histology aid for medical students

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2. microscopy
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3. Definition
▪ Tissues - Group of similarly
specialized cells and the
intercellular substance
▪ Tissues - building blocks of ;
– Organs (e.g. kidney, liver, ovary)
– Functional systems (e.g. digestive
system, urinary system, reproductive
system, etc.).
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4. Definition
• Greek root, histos, meaning “web” (tissue).
• Histology (microscopic anatomy) is that
branch of anatomy that studies tissues.
• No longer merely deals with the structure of
the body; it also concerns itself with the
body's function.
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5. Definition
• Histology is a science devoted to a detailed
study of the microscopic organization,
appearance, and function of cells, tissues, and
organs, as well as the fluids and extracellular
macromolecules found in the human body.
• Histology is the study of normal cells and
tissues, mainly using microscopes.
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6. ❑ Study of the tissues of the body and how these
tissues are arranged to constitute organs.
❑Organs= orderly and precisely combined several tissues
➢Provide a basis for understanding function and
abnormal changes that may occur.
Definition
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7. Introduction
• Tissues have two interacting components:
– cells and extracellular matrix (ECM).
• The ECM
– consists macromolecules (collagen fibrils and
basement membranes)
– supports the cells
– transports nutrients to and from the cells
• The cells
– Produce the ECM
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8. Introduction
• Cells and matrix interact extensively
– functions together
– reacts to stimuli and inhibitors together
• Several types of associations exist between
them
• Characteristics associations between them helps
to recognize tissue types.
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9. • Histology dependent on the use of microscopes
and molecular methods of study.
– Small size of structures
• Familiarity with the tools and methods of any
branch of science is essential for a proper
understanding of the subject.
• Advances in biochemistry, molecular biology,
physiology, immunology, and pathology are
essential for a better knowledge of tissue biology.
Introduction
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10. There are four basic types of tissues with a
specific function:
– Epithelial tissue – protection
– Connective tissue – support
– Muscular tissue – contraction
– Nervous tissue – conduction
HISTOLOGICAL TECHNIQUES
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11. For Light Microscopy
• Micrometer (micron)= (μm)
• 1 micrometer = 0.001 mm or 10 –6 m.
For Electron Microscopy
• Nanometer (nm) or angstrom (A°).
• 1 nanometer = 0.001 μm or 10 –9 m.
• 1 angstrom = 0.1 nm, 10−4 μm or 10 –10 m.
HISTOLOGICAL TECHNIQUES
Units of measurement used in histology
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12. INTERPRETATION OF A SECTION
• A section is a slice cut through parts of 3D
objects
• Histologic sections- thin, flat slices of fixed and
stained tissues or organs mounted on glass
slides.
• A section under microscope - is two-dimensional
image
HISTOLOGICAL TECHNIQUES
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13. INTERPRETATION OF A SECTION
• Often difficult to interpret the orientation of
structures in sectional view (either transversely or
longitudinally)
– the cells, fibres and tubes found in random orientation.
– shape and size may vary
– Variation in the appearance- different angle of the
plane of section
➢In order to comprehend, study sections cut in
different planes.
HISTOLOGICAL TECHNIQUES
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14. Planes of Section of a Round Object
e.g., in hard-boiled egg
Longitudinal
Transverse
Tangential
Tangential
Longitudinal
Transverse
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15. Planes of Section of a Tube
Most easily recognized when they are cut in transverse sections.
E.g., blood vessel, duct, or glandular structure
longitudinal
Transverse
Transverse
Tangential
Oblique
Transverse
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16. Section through a single coiled tube
E.g., Testes & kidneys consist- highly twisted or convoluted tubule
➔May appear as sections of many separate tubes
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17. Sections through a solid ball (A) and sections through a
solid cylinder (B) may be very similar.
(A)
(B)
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18. Preparation of Tissues
Four different approaches for microtechnique:
• Fresh preparation
– e.g., blood smear, connective tissue spread.
Fixed preparation
– e.g., paraffin, celloidin
• Frozen preparation
– e.g., frozen section
• Special techniques
– e.g., tissue culture, radioautography, in situ hybridization and
cell fractionation.
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19. • Most common procedure
• Under the light microscope, tissues are
examined visually in a beam of transmitted
light.
• Most tissues and organs are too thick for
light to pass through them
• They must be sliced to obtain thin,
translucent sections that are attached to
glass slides for microscopic examination
Preparation of Tissues for Light Microscopy
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20. Preparation of Tissues for Light Microscopy
• Tissues are processed by the following
procedure to obtain thin translucent sections
Tissue acquisition: Biopsy, surgical resection
– Purpose: Sampling tissue to examine
microscopically.
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21. Fixation: Placing tissue samples in a fixative
to get permanent section
– Formalin (4% formaldehyde) and Bouin's fluid.
• cross-link proteins, maintaining a lifelike image of the
tissue
– Mercuric chloride, acetic acid, picric acid and
glutaraldehyde
Preparation of Tissues for Light Microscopy
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22.  Fixation: Placing tissue samples in a fixative
➢Stopping tissue degradation, killing
microorganisms
• Preserve the morphology and chemical composition of
the tissue
• To preserve tissue’s ultrastructural detail (double
fixation)
• Prevent autolysis and putrefaction
• harden the tissue for easy manipulation
• solidify colloidal material, and
• influence staining.
Preparation of Tissues for Light Microscopy
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23.  Fixation: Placing tissue samples in a fixative
A combination of fixatives is often prepared to
get the maximum desirable effect. commonly
used are:
– Bouin’s fluid (formalin, acetic acid and picric acid)
– Formal sublimate (formalin and mercuric chloride)
– Helly’s fluid (formalin, mercuric chloride and
potassium dichromate)
– Zenker’s fluid (acetic acid, mercuric chloride and
potassium dichromate)
Preparation of Tissues for Light Microscopy
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24.  Fixation: Placing tissue samples in a fixative
– After fixation, some hard tissues which contain
large amount of calcium salts (bone and tooth)
require decalcification.
– makes the hard tissues soft, easy to be cut
– Decalcifying agents
• 10% nitric acid,
• 5% trichloroacetic acid and
• Ethylene diamine tetra acetic acid (EDTA).
Preparation of Tissues for Light Microscopy
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25. ❷ Dehydration
• Gradual removal of water from the tissues by
immersing in ascending grades of alcohol, viz.
50%, 70%, 90% and absolute alcohol
• Tissue remains in each of these grades for 30–
60 minutes.
Preparation of Tissues for Light Microscopy
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26. ❸ Clearing
• Tissue is treated with a paraffin solvent
(clearing agent) like xylene or toluene for 2-3
hours.
• These agents penetrate and replace the
alcohol from the tissue and make it
translucent (clear).
Preparation of Tissues for Light Microscopy
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27. ❹ Embedding ; Placing tissue into a hardening
agent (paraffin) in a tissue block
– infiltrated
– thin sections obtained with microtome,
• embedding media for light microscopy include;
– paraffin wax, celloidin, gelatin, plastic resins (for EM),
etc. Paraffin is the routinely used
• Involves two steps
– Impregnation
– casting or block making.
Preparation of Tissues for Light Microscopy
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28. Tissue embedding in melted paraffin wax
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29. Tissue embedding
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30. ❺ Section Cutting (Microtomy)
• Slicing the tissue into thin sections (1–10 μm)
with microtome.
– a machine equipped with a blade and an arm that
advances the tissue block in specific equal
increments
Preparation of Tissues for Light Microscopy
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31. Types of microtomes
①Standard microtome
– For paraffin sections
only
②Rotary (international)
microtome
– For paraffin and
celloidin
③Sliding microtome
– For celloidin and frozen
sections
④Freezing microtome
(cryostat)
⑤Ultramicrotome. 34
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32. ❺ Section Cutting (Microtomy)
• The cut paraffin sections are affixed to
albuminised glass microslides after flattening
the sections over warm water.
• The microslides with sections are either air
dried or dried in an incubator overnight at 37
°C and stored for staining at room
temperature.
Preparation of Tissues for Light Microscopy
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34. Unstained section on glass slide
Unstained slides in drying oven
Picking sections up from water bath
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35. ❻ Staining
• Most tissues are colorless
• Methods of staining make tissue components
conspicuous and also permit distinctions
between them.
• Staining is done routinely by using a basic and
an acidic dye that stain tissue components
selectively.
Preparation of Tissues for Light Microscopy
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36. ❻ Staining
• Tissue components that stain more readily
with basic dyes are termed basophilic (blue in
color)
– Haematoxylin, Toluidine blue and Methylene blue
• Tissue with an affinity for acid dyes are termed
acidophilic (pink/orange in colour.)
– Eosin, Orange G and Acid Fuchsin.
Preparation of Tissues for Light Microscopy
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37. ❻ Staining procedure
• Combination of Haematoxylin and Eosin (H&E)
is most commonly used
• Special stains like periodic acid Schiff reagent
(PAS), osmic acid, Mallory and Masson’s
trichrome stains are being used to selectively
identify certain tissue components.
Preparation of Tissues for Light Microscopy
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38. a) Deparaffinization
• To remove the paraffin from the section, the slides
are treated with xylol.
• Three changes are necessary, each for 3–5 minutes.
b) Hydration
• The slides are passed through the following series
to hydrate the sections:
– Absolute alcohol – 5 min (with 2 changes)
– 90% alcohol – 3 min
– 70% alcohol – 3 min
– 50% alcohol – 3 min
– [Wash in] Distilled water – 3 min
Preparation of Tissues for Light Microscopy
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39. C) Staining
• For differential staining (the commonly used
technique), following steps are involved:
• A staining with haematoxylin for 5–7 minutes.
• Washing well in running tap water until the section
becomes blue.
• Differentiation with 1% acid alcohol for 5 seconds.
• Washing in running tap water again, until the
section becomes blue.
• Staining with 1% eosin for 1 minute
Preparation of Tissues for Light Microscopy
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40. d) Dehydration
• The stained sections are dehydrated in the following
series:
– 50% alcohol – 10 sec
– 70% alcohol – 10 sec
– 90% alcohol – 30 sec
– Absolute alcohol – 5 min (with 2 changes)
e) Clearing and Mounting
– The final step before microscopic observation
– The sections are cleared in xylene
– Mounting a protective glass coverslip on the slide
Preparation of Tissues for Light Microscopy
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41. Cover slipping
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42. Interpretation of Structures Prepared by
Different Types of Stains
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43. Hematoxylin and
Eosin Stain
• Nuclei stain
blue
• Cytoplasm
stains pink or
red
• Collagen fibers
stain pink
• Muscles stain
pink
Interpretation of Structures Prepared by
Different Types of Stains
(A) Hematoxylin & (B)Eosin
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44. Other naturally occurring pigments in cells
• Melanin: Black-brown pigment
– E.g., in keratinocytes of the skin
• Lipofuscin: Yellow-brown pigment
– E.g., accumulate in cells such as cardiomyocytes,
neurons, and hepatocytes.
▪ Thought to be the residues of lysosomes
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45. Problems in the Interpretation of
Tissue Sections
1- Distortions & Artifacts Caused by Tissue Processing
– The shrinkage
• produced by the fixative, by the ethanol, and
• by the heat needed for paraffin embedding
– Artificial spaces
• shrinkage RESULTS the appearance of artificial spaces
between tissue components.
• ALSO loss of molecules (Glycogen and lipids )
Artificial spaces and other distortions caused by
preparation procedure are called Artifacts
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46. Problems in the Interpretation of
Tissue Sections
 Artifacts:
– Any artificial structures, defects, or observations
– introduced during preparatory steps
– not naturally present in vivo
– include
• dust particles
• separation or folding of tissue slice
• wrinkles of the section (which may be confused with blood
capillaries)
• precipitates of stain (which may be confused with cytoplasmic
granules)
• exaggeration of spaces between cells and tissues, and
• empty space effect in previously lipid-filled areas
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47. Problems in the Interpretation of
Tissue Sections
2- Totality of the Tissue
• impossibility to differentially staining all tissue
components on only one slide.
– Necessary to examine several preparations, each
one stained by a different method,
• However, transmission electron microscope,
allows the observation of a cell with all its
organelles and inclusions surrounded by the
components of the extracellular matrix.
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48. Problems in the Interpretation of
Tissue Sections
3- Two Dimensions & Three Dimensions
– When a three-dimensional volume is cut into very
thin sections, the sections seem to have only two
dimensions: length and width.
⧫Because many structures are thicker than the section
• Should imagine that something may be missing in front of
or behind
⧫ Remember that the structures within a tissue are
usually sectioned randomly
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