histology best note

1. Introduction to Histology
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2. Introduction
• Histology is the study of the tissues of the body and
how these tissues are arranged to constitute organs
• Greek: histo = "tissue" or "web"
• Histology involves all aspects of tissue biology, with the
focus on how cells' structure and arrangement optimize
functions specific to each organ
• A tissue is a group of similar cells that have a common
origin and function together to carry out specialized
activities
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3. • Tissues do not consist entirely of cells
– between the living cells is nonliving
extracellular material
• Tissues are made of two interacting components:
cells and extracellular matrix
• There are 4 principal types of tissues
– epithelial tissue
– connective tissue
– muscle tissue
– nervous tissue
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4. • Each of the fundamental tissues is formed by
several types of cells and typically by specific
associations of cells and extracellular matrix
• Tissues are organized into organs
– Most organs are formed by an orderly
combination of several tissues, but have one
predominant tissue
– The arrangement and proportion of tissues
present determines the function of the organ
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5. • The small size of cells and matrix components
makes histology dependent on the use of
microscopes
• Advances in chemistry, molecular biology,
physiology, immunology, and pathology—and the
interactions among these fields—are essential
for a better knowledge of tissue biology
• Familiarity with the tools and methods of any
branch of science is essential for a proper
understanding of the subject
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6. Preparation of Tissues for Study
• The most common procedure used in the study
of tissues is the preparation of histological
sections or tissue slices that can be studied
with the aid of the light microscope
• Because tissues and organs are usually too
thick for light to pass through them, they must
be sectioned to obtain thin, translucent
sections and then attached to glass slides
before they can be examined
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7. • The ideal microscope tissue preparation
should be preserved so that the tissue on
the slide has the same structure and
molecular composition as it had in the
body
• However, as a practical matter this is
seldom feasible and artifacts, distortions,
and loss of components due to the
preparation process are almost always
present
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8. The basic steps used in tissue preparation for
histology
8

9. 1.Fixation: Small pieces of fresh tissue are place
d in fixative solutions which generally cross-link
proteins, inactivating degradative enzymes and p
reserving cell structures
2.Dehydration: The fixed pieces then undergo de
hydration by being transferred through a series
of increasingly more concentrated alcohol soluti
ons, ending in 100% which effectively removes al
l water from the tissue
3.Clearing: The alcohol is then removed in a cleari
ng solution miscible in both alcohol and melted p
araffin
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10. 4. Infiltration: When the tissue is then placed i
n melted paraffin at 58°C it becomes compl
etely infiltrated with this substance
5. Embedding: After infiltration the tissue is pl
aced in a small mold containing melted paraffi
n, which is then allowed to harden. The result
ing paraffin block is trimmed to expose the ti
ssue for sectioning (slicing)
6. Sectioning: The hard blocks containing the ti
ssues are then placed in an instrument called
a microtome and are sliced by the microtome'
s steel or glass blade into sections 1 to 10 mic
rometers thick
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11. A microtome is used for sectioning paraffin-embedded tissues for light microscopy.
After mounting a trimmed block with the tissue specimen, rotating the drive wheel
moves the tissue-block holder up and down. Each turn of the drive wheel advances the
specimen holder a controlled distance, generally between 1 and 10 µm, and after each
forward move the tissue block passes over the steel knife edge, which cuts the sections
at a thickness equal to the distance the block advanced.
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12. • The sections are floated on water and
then then adhered to glass slides,
deparaffinized, and stained for
microscopic examination
12

13. • 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) and at the same time
become hard and thus ready to be sectioned
• A freezing microtome— the cryostat—is then used to
section the frozen block with tissue
• Because this method allows the rapid preparation of
sections without going through the long embedding
procedure described above, it is routinely used in
hospitals to study specimens during surgical procedures
• 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
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14. Staining
• 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
• The dyes stain tissue components more or less
selectively
• Most of these dyes behave like acidic or basic
compounds and have a tendency to form electrostatic
(salt) linkages with ionizable radicals of the tissues
• Tissue components with a net negative charge (anionic)
stain more readily with basic dyes and are termed
basophilic; cationic components, such as proteins with
many ionized amino groups, have affinity for acidic
dyes and are termed acidophilic
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15. • Examples of basic dyes are toluidine blue, alcian
blue, and methylene blue
• Hematoxylin behaves like a basic dye, that is, it
stains the basophilic tissue components
• The main tissue components that ionize and
react with basic dyes do so because of acids in
their composition (nucleic acids,
glycosaminoglycans, and acid glycoproteins)
• Acid dyes (eg, orange G, eosin, acid fuchsin)
stain the acidophilic components of tissues such
as mitochondria, secretory granules, and
collagen
• Of all dyes, the simple combination of
hematoxylin and eosin (H&E) is used most
commonly
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16. • Hematoxylin stains DNA of the cell nucleus and other
acidic structures (such as RNA-rich portions of the
cytoplasm and the matrix of cartilage) blue
• In contrast, eosin stains other cytoplasmic components
and collagen pink
• Many other dyes, such as the trichromes (eg, Mallory
stain, Masson stain), are used in different histologic
procedures
• The trichromes, besides showing the nuclei and
cytoplasm very well, help to distinguish extracellular
tissue components better than H&E
• A good technique for differentiating collagen is the use
of picrosirius
• In addition to tissue staining with dyes, metal
impregnation techniques usually using silver salts are a
common method of visualizing certain ECM fibers and
specific cellular elements in nervous tissue
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17. Hematoxylin & Eosin (H&E) and Periodic acid-Schiff (PAS) staining.
Micrographs of the columnar epithelium lining the small intestine. (a):
Micrograph stained with hematoxylin and eosin (H&E). With H&E, basophilic
cell nuclei are stained purple while cytoplasm stains pink. Cell regions with
abundant oligosaccharides on glycoproteins, such as the apical ends of the
cells or the scattered mucus-secreting goblet cells in the layer are poorly
stained. 17

18. (b): Micrograph stained by the 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) and
in the mucin-rich secretory granules of goblet cells. Cell surface glycoproteins
and mucin are PAS-positive due to their high content of oligosaccharides and
polysaccharides. The PAS-stained tissue was counterstained with hematoxylin
to show the cell nuclei. Both X300.
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19. • The whole procedure, from fixation to observing
a tissue in a light microscope, may take from 12
hours to 2 1/2 days, depending on the size of
the tissue, the fixative, the embedding medium,
and the method of staining
• The final step before observation is mounting a
protective glass cover slip on the slide with
adhesive mounting media
19

20. Light Microscopy
• Types
– Conventional bright-field microscopy
– Fluorescence
– Phase-contrast
– differential interference
– Confocal
– Polarizing microscopy
• All are based on the interaction of light
and tissue components and can be used
to reveal and study tissue features
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21. Bright-Field Microscopy
• With the bright-field microscope, widely used,
stained preparations are examined by means
of ordinary light that passes through the
specimen
• The microscope is composed of mechanical and
optical parts
• The optical components consist of three
systems of lenses
– The condenser collects and focuses light, producing
a cone of light that illuminates the object to be
observed
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22. – The objective lenses enlarge and project the
illuminated image of the object in the
direction of the eyepiece
• For routine histological studies objectives having three
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; and
X40 for high magnification of more detailed areas.
– The eyepiece or ocular lens further
magnifies this image (X10) and projects it
onto the viewer's retina, or photographic film
of a camera
• The total magnification is obtained by
multiplying the magnifying power of the
objective and ocular lenses 22

23. 23

24. • The critical factor in obtaining a detailed image
with a light microscope is its resolving power,
defined as the smallest distance between two
particles at which they can be seen as separate
objects
• The maximal resolving power of the light
microscope is approximately 0.2 µm; this power
permits good images magnified 1000–1500
times
• Objects smaller or thinner than 0.2 µm (such as
a ribosome, a membrane, or a filament of actin)
cannot be distinguished with this instrument
• Likewise, two objects such as mitochondria will
be seen as only one object if they are
separated by less than 0.2 µm
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25. • The quality of the image—its clarity and
richness of detail—depends on the
microscope's 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
• 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 magnification also have higher resolving
power 25

26. Problems in the Study of Tissue
Sections
• Artifacts
– Microscope preparations are the end result of a
series of processes that began with collecting the
tissue and ended with mounting a coverslip on the
slide
– Several steps of this procedure may distort the
tissues, producing minor structural abnormalities
called artifacts
– Structures seen microscopically then may differ
slightly from the structures present when they
were alive
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27. – Sources of artifact
• Shrinkage: minor shhrinkage 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 and other tissue
components
• Loss of molecules: molecules such as lipids,
glycogen, or low molecular weight substances are
not kept in the tissues by the fixative or removed
by the dehydrating and clearing fluids
• Cracks: slight cracks in sections also appear as
large spaces in the tissues
• Wrinkles: of the section may be confused with
linear structures such as blood capillaries
• Precipitates: of stain may be confused with cellular
structures such as cytoplasmic granules
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28. • It is impossible to differentially 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
tissue's complete composition and structure
– The TEM, on the other hand, allows the
observation of cells with all organelles and
inclusions, surrounded by the components of
the ECM
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29. • Interpretation of 3-D structures in 2-D
tissue sections
– When a three-dimensional tissue volume is cut
into very thin sections, the sections appear
microscopically to have only two dimensions:
length and width
– 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
– Round structures seen microscopically may be
sections through spheres or cylinders and
tubes in cross-section look like rings
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30. – Also since structures within a tissue have
different orientations, their two-dimensional
appearance will vary depending on the plane of
section
• A single convoluted tube will appear
histologically as several rounded structures
• To understand the architecture of an organ,
one often must study sections made in
different planes
• Examining many parallel sections (serial
sections) and reconstructing the images
three-dimensionally provides better
understanding of a complex organ or
organism 30

31. Sections through a hollow swelling on a tube produce large
and small circles, oblique sections through bent regions of
the tube produce ovals of various dimensions. 31

32. A single section through a highly coiled tube shows many small,
separate round or oval sections. 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. 32

33. 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. 33