidation of certain substrates w/ the transfer of hydrogen ions to hydrogen peroxide, forming molecules of water.
155. 155. Examples of enzymes that can be detected histochemically include the following: Peroxidase, In this method, sections of adequately fixed tissue are incubating in a solution containing hydrogen peroxide & 3,3- diamino-azobenzidine (DAB).
156. 156. Examples of enzymes that can be detected histochemically include the following: Peroxidase, The latter compound is oxidized in the presence of Peroxidase, resulting in an insoluble, brown, electron-dense precipitate that permits the localization of Peroxidase activity by Light & electron microscopy.
157. 157. Examples of enzymes that can be detected histochemically include the following: Peroxidase, Peroxidase staining in White blood cells is important in the diagnosis of certain Leukemias.
158. 158. Figure 1-10a (a): Micrograph of cross sections of Kidney tubules treated histochemically by the Gomori method for Alkaline phosphatases show strong activity of this enzyme at the apical surfaces of the cells at the lumen of the tubules.
159. 159. Figure 1-10b (b): TEM image of a Kidney cell in w/c acid phosphatases has been localized histochemically in 3 lysosomes (Ly) near the nucleus (N). The dark material w/in these structures is Lead phosphate that precipitated in places w/ acid phosphatase activity. (Figure 110b, with permission, from Eduardo Katchburian, Department of Morphology, Federal University of Sao Paulo, Brazil.)
160. 160. Many histochemical procedures are used frequently in laboratory diagnosis, including: Perls Prussian blue reaction for Iron ( used to detect the Iron storage diseases, hemochromatosis &, Hemosiderosis), the PAS-amylase & Alcian blue reactions for Glycogen & Glycosaminoglycans ( to detect glycogenosis & Mucopolysaccharides), & reactions for lipids & sphingolipids (to detect Sphingolipidosis)
161. 161. Figure 1-11 A specific macromolecules present in a tissue section may sometimes be identified by using tagged compounds or macromolecules that specifically interact w/ the material of interest.
162. 162. The compounds that will interact w/ the molecule must be tagged w/ a label that can be detected under the light or electron microscope.
163. 163. The most commonly used labels are: fluorescent compounds - w/c can be seen w/ a Fluorescence or laser microscope, radioactive atoms - w/c can be detected w/ Autoradiography, Molecules of Peroxidase or other enzymes w/c can be detected w/ Histochemistry, & metal (usually GOLD) particles that can be observed w/ Light & electron microscopy.
164. 164. These methods can be used for detecting & localizing specific sugars, proteins, & nucleic acids.
165. 165. Labeling by Specific, High-affinity interactions : Compounds or macromolecules that have affinity toward certain cell or tissue macromolecules can be tagged w/ a label & used to identify that component & determine its location in cells & tissues.
166. 166. Figure 1-11 (1) Labeling by Specific, High-affinity interactions: (1) Molecule A has a high & specific affinity toward a portion of molecule B. Examples: Antibody that recognizes specific antigens, usually Proteins, or a segment of single-stranded DNA w/ sequence-specific complementarity to RNA molecules in a cell.
167. 167. Figure 1-11 (1) Labeling by Specific, High-affinity interactions: (1) Molecule A can also be a small compound like Phalloidin, w/c specifically binds actin filaments, or a protein as protein A w/c binds all Immunoglobulins.
168. 168. Figure 1-11 (2) Labeling by Specific, High-affinity interactions: (2) When A & B are mixed, A binds to the portion of B it recognizes.
169. 169. Figure 1-11 (3) Labeling by Specific, High-affinity interactions: (3) Molecule A may be tagged w/ a label that can be visualized / a light or electron microscope. The label can be a Fluorescent compound, a enzyme such as Peroxidase, an electron- dense particle, or radioisotope.
170. 170. Figure 1-11 (4) Labeling by Specific, High-affinity interactions: (4) If molecule B is present in a cell or extracellular matrix that is incubated w/ labeled molecule A, molecule B can be detected & localized by visualizing the labeled molecule A & bound to it.
171. 171. Example of molecules that interact specifically w/ other molecules include the following: Phalloidin is a compound extracted from the mushroom Amanita phalloides & interacts strongly w/ actin. Tagged w/ Fluorescent dyes, Is commonly used to demonstrate actin filaments in cells.
172. 172. Example of molecules that interact specifically w/ other molecules include the following: Protein A is obtained from Staphylococcus aureus & binds to the Fc of immunoglobulin (Antibody) molecules. Labeled protein A can therefore be used to localize naturally occurring or applied antibodies bound to cell structures.
173. 173. Example of molecules that interact specifically w/ other molecules include the following: Lectins are proteins or glycoproteins, derived mainly from plant seeds & that bind to Carbohydrates w/ high affinity & specificity. Different Lectins binds to specific sugars or sequence of sugar residues.
174. 174. Example of molecules that interact specifically w/ other molecules include the following: Lectins Fluorescent labeled lectins are used to stain specific Glycoproteins, proteoglycans, & glycolipids and are used to characterize membrane components w/ specific sequence of sugar residues.
175. 175. A highly specific interaction between molecules is that between an antigen & its antibody. For this reason, methods using labeled antibodies have become extremely useful in identifying & localizing many specific proteins, Not just those w/ enzymatic activity that can be demonstrated by histochemistry.
176. 176. The bodys immune cells are able to discriminate its own molecules (Self) from foreign ones. When exposed to foreign molecules called Antigens the body responds by producing antibodies that react specifically & bind to the antigen thus helping to eliminate the foreign substance.
177. 177. Antibodies belong to the Immunoglobulin family of glycoproteins, produced by lymphocytes. In Immunohistochemistry, a tissue section (or cells in culture) that one believes contains the protein of interest is incubated in a solution containing an antibody to this protein.
178. 178. The antibody binds specifically to the protein, whose location in the tissue or cell can than be seen w/ either the light or electron microscope, depending on the type of compound used to label the antibody.
179. 179. Antibodies are commonly tagged w/ fluorescent compounds, w/ Peroxidase or alkaline phosphatase for histochemical detection, or w/ electron-dense gold particles.
180. 180. For immunohistochemistry, one must have an antibody against the protein that is to be detected. This means that the protein must have been previously purified using biochemical or molecular approaches so that antibodies against it can be produced.
181. 181. To produced antibodies against protein x of a certain animal species (eg. A Human or Rat ), the protein is first isolated & then injected into an animal of another species (eg. A rabbit or a goat ). If the proteins amino acid sequence is sufficiently different for this animal to recognize it as foreign, that is, an antigen , the animal will produce antibodies against the protein.
182. 182. Different groups (Clones) of lymphocytes in the animal that was injected recognize different parts of protein x and each clone produces an antibody against that part. These antibodies are collected from the animals plasma & constitute a mixture of Polyclonal antibodies, each capable of binding a different region of protein x.
183. 183. It is also possible, however, to inject protein x into a mouse & then days later to isolate the activated lymphocytes & place them into culture. Growth & activity of these cells can be prolonged indefinitely by fusing them w/ lymphocytic tumor cells to produce Hybridoma cells.
184. 184. Different Hybridoma clones produce different antibodies against the several parts of protein x & each clone can be isolated & cultured separately so that the different antibodies against protein x can be collected separately. Each of these antibodies is a Monoclonal antibody.
185. 185. An advantage to using a monoclonal antibody rather than polyclonal antibodies is that it can be selected to be highly specific & to bind strongly to the protein to be detected, producing less nonspecific binding to other proteins similar to the one of interest.
186. 186. In the direct method of immunocytochemistry, the antibody (either monoclonal or polyclonal) is tagged itself w/ an appropriate label. A tissue section is incubated w/ the antibody for some time so that the antibody interacts with & binds to protein x.
187. 187. Figure 1-12 The section is then washed to remove the unbound antibody, processed by the appropriate method & examined microscopically to study the location or other aspects of protein x.
188. 188. Figure 1-12 Can be direct or indirect.
189. 189. Figure 1-12 Direct immunocytochemistry - uses an antibody made against the tissue protein of interest & tagged directly w/ a label such as a Fluorescent compound or Peroxidase.
190. 190. Figure 1-12 Direct immunocytochemistry -When placed w/ the tissue section on a slide, these labeled antibodies bind specifically to the protein (Antigen) against w/c they were produced & can be visualized by the appropriate method.
191. 191. Figure 1-12 Indirect immunocytochemistry uses 2 different antibodies. A Primary antibody - is made against the protein (Antigen) of interest & applied to the tissue section first to bind its specific antigen.
192. 192. Figure 1-12 Indirect immunocytochemistry Then a labeled Secondary antibody is obtained that was : (1) Made in another vertebrae species against Immunoglobulin proteins (antibodies) from the species in w/c the primary antibodies were made & then
193. 193. Figure 1-12 Indirect immunocytochemistry Then a labeled Secondary antibody is obtained that was : (2) Labeled w/ a Fluorescent compound or Peroxidase. When this labeled secondary antibody is applied to the tissue section it specifically binds the primary antibodies, indirectly labeling the protein of interest on the slide.
194. 194. Figure 1-12 Indirect immunocytochemistry Then a labeled Secondary antibody is obtained that was : (2) Since more than one labeled secondary antibody can bind each Primary antibody molecule, labeling of the protein of interest is amplified by the direct method.
195. 195. The Indirect method of immunocytochemistry is more sensitive but requires 2 antibodies & additional steps. Instead of labeling the (Primary) antibody specific for protein, the detectable tag is conjugated to a secondary antibody made in a different Foreign species against the immunoglobulin class to w/c the Primary antibody belongs.
196. 196. Figure 1-12 Indirect immunocytochemistry detection is performed by initially incubating a section of a human tissue believed to contain protein x w/ mouse anti-x antibody. After washing, the tissue sections are incubated w/ labeled rabbit or goat antibody against mouse antibodies.
197. 197. Figure 1-12 Indirect immunocytochemistry detection This secondary antibodies will recognize the mouse antibody that had recognized protein x . Protein x can then be detected by using a microscopic technique appropriate for the label used for the secondary antibody.
198. 198. The Indirect method of immunocytochemistry : There are other indirect methods that involve the use of other intermediate molecules, such as the Biotin-avidin technique.
199. 199. Figure 1-13 Examples of Indirect immunocytochemistry, demonstrating the use of labeling methods w/ cells in culture or after sectioning for both light microscopy & TEM.
200. 200. Figure 1-13a Immunocytochemical methods to localize specific proteins in cells can be applied to either light microscopic or TEM preparations using a variety of labels: (a) A decidual cell grown in vitro stained to reveal a mesh of intermediate filaments throughout the cytoplasm.
201. 201. Figure 1-13a Immunocytochemical methods : (a) Primary antibodies against the protein Desmin, w/c forms these intermediate filament, & FITC-labeled secondary antibodies were used in an indirect immunofluorescence technique. The Nucleus is counterstained light blue w/ DAPI.
202. 202. Figure 1-13b Immunocytochemical methods : (b): A section of small intestine stained w/ an antibody against the enzyme lysozyme. The secondary antibody labeled w/ Peroxidase was then applied & the localized brown color produced histochemically w/ the Peroxidase substrate DAB.
203. 203. Figure 1-13b Immunocytochemical methods : (b): A section of small intestine The method demonstrates lysozyme containing structures in scattered microphages & in the clustered Paneth cells. Nuclei were counterstain w/ hematoxylin.
204. 204. Figure 1-13c Immunocytochemical methods : (c): A section of Pancreatic acinar cells in a TEM preparation incubated w/ antibody against the enzyme Amylase antibody & then w/ protein A coupled w/ Gold particles. Protein A has high affinity toward antibody molecules & the resulting image reveals the presence of Amylase w/ the Gold particles
205. 205. Figure 1-13c Immunocytochemical methods : (c): A section of Pancreatic acinar cells Protein A has high affinity toward antibody molecules & the resulting image reveals the presence of Amylase w/ the Gold particles localized as very small black dots over dense secretory granules & developing granules (left).
206. 206. Figure 1-13c Immunocytochemical methods : (c): A section of Pancreatic acinar cells With specificity for Immunoglobulin molecules, labeled protein A can be used to localize any Primary antibody. (Figure 113c, with permission, from Moise Bendayan, Departments of Pathology and Cell Biology, University of Montreal.)
207. 207. Immunocytochemistry has contributed significantly to research in Cell biology & to the improvement of medical diagnostic procedures. Table 1-1 shows some of the routine applications of Immunocytochemical procedures in clinical practice.
208. 208. Table 1-1: Many pathologic conditions are diagnosed by localizing specific markers of the disorder using antibodies against those antigens in immune-histochemical staining. Antigens Diagnosis Specific cytokeratins Tumors of epithelial origin Protein & polypeptide hormones Protein or Polypeptide hormone- producing endocrine tumors Carcinoembryonic antigen (CEA) Glandular tumors, mainly of the digestive tract & breast Steroid hormone receptors Breast duct cell tumors Antigens produced by Viruses Specific virus infections
209. 209. The central challenge in modern cell biology is to understand the workings of the cell in molecular detail. This goal requires techniques that permit analysis of the molecules involved in the process of information flow from DNA to Protein. Many techniques are based on Hybridization.
210. 210. Hybridization is the binding between 2 single strands of nucleic acids (DNA w/ RNA, RNA w/ RNA, or RNA w/ DNA) that recognize each other if the strands are complementary. The greater the similarities of the sequences, the more readily complementary strands form hybrid double-strand molecules.
211. 211. Hybridization thus allows the specific identification of sequences of DNA or RNA. This is commonly performed w/ nucleic acids in solution, but hybridization also occurs when solution of nucleic acid are applied directly to cells & tissue sections, a procedure called in situ hybridization (ISH).
212. 212. This technique is ideal for : (1) determining if a cell has a specific sequence of DNA (such as a Gene or part of a gene), (2) identifying the cells containing specific mRNAs ( in w/c the corresponding gene is being transcribed), or (3) determining the localization of a gene in a specific chromosome.
213. 213. DNA & RNA of the cells must be initially denatured by Heat or other agents to become completely single-stranded. They are then ready to be hybridized w/ a segment of single-stranded DNA or RNA (called a Probe) that is complementary to the sequence one wishes to detect.
214. 214. The Probe may be obtained by cloning, by PCR amplification of the target sequence, or by chemical synthesis if the desired sequence is short. The probe is tagged w/ nucleotides containing a radioactive isotope (w/c can be localized by autoradiography) or modified w/ a small compound such as Digoxygenin (w/c can be identified by Immunocytochemistry).
215. 215. Figure 1-14 A solution containing the probe is placed over the specimen for a period of time necessary for Hybridization. After washing off the excess unbound probe, the localization of the hybridized probe is revealed through its label.
216. 216. Figure 1-14 In situ hybridization shows that many of the epithelial cells in this section of a Genital wart contain the Human papillomavirus (HPV), w/c causes this benign proliferative condition.
217. 217. Figure 1-14 In situ hybridization: The section was incubated w/ a solution containing a Digoxygenin-labeled cDNA probe for the HPV DNA. The probe was then visualized by direct immunohistochemistry using Peroxidase- labeled antibodies against digoxgenin.
218. 218. Figure 1-14 In situ hybridization: This procedure stains brown only those cells containing HPV. X400. H&E counterstain. (With permission, from Jose E. Levi, Virology Lab, Institute of Tropical Medicine, University of Sao Pulo, Brazil.)
219. 219. A key point to be remembered in studying & interpreting stained tissue sections is that : (1) Microscope preparations are the end result of a series of processes that began w/ collecting the tissue & ended w/ mounting a coverslip on the slide. Several steps of this procedure may distort the tissues, producing minor structural abnormalities called Artifacts.
220. 220. Structures seen microscopically then may differ slightly from the structures present when they were alive. One such distribution is minor shrinkage of cells or tissue regions produced by the Fixative, by the ethanol, or by the Heat needed for Paraffin embedding. Shrinkage can produce the appearance of artificial spaces between cells & other tissue components.
221. 221. Another source of artificial spaces is the loss of molecules such as Lipids, Glycogen, or low molecular weight substances that are not kept in he tissues by the Fixative or removed by the dehydrating & clearing fluids. Slight cracks in sections also appear as large spaces in the tissues.
222. 222. Other artifacts may include: Wrinkles of the section w/c may be confused w/ linear structures such as Blood capillaries & Precipitates of stain w/c may be confused w/ cellular structures such as Cytoplasmic granules. Students must be aware of the existence of artifacts & able to recognize them.
223. 223. A key point to be remembered in studying & interpreting stained tissue sections is that : (2) Impossibility of differentiating staining all tissue components on a slide stained by a single procedure. With the Light microscope it is necessary to examine several preparations stained by different methods to obtain an idea of the tissues complete composition & structure.
224. 224. The TEM, on the other hand, allows the observation of cells w/ all organelles & inclusions, surrounded by the components of the ECM.
225. 225. A key point to be remembered in studying & interpreting stained tissue sections is that : (3) Finally, when a three-dimensional tissue volume is cut into very thin sections, the sections appear microscopically to have only 2 dimensions: Length & Width
226. 226. When examining a section under the microscope, one must always keep in mind that something may be missing in front of or behind that section because many tissue structures are thicker than the section.
227. 227. Figure 1-15 Round structures seen microscopically may be sections through spheres or cylinders & tubules in cross- section look like rings. Also since structures w/in a tissue have different orientations, their two-dimensional appearance will vary depending on the plane of section.
228. 228. Figure 1-15 A single convoluted tube will appear histologically as several rounded structures.
229. 229. Figure 1-15a 3-D structures appear to have only 2-D in thin sections: (a) Sections through a hollow swelling on a tube produce large & small circles, oblique sections through bent regions of the tube produce ovals of various dimensions.
230. 230. Figure 1-15b 3-D structures appear to have only 2-D in thin sections: (b) A single section through a highly coiled tube shows many small, separate round or oval sections.
231. 231. Figure 1-15b 3-D structures appear to have only 2-D in thin sections: (b) On first observation it may be difficult to realize that these represent a coiled tube, but it is important to develop such interpretive skill in understanding histological preparations.
232. 232. Figure 1-15c 3-D structures appear to have only 2-D in thin sections: (c) Round structures in sections may be portions of either spheres or cylinders. Additional sections or the appearance of similar nearby structures help reveal a more complete picture.
233. 233. To understand the architecture of an organ, one often must study sections made in different planes. Examining many parallel sections (Serial sections) & reconstructing the images 3- dimensionally provides better understanding of a complex organ or organism.