Histological Techniques 2

UNIVERSITY OF ZAMBIA
SCHOOL OF MEDICINE
• HUMAN HISTOLOGY
• HISTOLOGICAL TECHNIQUES
Dr. Mukape Mukape:

- BSc.HB, MBChB, MSc (UNZA)
2/13/20 Dr. Mukape Mukape 1

Introduction
• Histology is the study of the tissues of the body and how these tissues are
arranged to constitute organs
• Greek root histo can be translated as either “tissue” or “web,”
• Tissues are usually webs of interwoven filaments and fibres, both cellular
and noncellular, with membranous linings
• Histology involves all aspects of tissue biology, with the focus on how cells’
structure and arrangement optimize functions specific to each organ
• Tissues have two interacting components:
- Cells
- Extracellular matrix (ECM)

Introduction
• Extracellular matrices consist of:
- Protein fibers
vCollagen (most abundant of all protein fibres)
vReticular
vElastic
- Ground substance

Introduction
• ECM consists of many kinds of macromolecules, most of which form
complex structures, such as collagen fibrils and basement membranes
• It supports the cells and the fluid that transports nutrients to the cells, and
carries away their catabolites and secretory products
• Cells produce the ECM and are also influenced and sometimes controlled by
matrix molecules
• Cells and matrix interact extensively, with many components of the matrix
recognized by and attaching to cell surface receptors
• Many of these protein receptors span the cell membranes and connect to
structural components inside the cells

Introduction

Introduction
• Thus, cells and ECM form a continuum that functions together and reacts to
stimuli and inhibitors together
• The fundamental tissues of the body are each formed by several types of
cell-specific associations between cells and ECM
• These characteristic associations facilitate the recognition of the tissue types
• Organs are formed by an orderly combination of several tissues, and the
precise combination of these tissues allows the functioning of each organ
and of the organism as a whole
• The small size of cells and matrix components makes histology dependent on
the use of microscopes and molecular methods of study

Preparation of Tissues
• The most common procedure used in histologic research is the preparation
of tissue sections or slices that can be studied with the light microscope
• 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
• Thus must be sliced using a microtome to obtain thin, translucent sections
that are attached to glass slides for microscopic examination
• Ideal microscopic preparation is preserved so that the tissue on the slide
has the same structure and molecular matter
• This is seldom feasible: artifacts, distortions, and loss of components due
to the preparation process are often present

• Steps:
1. Obtain fresh specimen
2. Fixation: preservation of tissues in its original condition
3. Dehydration: removal of water from tissues
4. Clearing: infiltration of paraffin solvent
5. Embedding: infiltration of paraffin wax
6. Trimming: putting paraffin block in orderly condition by clipping, pruning
7. Sectioning: cutting thin slides with microtome
8. Staining: colouring of tissues
9. Mounting: arranging tissues on slides
Fixation
• Is the process of treating pieces of organs as soon as possible after removal
from the body with solutions of stabilizing or crosslinking compounds
called fixatives
• These fixatives avoids autolysis and preserves cell and tissue structure
• Autolysis is basically tissue digestion by enzymes present within the cells
• Because a fixative must fully diffuse through the tissues to preserve all
cells, tissues are usually cut into small fragments before fixation to
facilitate penetration and better ensure tissue preservation
• Intravascular perfusion of fixatives can be used with some organs or
laboratory animals
• Because the fixative in this case rapidly reaches the tissues through the
blood vessels, fixation is improved

Fixation
• Examples of fixatives are:
- Light microscopy: formalin, a buffered isotonic solution of 37%
formaldehyde
- Electron microscopy: glutaraldehyde
• Both formaldehyde and glutaraldehyde react with the amine groups (NH2)
of tissue proteins, preventing their degradation

Fixation
• With the greater magnification and resolution of very small structures in
the electron microscope, fixation must be done carefully to preserve
“ultrastructural” detail
• Toward that end, a double-fixation procedure, using a buffered

glutaraldehyde solution followed by immersion in buffered osmium
tetroxide, is a standard method to prepare tissue for such studies
• Osmium tetroxide preserves (and stains) membrane lipids as well as

proteins

Embedding and Sectioning
• Tissues are embedded in a solid medium to facilitate sectioning
• In order to cut very thin sections, tissues must be infiltrated after fixation
with embedding material that imparts a rigid consistency to the tissue
• Embedding materials include paraffin and plastic resins
• Paraffin is used routinely for light microscopy, resins for both light and
electron microscopy

• Paraffin embedding, or tissue impregnation, is preceded by two other main
steps: dehydration and clearing
• In dehydration, water is extracted from the fixed tissues by successive
transfer through a graded series of ethanol and water mixtures, usually
from 70% to 100% ethanol
• The ethanol is then replaced by an organic solvent miscible with both alcohol
and the embedding medium
• As the solvent infiltrates the tissues, they become more transparent
(undergo clearing)
• The fully cleared tissue is then placed in melted paraffin in an oven at 52℃-
60℃

• At such temperatures the clearing solvent evaporates and the tissue is filled
with liquid paraffin
• The impregnated tissue then hardens in a small container of paraffin at
room temperature
• Tissues to be embedded with plastic resin are also dehydrated in ethanol
• Depending on the kind of resin used, the infiltration is done with plastic
solvents
• The ethanol or solvents are later replaced by plastic solutions that harden
with the addition of cross-linking polymerizers
• Plastic embedding avoids the higher temperatures needed for paraffin
embedding, which helps avoid shrinkage and major distortion of the tissue
• A hardened block containing tissue and paraffin is placed in an instrument
called a microtome and sliced by the steel blade into extremely thin
sections
• Paraffin sections are generally cut at 1-10 μm thickness, while the glass or
diamond knives of ultramicrotomes produce sections of less than 1 μm for
electron microscopy
• Very thin sections are placed on glass slides and stained for light microscopy
or on special grids for electron microscopic staining and examination

Staining
• Most cells and extracellular material are completely colourless, and to be studied
microscopically sections must typically be stained (dyed)
• Methods of staining have been devised that not only make the various tissue
components conspicuous but also permit distinctions to be made between them
• Dyes stain tissue components more or less selectively, with many behaving like
acidic or basic compounds and forming electrostatic (salt) linkages with ionizable
radicals of molecules in tissues
• Cell components such as:
- Anionic components: e.g. nucleic acids 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
Staining
• Examples of basic dyes are toluidine blue, alcian blue, and methylene blue
• Hematoxylin behaves like a basic dye, staining basophilic tissue components
• The main tissue components that ionize and react with basic dyes do so
because of acids in their composition (DNA, RNA, and glycosaminoglycans)
• Acid dyes (eg, eosin, orange G, and acid fuchsin) stain the acidophilic
components of tissues such as mitochondria, secretory granules, and
collagen
• Of all staining methods, the simple combination of hematoxylin and eosin
(H&E) is used most commonly
• Hematoxylin produces a dark blue or purple colour, staining DNA in the cell
nucleus and other acidic structures (such as RNA-rich portions of the
cytoplasm and the matrix of cartilage)
• In contrast, eosin stains other cytoplasmic components and collagen pink
Staining

Staining
• Other dyes, such as the trichromes (eg, Mallory stain, Masson stain), are
used in more complex histologic procedures
• Trichromes, besides showing the nuclei and cytoplasm very well, help to
distinguish extracellular tissue components better than H&E
• The chemical basis of other staining procedures is more complicated than
that of the electrostatic interactions underlying basophilia and acidophilia
• DNA can be specifically identified and quantified in nuclei using the Feulgen
reaction, in which deoxyribose sugars are hydrolyzed by mild hydrochloric
acid, followed by treatment with periodic acid-Schiff (PAS) reagent
• This PAS reaction is based on the transformation of 1,2-glycol groups present
in the sugars into aldehyde residues, which then react with Schiff reagent to
produce a purple or magenta colour
Staining
• Polysaccharides constitute a heterogeneous group in tissues, occurring
either in a free state or bound to proteins and lipids
• Because of their hexose sugar content, many polysaccharides can also be

demonstrated by the PAS reaction
• A very common free polysaccharide in animal cells is glycogen, which can be

demonstrated by PAS in liver, striated muscle, and other tissues where it
accumulates
• Short branched chains of sugars (oligosaccharides) are attached to specific

amino acids of glycoproteins, making most glycoproteins PAS-positive
Staining
• Glycosaminoglycans (GAGs) are anionic, unbranched long-chain
polysaccharides containing aminated sugars (hence are proteoglycans) which
make part of ECM
• GAGs and many acidic glycoproteins do not undergo the PAS reaction, but
because of their high content of anionic carboxyl and sulfate groups, they
show a strong electrostatic interaction with alcian blue and other basic stains
• Basophilic or PAS-positive material can be further identified by enzyme

digestion, pretreatment of a tissue section with an enzyme that specifically
digests one substrate

Staining
• In many staining procedures certain structures such as nuclei become visible,
but other parts of cells remain colourfree
• In such cases a counterstain is used to give additional information
• A counterstain is usually a single stain that is applied separately to allow
better recognition of nuclei and other structures
• In H&E staining, eosin is the counterstain to hematoxylin
• Lipid-rich structures of cells are best revealed with lipid soluble dyes and
avoiding the processing steps that remove lipids such as treatment with
heat, organic solvents, or paraffin
• Typically, frozen sections are stained in alcohol solutions saturated with a
lipophilic dye such as Sudan black, which dissolves in lipid-rich structures of
cells

Staining
• Specialized methods for the localization of cholesterol, phospholipids, and
glycolipids are useful in diagnosis of metabolic diseases in which there are
intracellular accumulations of these different lipids
• In addition to staining tissues with dyes, metal impregnation
techniques usually using solutions of silver salts are a common method of
visualizing certain ECM fibers and specific cellular elements in nervous tissue
• 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, and the method of staining
• The final step before microscopic observation is mounting a protective glass
coverslip on the slide with clear adhesive

What is a Microscope?
• A microscope is an instrument used to
see objects that are too small for the
naked eye
• The science of investigating small objects
using such an instrument is called
microscopy
• Microscopic means invisible to the eye
unless aided by a microscope

Uses of Microscopes
• Cell and tissue Analysis
- It is common for histologists to study cells and tissues using the microscope
- For example, if a section of tissue is taken for analysis, histologists can use a
microscope in combination with other tools to determine if the sample is
cancerous.
• Examining Forensic Evidence
- Evidence collected at a crime scene may contain information that is not visible
to the naked eye
- For example, striations in bullets can be examined under a microscope to see
if they match bullets shot from a particular gun (ballistic fingerprinting)

Uses of Microscopes
• Determining the Health of an Ecosystem
- It is common for field biologists to monitor the health of a particular
ecosystem, such as a stream, by using microscopes to identify the number
and diversity of organisms in a particular region over time
• Studying the Role of a Protein within a Cell
- Research scientists find microscopes an invaluable tool when they study the
function of proteins within cells
- With today's technology, many proteins can be labelled with a tag and studied
in live cells
• Studying atomic structures
- Powerful microscopes such as atomic force microscopes have aided scientists
in studying the surfaces of individual atoms

Types of Microscopes
• Microscopes can be separated into several different classes
• One grouping is based on what interacts with the sample to generate the
image, i.e., light or photons (optical microscopes), or electrons (electron
microscopes)
• Alternatively, microscopes can be classed on whether they analyse the

sample via a scanning point (confocal optical microscopes, scanning
electron microscopes and scanning probe microscopes) or analyse the
sample all at once (wide field optical microscope and transmission electron
microscopes)
Types of Microscopes
• Based on what interacts with the sample to generate an image (mostly used classification):
1. Optical microscopes
Ø Conventional bright-field microscope
Ø Fluorescence microscope
Ø Phase-contrast microscope
Ø Differential interference microscope
Ø Polarizing microscope
Ø Confocal microscope
2. Electron microscopes
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)

Bright-Field Microscopy
• Widely used by students of histology
• Stained preparations are examined by means of ordinary light that passes
through the specimen
• Is composed of optical and mechanical parts to move and focus the
specimen
• The optical components consist of three systems of lenses:
1. Condenser
2. Objective
3. Eyepiece

• Condenser:
- Collects and focuses a cone of light onto the specimen to be observed
- Condenser lenses are most useful at the highest powers (400X and above)
- Microscopes with stage condenser lenses render a sharper image than those with
no lens (at 400X)
• Objective lens:
- Enlarges and projects the image of the object in the direction of the eyepiece
- Usually you will find 3 or 4 objective lenses on a microscope
- They almost always consist of 4X, 10X, 40X and 100X powers
- When coupled with a 10X (most common) eyepiece lens, we get total magnifications
of 40X (4X times 10X), 100X , 400X and 1000X

• Eyepiece or ocular lens:
- Further magnifies this image and projects it onto the viewer’s retina or a
charge-coupled device (CCD) highly sensitive to low light levels with a
monitor and camera
- They are usually 10X or 15X power
• The total magnification is obtained by multiplying the magnifying power

of the objective and ocular lenses

• The critical factor in obtaining a crisp, detailed image with a light microscope is its
resolving power
• Resolving power is 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 0.2μm, a

power that 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 structures such as mitochondria will be seen as only one object if
they are separated by less than 0.2 μm
• The quality of the image—its clarity and richness of detail—depends on the
microscope’s resolving power
• Magnification is of value only when accompanied by high resolution
• Resolving power of a microscope depends mainly on the quality of its objective lens
• Eyepiece lens enlarges only the image obtained by the objective; it does not
improve resolution
• For this reason, when objectives of different magnifications are compared, those
providing higher magnification also have higher resolving power

Mechanical parts
• Tube: Connects the eyepiece to the objective lenses
• Arm: Supports the tube and connects it to the base
• Base: The bottom of the microscope, used for support
• Illuminator: A steady light source (110 volts)
• Stage: The flat platform where you place your slides. Stage clips hold the
slides in place. A microscope with a mechanical stage, enables one to move
the slide around by turning two knobs. One moves it left and right, the other
moves it up and down.
• Revolving Nosepiece or Turret: This is the part that holds two or more
objective lenses and can be rotated to easily change power.

• Rack Stop:
- This is an adjustment that determines how close the objective lens can get to the
slide
- You would only need to adjust this if you were using very thin slides and you weren't
able to focus on the specimen at high power
• Diaphragm or Iris:
- Many microscopes have a rotating disk under the stage
- This diaphragm has different sized holes and is used to vary the intensity and size of
the cone of light that is projected upward into the slide
- There is no set rule regarding which setting to use for a particular power
- Rather, the setting is a function of the transparency of the specimen, the degree of
contrast you desire and the particular objective lens in use
• How to Focus The Microscope:
- The proper way to focus a microscope is to start with the lowest power
objective lens first and while looking from the side, crank the lens down as
close to the specimen as possible without touching it
- Now, look through the eyepiece lens and focus upward only until the image is
sharp
- If you can't get it in focus, repeat the process again
- Once the image is sharp with the low power lens, you should be able to simply
click in the next power lens and do minor adjustments with the focus knob
- If your microscope has a fine focus adjustment, turning it a bit should be all
that's necessary
- Continue with subsequent objective lenses and fine focus each time
• Digital cameras highly sensitive to light enhance the power of the bright-field and
other light microscopes by allowing the capture of images suitable for quantitative
analysis and immediate printing
• The frontiers of light microscopy have been redefined by the use of digital cameras,
and image-enhancement programs (eg, to improve contrast) allow objects that may
not be directly visible through the eyepieces to be analysed on the video screen
• Such systems are also useful for studying living cells for long periods of time because
they use low-intensity light that avoids damaging the cells with heat from more
intense illumination
• Software developed for image analysis allows rapid measurements and quantitative
study of microscopic structures
Fluorence Microscopy
• When certain cellular substances are irradiated by light of a proper wavelength,
they emit light with a longer wavelength—a phenomenon called fluorescence
• In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet

(UV) light and the emission is in the visible portion of the spectrum
• The fluorescent substances appear brilliant on a dark background
• For this method, the microscope has a strong UV light source and special filters that
select rays of different wavelengths emitted by the substances
• Fluorescent compounds with affinity for specific cell macromolecules may be used
as fluorescent stains

• Acridine orange, which binds both DNA and RNA, is an example
• When observed in the fluorescence microscope, these nucleic acids emit slightly
different fluorescence, allowing them to be localized separately in cells
• Other compounds such as DAPI and Hoechst stain specifically bind DNA and are
used to stain cell nuclei, emitting a characteristic blue fluorescence under UV
• Another important application of fluorescence microscopy is achieved by coupling

compounds such as fluorescein to molecules that will specifically bind to certain
cellular components and thus allow the identification of these structures under the
microscope
• Antibodies labelled with fluorescent compounds are extremely important in

immunohistologic staining
Phase-Contrast Microscopy
• Unstained cells and tissue sections, which are usually transparent and
colourless, can be studied with these modified light microscopes
• Cellular detail is normally difficult to see in unstained tissues because all parts
of the specimen have roughly similar optical densities
• Phase-contrast microscopy, however, uses a lens system that produces visible

images from transparent objects and, importantly, can be used with living,
cultures cells

• Phase-contrast microscopy is based on the principle that light changes its speed
when passing through cellular and extracellular structures with 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
• Because they allow the examination of cells without fixation or staining, phase-
contrast microscopes are prominent tools in all cell culture laboratories
• A modification of phase-contrast microscopy is differential interference

microscopy with Nomarski optics, which produces an image of living cells with a
more apparent three-dimensional all cell culture laboratories


Confocal Microscopy
• With a regular bright-field microscope, the beam of light is relatively large and
fills the specimen
• Stray (excess) light reduces contrast within the image and compromises the
resolving power of the objective lens
• Confocal microscopy avoids these problems and achieves high resolution and
sharp focus by using a:
1. Small point of high-intensity light, often from a laser
2. Plate with a pinhole aperture in front of the image detector

Confocal Microscopy

Confocal Microscopy
• The point light source, the focal point of the lens, and the detector’s pinpoint
aperture are all optically conjugated or aligned to each other in the focal
plane (confocal), and unfocused light does not pass through the pinhole
• This greatly improves resolution of the object in focus and allows the
localization of specimen components with much greater precision than with
the bright-field microscope
• Confocal microscopes include a computer-driven mirror system (the beam
splitter) to move the point of illumination across the specimen automatically
and 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
• Creating such allows them to be digitally reconstructed into a 3D image

Polarizing Microscopy
• Polarizing microscopy allows the recognition of stained or unstained structures made of
highly organized subunits
• When normal light passes through a polarizing filter, it exits vibrating in only one
direction
• If a second filter is placed in the microscope above the first one, with its main axis
perpendicular to the first filter, no light passes through
• If, however, tissue structures containing oriented macromolecules are located between
the two polarizing filters, their repetitive structure rotates the axis of the light emerging
from the polarizer and they appear as bright structures against a dark background
• The ability to rotate the direction of vibration of polarized light is called

birefringence and is a feature of crystalline substances or substances containing highly
oriented molecules, such as cellulose, collagen, microtubules, and actin filaments

Electron Microscopy
• Two types:
1. Transmission electron microscope (TEM)
2. Scanning electron microscope (SEM)
• Transmission and scanning electron microscopes are based on the interaction

of tissue components with beams of electrons
• The wavelength in the electron beam is much shorter than that of light,
allowing a 1000-fold increase in resolution

Electron Microscopy

Electron Microscopy

Transmission Electron Microscopy
• Is an imaging system that theoretically permits very high resolution (0.1 nm)
• Passes electrons through the sample, analogous to basic optical microscopy
• In practice, however, the resolution obtained by most good instruments is
around 3 nm
• This high resolution allows magnifications of up to 400,000 times to be
viewed in detail
• Unfortunately, this level of magnification applies only to isolated
macromolecules or particles
• Very thin tissue sections can be observed with details at magnifications of up
to about 120,000 times


• TEM a metallic filament cathode emits electrons that move toward an
anode, a metal plate with a central hole that forms a beam of electrons
passing through it
• The voltage difference between cathode and anode can be varied between
roughly 60 and 120 kV, producing electron beams of different wavelengths
• Electrons transmitted through the specimen reach the objective lens, which
forms a focused, magnified image that is then magnified further through
other lenses and captured on a viewing screen

Scanning Electron Microscopy
• Scanning electron microscopy (SEM) provides a high resolution view of the surfaces
of cells, tissues, and organs
• Like the TEM, this microscope produces and focuses a very narrow beam of
electrons, but in this instrument the beam does not pass through the specimen
• Instead, the surface of the specimen is first dried and spray-coated with a very thin
layer of heavy metal (often gold) through which electrons do not pass readily
• When the beam is scanned from point to point across the specimen, it interacts
with the metal atoms and produces reflected electrons or secondary electrons
emitted from the metal
• These are captured by a detector, and the resulting signal is processed to produce a
black-and-white image on a monitor
• SEM images are usually easy to interpret because they present a 3D view that
appears to be illuminated from above, in the same way that large objects are seen
with highlights and shadows caused by light from above
Scanning Electron Microscopy

Scanning Electron Microscope

The End!

Histological Techniques 2

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UNIVERSITY OF ZAMBIA

Dr. Mukape Mukape:

2/13/20 Dr. Mukape Mukape 1

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• Extracellular matrices consist of:

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• Toward that end, a double-fixation procedure, using a buffered

• Osmium tetroxide preserves (and stains) membrane lipids as well as

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• Embedding materials include paraffin and plastic resins

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• Because of their hexose sugar content, many polysaccharides can also be

• A very common free polysaccharide in animal cells is glycogen, which can be

• Short branched chains of sugars (oligosaccharides) are attached to specific

• Basophilic or PAS-positive material can be further identified by enzyme

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• Alternatively, microscopes can be classed on whether they analyse the

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• The total magnification is obtained by multiplying the magnifying power

2/13/20 Dr. Mukape Mukape 32

• The maximal resolving power of the light microscope is approximately 0.2μm, a

• Objects smaller or thinner than 0.2 μm (such as a ribosome, a membrane, or a

• Magnification is of value only when accompanied by high resolution

2/13/20 Dr. Mukape Mukape 34

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• In fluorescence microscopy, tissue sections are usually irradiated with ultraviolet

• The fluorescent substances appear brilliant on a dark background

2/13/20 Dr. Mukape Mukape 41

• Another important application of fluorescence microscopy is achieved by coupling

• Antibodies labelled with fluorescent compounds are extremely important in

• Phase-contrast microscopy, however, uses a lens system that produces visible

2/13/20 Dr. Mukape Mukape 43

• A modification of phase-contrast microscopy is differential interference

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• The ability to rotate the direction of vibration of polarized light is called

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• Transmission and scanning electron microscopes are based on the interaction

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