BASICS OF

HISTOLOGY
DR NOOR KAMIL
ILOS

• After the completion of the lecture students will be able to

• Define histology
• Describe various microscopic methods used in histology
• Explain the significance of histology
HISTOLOGY & ITS METHODS OF STUDY

• PREPARATION OF TISSUES FOR • ELECTRON MICROSCOPY

STUDY • Transmission electron Microscopy
• Fixation • Scanning electron Microscopy
• embedding & Sectioning • AUTORADIOGRAPHY
• Staining
• CELL & TISSUE CULTURE
• LIGHT MICROSCOPY
• ENZYME HISTOCHEMISTRY
• Bright-Field Microscopy
• VISUALIZING SPECIFIC MOLECULES
• Fluorescence Microscopy
• Phase-Contrast Microscopy • Immunohistochemistry
• Confocal Microscopy • Hybridization Techniques
• Polarizing Microscopy • Interpretation of Structures in Tissue Sections
HISTOLOGY

• Study of the tissues of the body and how these tissues are arranged to
constitute organs
• The Greek root histo can be translated as either “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.
• Tissues have two interacting components: cells and extracellular matrix
(ECM).
• The ECM consists
• Macromolecules, most of which form complex structures such as collagen
fibrils and basement membranes
• Supports the cells and the fluid that transports nutrients to the cells, and
carries away their catabolites and secretory products
• The 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
PREPARATION OF TISSUES FOR STUDY

• Most common procedure used light microscope

• Fixation:
• Small pieces of tissue are placed in solutions of chemicals that preserve by cross-linking
proteins and inactivating degradative enzymes.
• Dehydration:
• The tissue is transferred through a series of increasingly concentrated alcohol solutions, ending
in 100%, which removes all water.
• Clearing:
• Alcohol is removed in toluene or other agents 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.
• Similar steps are used in preparing tissue for transmission electron microscopy (TEM), except special
fixatives and dehydrating solutions are used with smaller tissue samples and embedding involves
epoxy resins which become harder than paraffin to allow very thin sectioning.
A. Staining technique
B. Microtome
MICROTOME

• 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
between 1 and 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.
For TEM, sections less than 1 μm thick are prepared from resin-embedded cells using an
ultramicrotome with a glass or diamond knife.
 STAINING
• Cell components such as nucleic acids 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
• Most commonly hematoxylin and eosin (H&E) is used. Hematoxylin produces a dark blue or purple color,
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
• 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 color
• Counterstain is used to give additional information
• Lipophilic dye such as Sudan black, which dissolves in lipid-rich structures of cells
• 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
 ❯❯ MEDICAL APPLICATION

• Biopsies are tissue samples removed during surgery or routine medical procedures.
• In the operating room or medical center, biopsies are fixed in vials of formalin for later processing and microscopic
analysis in a pathology laboratory.
• If results of such analyses are required before the medical procedure is completed, for example to know whether a growth
is malignant before the patient is closed, a much more rapid processing method is used.
• The biopsy is rapidly frozen in liquid nitrogen, preserving cell structures and at the same time making the tissue hard and
ready for sectioning. A microtome called a cryostat in a cabinet at subfreezing temperature is used to section the block
with tissue, and the frozen sections are placed on slides for rapid staining and microscopic examination by a pathologist.
• Freezing of tissues is also effective in the histochemical study of very sensitive enzymes or small molecules, because
freezing, unlike fixation, does not inactivate most enzymes. Finally, because clearing solvents such as toluene dissolve
cell lipids in fixed tissues, frozen sections are also useful when structures containing lipids are to be studied
histologically.
Micrograph of epithelium lining the small intestine, (a) stained with H&E, and (b) stained with the PAS reaction for
glycoproteins.
With H&E, basophilic cell nuclei are stained purple while cytoplasm stains pink. Cell regions with abundant
oligosaccharides on glycoproteins, such as the ends of the cells at the lumen (L) or the scattered mucus-secreting goblet
cells (G), are poorly stained. X300
❯ LIGHT MICROSCOPY

• Conventional bright-field microscopy, as well as

fluorescence, phase-contrast, differential interference,
confocal, and polarizing microscopy are all based on
the interaction of light with tissue components and are
used to reveal and study tissue features in different
ways.
BRIGHT-FIELD MICROSCOPY

• With the bright-field microscope, widely used for 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, 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.
 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 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.
• Antibodies labeled with fluorescent compounds are extremely important in immunohistologic staining.
Components of cells are often stained with compounds visible by fluorescence microscopy.
(a) Acridine orange binds nucleic acids and causes DNA in cell nuclei (N) to emit yellow light and the RNA-rich cytoplasm (R) to appear
orange in these cells of a kidney tubule. (b) Cultured cells stained with DAPI (4’,6-diamino-2-phenylindole) that binds DNA and with
fluorescein-phalloidin that binds actin filaments show nuclei with blue fluorescence and actin filaments stained green. Both X500.
 PHASE-CONTRAST MICROSCOPY

• 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 (3D) aspect
Living neural crest cells growing in culture appear differently with various techniques of light microscopy. Here the same field of unstained
cells, including two differentiating pigment cells, is shown using three different methods (all X200):

(a) Bright-field microscopy: Without fixation and staining, only the two pigment cells can be seen.
(b) Phase-contrast microscopy: Cell boundaries, nuclei, and cytoplasmic structures with different refractive indices affect in-phase light
differently and produce an image of these features in all the cells.
(c) Differential interference microscopy: Cellular details are highlighted in a different manner using Nomarski optics.

Phase-contrast microscopy, with or without differential interference, is widely used to observe live cells grown in tissue culture.
CONFOCAL MICROSCOPY

• Confocal microscopy avoids various problems and achieves high resolution and sharp
focus by 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
• 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
• Confocal microscopes include a computer-driven mirror system (the beam splitter) to
move the point of illumination across the specimen automatically and rapidly
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.
• 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.
Polarizing light microscopy produces an image only of material having repetitive, periodic macromolecular structure;
features without such structure are not seen. Pieces of thin, unsectioned mesentery were stained with red picrosirius,
orcein, and hematoxylin, placed on slides and observed by bright-field (a) and polarizing (b) microscopy. (a) With
bright-field microscopy collagen fibers appear red, with thin elastic fibers and cell nuclei darker. (b) With polarizing
microscopy, only the collagen fibers are visible and these exhibit intense yellow or orange birefringence (a: X40; b:
X100).
❯ ELECTRON MICROSCOPY

• 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.
TRANSMISSION ELECTRON MICROSCOPY

• The transmission electron microscope (TEM) is an imaging

system that permits resolution 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.
 TRANSMISSION ELECTRON MICROSCOPY
• Schematic view of the major components of a transmission electron microscope (TEM), which is configured rather
like an upside-down light microscope.
• With the microscope column in a vacuum, a metallic (usually tungsten) filament (cathode) at the top emits
electrons that travel to an anode with an accelerating voltage between 60 and 120 kV.
• Electrons passing through a hole in the anode form a beam that is focused electromagnetically by circular electric
coils in a manner analogous to the effect of optical lenses on light. The first lens is a condenser focusing the beam
on the section.
• Some electrons interact with atoms in the section, being absorbed or scattered to different extents, while others are
simply transmitted through the specimen with no interaction.
• Electrons reaching the objective lens form an image that is then magnified and finally projected on a fluorescent
screen or a charge-coupled device (CCD) monitor and camera.
• In a TEM image areas of the specimen through which electrons passed appear bright (electron lucent), while
denser areas or those that bind heavy metal ions during specimen preparation absorb or deflect electrons and
appear darker (electron dense). Such images are therefore always black, white, and shades of gray.
SCANNING ELECTRON MICROSCOPY
• The scanning electron microscope (SEM) has many similarities to a TEM.
• However, here the focused electron beam does not pass through the specimen, but rather is
moved sequentially (scanned) from point to point across its surface similar to the way an
electron beam is scanned across a television tube or screen.
• For SEM specimens are coated with metal atoms with which the electron beam interacts,
producing reflected electrons and newly emitted secondary electrons.
• All of these are captured by a detector and transmitted to amplifiers and processed to
produce a black-and-white image on the monitor.
• The SEM shows only surface views of the coated specimen but with a striking 3D,
shadowed quality. The inside of organs or cells can be analyzed after sectioning to expose
their internal surfaces.
OTHER TECHNIQUES
❯ AUTORADIOGRAPHY

• Microscopic autoradiography is a method of localizing newly synthesized

macromolecules (DNA, RNA, protein, glycoproteins, and polysaccharides) in cells or
tissue sections.
• Radioactively labeled metabolites (nucleotides, amino acids, sugars) incorporated into the
macromolecules emit weak radiation that is restricted to the specific regions where the
molecules are located.
Autoradiographs are tissue preparations in which particles called silver grains indicate the cells or regions of cells in which
specific macromolecules were synthesized just prior to fixation. Shown here are autoradiographs from the salivary gland of a
mouse injected with 3H-fucose 8 hours before tissue fixation. Fucose was incorporated into oligosaccharides, and the free 3H-
fucose was removed during fixation and sectioning of the gland. Autoradiographic processing and microscopy reveal locations
of newly synthesized glycoproteins containing that sugar.(a) Black grains of silver from the light-sensitive material coating the
specimen are visible over cell regions with secretory granules and the duct indicating glycoprotein locations. X1500. (b) The
same tissue prepared for TEM autoradiography shows silver grains with a coiled or amorphous appearance again localized
mainly over the granules (G) and in the gland lumen (L). X7500. (Figure 1–9b, with permission, from Drs Ticiano G. Lima
and A. Antonio Haddad, School of Medicine, Ribeirão Preto, Brazil.)
 ❯ CELL & TISSUE CULTURE
• Live cells and tissues can be maintained and studied outside the body in culture (in vitro).
• In the organism (in vivo) cells are bathed in fluid derived from blood plasma, containing many different
molecules required for survival and growth.
• Cell culture has been invaluable in studying the functions of these molecules. It also allows the direct
observation of cellular behavior under a phase-contrast microscope.
• Many experiments technically not possible to perform in the living animal can be accomplished in vitro.
• The cells and tissues are grown in complex solutions of known composition (salts, amino acids, vitamins)
to which serum components or specific growth factors are added.
• Cultures of cells isolated in this way are called primary cell cultures.
• Many cell types, once isolated from normal or pathologic tissue, can be maintained in vitro for long
periods because they become immortalized and constitute a permanent cell line.
• Most cells obtained from normal tissues have a finite, genetically programmed life span. Certain changes
with oncogenes, can promote cell immortality, a process called transformation and are similar to the
initial changes in a normal cell’s becoming a cancer cell.
❯❯ MEDICAL APPLICATION

Cell culture is very widely used to study molecular changes that occur in
cancer; to analyze infectious viruses, mycoplasma, and some protozoa; and
for many routine genetic or chromosomal analyses.
Cervical cancer cells from a patient later identified as Henrietta Lacks, who
died from the disease in 1951, were used to establish one of the first cell
lines, called HeLa cells, which are still used in research on cellular structure
and function throughout the world.
❯ ENZYME HISTOCHEMISTRY

• Enzyme histochemistry (cytochemistry) is a method for localizing cellular structures

using a specific enzymatic activity present in those structures.
• To preserve these enzymes, histochemical procedures usually use unfixed or mildly fixed
tissue, which is sectioned on a cryostat to avoid adverse effects of heat and organic
solvents on enzymatic activity
• Phosphatases
• Dehydrogenases
• Peroxidase
(a) Micrograph of cross sections of kidney tubules treated
histochemically to demonstrate alkaline phosphatases
shows strong activity of this enzyme at the apical surfaces
of the cells at the lumens (L) of the tubules. X200.
(b) TEM image of a kidney cell in which acid phosphatase
has been localized histochemically in three lysosomes (Ly)
near the nucleus (N). The dark material within these structures
is lead phosphate that precipitated in places with acid
phosphatase activity. X25,000.
❯ VISUALIZING SPECIFIC MOLECULES

• A specific macromolecule present in a tissue section may sometimes be identified by using

tagged compounds or macromolecules that bind specifically with the molecule of interest.
• Phalloidin is a compound extracted from a mushroom commonly used to demonstrate actin
filaments in cells.
• Protein A is obtained from Staphylococcus aureus bacteria and binds to the Fc region of
immunoglobulin (antibody) molecules. Labeled protein A can therefore be used to localize
naturally occurring or applied antibodies bound to cell structures.
• Lectins are proteins or glycoproteins, derived mainly from plant seeds, that bind to
carbohydrates with high affinity and specificity.
• Different lectins bind to specific sugars or sequences of sugar residues
• Fluorescently labeled lectins are used to stain specific glycoproteins, proteoglycans, and glycolipids
IMMUNOHISTOCHEMISTRY

• A highly specific interaction between molecules is that between an antigen

and its antibody. For this reason, histologic methods using labeled
antibodies are extremely useful in identifying and localizing many specific
proteins, not just those with enzymatic activity that can be demonstrated
by histochemistry.
• For both diagnostic and research purposes, immunohistochemistry is very
widely used to detect specific proteins (or other molecules) of interest in
cells and tissues.
Immunocytochemistry (or immunohistochemistry) can be direct or indirect.

Direct immunocytochemistry (left) uses an antibody made against the tissue protein of interest and tagged
directly with a label such as a fluorescent compound or peroxidase.

Indirect immunocytochemistry (right) uses first a primary antibody made against the protein (antigen) of
interest and applied to the tissue section to bind its specific antigen. Then a labeled secondary antibody is
obtained that was (1) against immunoglobulin proteins (antibodies) and (2) labeled with a fluorescent
compound or peroxidase.
❯❯ MEDICAL APPLICATION

Because cells in some diseases, including many cancer cells, often produce
proteins unique to their pathologic condition, immunohistochemistry can be
used by pathologists to diagnose many diseases, including certain types of
tumors and some virus-infected cells.
Immunocytochemical methods to localize specific proteins can be applied to either light microscopic or TEM preparations
using a variety of labels.

(a) A single cultured uterine cell stained fluorescently to reveal a meshwork of intermediate filaments (green)
(b) A section of small intestine treated with an antibody against the enzyme lysozyme, demonstrates lysozyme-containing
structuresproduced brown color histochemically.
(c) A section of pancreatic cells in a TEM preparation incubated with an antibody against the enzyme amylase and then
with protein A coupled with gold particles. amylase with the gold particles localized as very small black dots.
HYBRIDIZATION TECHNIQUES

• Hybridization usually implies the specific binding between two single strands of nucleic
acid, which occurs under appropriate conditions if the strands are complementary
• This technique is ideal for
• (1) determining if a cell has a specific sequence of DNA, such as a gene or part of a gene
• (2) identifying the cells containing specific messenger RNAs (mRNAs) (in which the
corresponding gene is being transcribed), or
• (3) determining the localization of a gene in a specific chromosome.
INTERPRETATION OF STRUCTURES
IN TISSUE SECTIONS
• In studying and interpreting stained tissue sections, it is important to
remember that microscopic 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.
• Certain steps in this procedure may distort the tissues slightly, producing
minor structural abnormalities called artifacts not present in the living
tissue.
In thin sections 3D structures appear to have only
two dimensions. Such images must be interpreted
correctly to understand the actual structure of
tissue and organ components.