1. Maria Mirasol L. Tuao, MD, DPPS Pediatric Consultant
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. Tissues are made of 2 interacting components: Cells &
Extracellular matrix
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. However, as a practical matter this is seldom feasible
& artifacts, distortions, & loss of components due to the
preparation process are almost always present.
15. The Basic steps used in tissue preparation for Histology
are shown in Figure 1-1.
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. 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. 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. The Basic steps used in tissue preparation for Histology
are shown in Figure 1-1.
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. 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. 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. 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. 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. In CHEMICAL FIXATION: Because the fixative in this case
rapidly reaches the tissues through the blood vessels, fixation is
greatly improved.
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. 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. 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. The effect of osmium tetroxide is to preserve & stain
lipids & proteins.
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. Embedding materials include: Paraffin - used routinely for
light microscopy Plastic resins used for both light & electron
microscopy
32. The process of Embedding, or tissue impregnation = is
ordinarily preceded by 2 main steps: Dehydration &
Clearing
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. The main tissue component that ionize & react w/ basic
dyes do so because of acids in their composition: Nucleic acids
Glycosaminoglycans & acid glycoproteins
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. 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. 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. 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. 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. A good technique for differentiating Collagen is the use of
PICROSIRIUS, especially when associated w/ polarized light.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Figure 1-3: Bright-Field Microscope The total magnification
is obtained by multiplying the magnifying power of the objective
& ocular lenses.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Figure 1-4b. Antibodies labeled w/ fluorescent compounds
are extremely important in Immunohistological staining.
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. 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. 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. Figure 1-5 Phase- contrast microscopy, however, uses a lens
system that produces visible images from transparent objects.
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. 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. Figure 1-5a (a): Bright-field microscopy: w/o fixation
& staining, only the 2 pigment cells can be seen
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. 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. Figure 1-5c (c): Differential interference microscopy:
Cellular details are highlighted in a different manner using
Nomarski optics.
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. 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. 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. 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. 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. 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. 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. 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. 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. Figure 1-6.
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. 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. 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. 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. 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. 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. Polarizing Light Microscopy: Produces an image only of
material having repetitive, periodic macromolecular structure;
features w/o such structure are Not seen.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Figure 1-9 This general procedure can be used in
preparations for both Light microscopy & TEM.
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. 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. 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. 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. 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. 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. Figure 1-9a (a): Black silver grains are visible over
regions w/ secretory granules & the duct indicating
glycoprotein locations. X1500.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Enzyme histochemistry usually works in the following way :
(4) This compound reacts w/ a molecule produced by enzymatic action
on the substrate;
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. 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. Figure 1-10 Phosphatases Both alkaline phosphatases w/c
have their maximum activity at an alkaline pH & acid
phosphatases can be detected.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. These methods can be used for detecting & localizing
specific sugars, proteins, & nucleic acids.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. Antibodies are commonly tagged w/ fluorescent compounds,
w/ Peroxidase or alkaline phosphatase for histochemical detection,
or w/ electron-dense gold particles.
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. 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. 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. 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. 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. 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. 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. 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. Figure 1-12 Can be direct or indirect.
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. The TEM, on the other hand, allows the observation of
cells w/ all organelles & inclusions, surrounded by the
components of the ECM.
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. 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. 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. Figure 1-15 A single convoluted tube will appear
histologically as several rounded structures.
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. 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. 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. 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. 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.