Methods of Histological Study

(I)

Dr. Ramada Khasawneh

2023
 Components of Tissues
Components of Tissues
Fibers

Organic and
Cells Extracellular matrix (ECM) inorganic
molecules
Water

Ground substance
PREPARATION OF TISSUES
FOR STUDY
• The most common procedure used in histologic
research is the preparation of tissue sections or
slices that can be studied with the light microscope.

• Tissues are examined visually in a beam of

transmitted light.

• Because 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.
• Most tissues studied histologically are
prepared as shown, with this sequence
of steps:
– Fixation
– Embedding & Sectioning
– Staining
 Fixation
• The main functions of fixation are:
– To avoid tissue digestion by enzymes
present within the cells (autolysis) or
bacteria
– It will prevent the protein enzymes
from functioning
– To preserve cell and tissue structure,
pieces of organs begin to be treated as
soon as possible after removal from
the body.
 Fixation
• fixation involves immersion in solutions of
stabilizing or crosslinking compounds called
fixatives.
• Because a fixative must fully diffuse through
the tissues to preserve all cells, tissues of
human sample and animals are usually cut into
small fragments before fixation to facilitate
penetration and better ensure tissue
preservation.
 Fixation
• 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.
• For the embryos of small animals (mouse)  slits made in the skin beneath
the forelimbs to allow sufficient penetration of the fixative (which mean you
can fix the whole embryo)

E15.5

Mice embryos Heart

 Fixation
• For light microscopy  a buffered
isotonic solution of 37% formaldehyde
or 4% paraformaldehyde.

• For electron microscopy  both

formaldehyde and glutaraldehyde, a
fixative often used. Also 4%
paraformaldehyde is usable for electron
microscopy.
– Osmium Tetroxide is traditionally used in
electron microscopy both as a fixative and
a heavy metal stain. Osmium Tetroxide is a
good fixative and excellent stain for lipids
in membranous structures and vesicles.
 Embedding & Sectioning
This step included the followings:
1. Dehydration
2. Clearing
3. Infiltration
4. Embedding
5. Trimming
 Embedding & Sectioning
dehydration, clearing and infiltration
• Dehydration  water is extracted from the fixed tissues by successive transfer through a
graded series of ethanol and water mixtures, usually from 50% to 100% ethanol (50%,
70% X2 – you can leave the sample in the second 70% forever, 90%, 100% X2).

• The ethanol is then replaced by histoclear which use to clear the tissue

histoclear

histoclear
50% 70% 70% 90% 100% 100%
 Embedding & Sectioning
• Tissues are embedded in a solid medium to
facilitate sectioning.
 Embedding & Sectioning
dehydration, clearing and infiltration
• Embedding materials include:
– paraffin
– plastic resins
– Optimal cutting temperature
compound (OCT compound)

• In case of paraffin
– The tissue incubated in a 1:1 solution of
histoclear : paraffin wax at 52-60 ̊C,
then placed in melted paraffin wax in an
oven at 52°-60°C
– At such temperatures the clearing
solvent evaporates and the tissue is
filled with liquid paraffin.
 Embedding & Sectioning
dehydration, clearing and infiltration

• In case of plastic resins

– Tissues to be embedded with plastic resin are also
dehydrated in ethanol and depending on the kind of
resin used subsequently infiltrated with plastic
solvents.
– Plastic embedding avoids the higher temperatures
needed for paraffin embedding, which helps avoid
shrinkage and major distortion of the tissue.
 Embedding & Sectioning
dehydration, clearing and infiltration

• In cause of OCT fixative:

– is a water-soluble blend of glycols and resins that
provides a convenient specimen matrix for
cryostat sectioning at temperatures of -10˚C and
below

– cryosections are physically less stable than

paraffin or resin embedded sections, but they are
generally superior for the preservation of
antigenicity and therefore the detection of
antigens by microscopy.

– preparation of cryosections does not involve the

dehydration steps typical of other sectioning
methods.
 Embedding & Sectioning
Embedding

• Tissue embedded in a paraffin or plastic resin

• The block that contain the tissue leave overnight at room temperature to
allowed to harden.
• The resulting paraffin block is trimmed to expose the tissue for sectioning.
 Embedding & Sectioning
Sectioning
• A hardened block containing tissue and embedding
material is placed in an instrument called a
microtome and sliced by the steel blade into
extremely thin sections.

• sections are generally cut at 1-10 μm thickness.

• The 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 colorless, and to be easily visible the
sections must be stained.

• Dyes stain tissue components more or less

selectively, with many behaving like acidic or basic
compounds.
 Staining
• Components of the cells with a net of negative charge
react with basic dyes (which are usually blue). These
components are called basophilic. Example: DNA, RNA,
Glycosaminoglycans and others.

• Components of the cells with a net positive charge

react with acidic dyes (which are usually red). These
components are called acidophilic. Example: proteins
(as in collagen fibers and mitochondria) and others.
 Staining
• Examples of basic dyes are:
– Toluidine blue  staining tissues rich in DNA and
RNA
– Alcian blue  used to stain polysaccharides such as
glycosaminoglycans in cartilages and other body
structures etc.
– Methylene blue  binds with negatively charged
part of the cell such as nucleas.
– Hematoxylin behaves like a basic dye, staining
basophilic tissue components like nuclei blue.
 Staining
• Examples of acid dyes :
– Eosin  stains the extracellular matrix and
cytoplasm pink.
– Orange G  use to stain keratin
– Acid fuchsin  used to stain cytoplasm of tissue
sections in the histology laboratory in order to
distinguish muscle from collagen.
 Staining
• Hematoxylin and eosin
(H&E) stain is used
most commonly  Hematoxyline stains
Hematoxylin produces a basic structure blue

dark blue or purple Eosin stain acidic

structures pink
color, staining DNA in
the cell nucleus. In
contrast, eosin stains
other cytoplasmic
components and
collagen pink.
 Staining
• The trichromes stain (eg,
Mallory stain, Masson
stain) besides showing
the nuclei and cytoplasm
very well, help to
distinguish extracellular
tissue components
better than H&E.
– It use three dyes to
differentiate intracellular
structures
 Staining
• Polysaccharide in animal cells can be identified
using periodic acid-Schiff (PAS) reagent.

• Lipid-rich structures of cells are best revealed with

lipidsoluble dyes such as Sudan black.

• Silver salts are a common method of visualizing

certain ECM fibers and specific cellular elements in
nervous tissue.
Microscopes
• Light Microscopy: which use the ordinary beam of
light
– Bright-Field Microscopy
– Fluorescence Microscopy
– Phase-Contrast Microscopy
– Confocal Microscopy
– Polarizing Microscopy

• Electron Microscopy: which use a narrow beam of

electrons
– Transmission Electron Microscopy
– Scanning Electron Microscopy
Light Microscopy
 Bright-Field Microscopy
• Widely used by students of histology.

• Stained preparations are examined by

means of ordinary light that passes
through the specimen.

• The maximal resolving power of the light

microscope is approximately 0.2 μm.

• Resolving power: the minimum distance

between two points that enable the device
to recognize as two points

• Images magnified 1000-1500 times.

 Fluorescence Microscopy
• In fluorescence microscopy, tissue
sections are usually irradiated by a ray of
certain wavelength, they emit an
electromagnetic wave of a longer
wavelength.

• When ultraviolet (UV) light is used, the

emission is in the visible spectrum.

• The fluorescent substances appear

brilliant on a dark background.

• Fluorescent compounds with affinity for

specific cell macromolecules may be
used as fluorescent stains.
 Confocal Microscopy
• Confocal microscopy achieves high resolution and sharp focus
by using
– small point of high-intensity light, often from a laser
– plate with a pinhole aperture in front of the image
detector
Fluorescence Microscopy Confocal Microscopy
 Phase-Contrast Microscopy

• Unstained cells and tissue

sections, which are usually
transparent and colorless, can be
studied with these modified light
microscopes.

• Phase-contrast microscopy, uses

a lens system that produces
visible images from transparent
objects and, importantly, can be
used with living, cultures cells.
 Polarizing Microscopy
• Polarizing microscopy allows the recognition
of stained or unstained structures made of
highly organized subunits.
Electron Microscopy
 Transmission Electron Microscopy
• Uses a beam of electrons instead of light
photons, which have a shorter wavelengths
than visible light.

• The transmission electron microscope (TEM)

is permits resolution around 3 nm.

• This high resolution allows magnifications of

up to 400,000 times to be viewed in detail.

• The level of magnification applies only to

isolated macromolecules or particles.

• TEM normally requires very thin sections

(40-90 nm).
 Scanning Electron Microscopy
• provides a high resolution view of
the surfaces of cells, tissues, and
organs, since the specimen is first
coated with a metal that reflects
electrons.
• The electron beam scans the
specimen from end to end
• The reflect electrons are captured
to produce a pseudo 3D image of
the coated surface