INTRODUCTION TO
HISTOLOGY & CELL
STUDY METHODS
Dr. Samah Rjoub
Basic Terminology
• Histology :
– Histos or hist- is a prefix used in medical terminology
that refers to body tissue ( cells and ECM)
– logy - study of
• =study of body tissue.
PREPARATION OF TISSUES FOR STUDY
sequence of tissue preparation steps
• ■ Fixation: Small pieces of tissue are placed in solutions
of chemicals that cross-link proteins and , inactivating
enzymes, and preventing cell autolysis or self-digestion.
which preserve cell and tissue structure.
• ■ Dehydration: The tissue is transferred through a series
of increasingly concentrated alcohol solutions, ending in
100%, which removes all water.
Dehydration
• ■ Clearing: Alcohol is removed in organic solvents in which
both alcohol and paraffin are miscible
• ■ Infiltration: The tissue is then placed in melted paraffin until
it becomes completely infiltrated with this substance.
• ■ Embedding: The paraffin-infiltrated tissue is placed in a
small mold with melted paraffin and allowed to harden
• ■ Trimming: The resulting paraffin block is trimmed to expose
the tissue for sectioning (slicing) on a microtome.
Trimming
• (b) A microtome is used for sectioning paraffin-embedded
tissues for light microscopy.
• The trimmed tissue specimen is mounted in the paraffin block
holder, and each turn of the drive wheel by the histologist
advances the holder a controlled distance, generally from 1 to
10 μm.
• After each forward move, the tissue block passes over the
steel knife edge and a section is cut at a thickness equal to
the distance the block advanced. The paraffin sections are
placed on glass slides and allowed to adhere, deparaffinized,
and stained for light microscope study.
• . ■ Sections are mounted on glass slides for staining,
which is required to reveal specific cellular and tissue
components with the microscope.
• ■ The most commonly used staining method is a
combination of the stains H&E, which act as basic and
acidic dyes, respectively.
• ■ Cell substances with a net negative (anionic) charge,
such as DNA and RNA, react strongly with hematoxylin
and basic stains; such material is said to be “basophilic.”
• ■ Cationic substances, such as collagen and many
cytoplasmic proteins react with eosin and other acidic
stains and are said to be “acidophilic
•
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Infiltration and embedding
Drive wheel
Block holder
Parafiin block
Tissue
Steel knife
Staining
• Staining Most cells and extracellular material are
completely colorless
• Methods of staining have been devised that make various
tissue components not only conspicuous but also
distinguishable from one another.
• Cell components, such as nucleic acids with a net negative
charge (anionic), have an affinity for basic dyes and are
termed basophilic
• Cationic components, such as proteins with many ionized
amino groups, stain more readily with acidic dyes and are
termed acidophilic.
• Examples of basic dyes include 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.
• the simple combination of hematoxylin and eosin (H&E) is used
most commonly.
• Hematoxylin stains DNA in the cell nucleus, RNA-rich portions
of the cytoplasm, and the matrix of cartilage, producing a dark
blue or purple color.
• In contrast, eosin stains other cytoplasmic structures and
collagen pink.
• More complex procedures, such as trichrome stains (eg,
Masson’s trichrome), allow greater distinctions among various
extracellular tissue components.
• The periodic acid–Schiff (PAS) reaction utilizes the hexose
rings of polysaccharides and other carbohydrate-rich tissue
structures and stains such macromolecules distinctly purple or
magenta.
H&EStaining
Hematoxyline stains
basic structure blue
Eosin stain acidic
structures pink
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
Light Microscopy
• Light Microscopy :Bright-field microscopy, the method most
commonly used by both students and pathologists, uses
ordinary light and the colors are imparted by tissue staining
• Fluorescence 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 bright on a dark background
• Phase-contrast microscopy :Unstained cells and tissue
sections, which are usually transparent and colorless, can
be studied with these modified light [Link]-
contrast microscopy is based on the principle that light
changes its speed when passing through cellular and
extracellular structures with different refractive indices.
• Confocal microscopy using (1) a small point of high
intensity light, often from a laser and (2) a plate with a
pinhole aperture in front of the image detector. 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.
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
Electron Microscopy
• Uses a beam of electrons instead of light photons, which
have a shorter wavelengths than visible light.
•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
*AUTORADIOGRAPHY :Microscopic autoradiography is a
method of localizing newly synthesized macromolecules in
cells or tissue sections. Radioactively labeled metabolites
(nucleotides, amino acids, sugars) provided to the living
cells are incorporated into specific macromolecules (DNA,
RNA, protein, glycoproteins, and polysaccharides) and emit
weak radiation that is restricted to those regions where the
molecules are located.
*ENZYME HISTOCHEMISTRY :Enzyme histochemistry (or
cytochemistry) is a method for localizing cellular structures
using a specific enzymatic activity present in those
structures.
*Immunohistochemistry :A highly specific interaction
between macromolecules is that between an antigen and
its antibody. For this reason, labeled antibodies are
routinely used in immunohistochemistry to identify and
localize many specific proteins, not just those with
enzymatic activity that can be demonstrated by
histochemistry
The End