INTRODUCTION TO HISTOLOGY

PREPARATION OF TISSUE FOR

HISTOLOGY
Dr. Okolo
 INTRODUCTION TO HISTOLOGY
• Cytology (cyto Cell + logos study)
• Study of the cell
– Structure
– Constituents
• Cells make up tissues
• Histology (Gr. histo, web or tissue, + logos, study)
• Study of the tissues of the body and of how
these tissues are arranged to constitute organs.
 TISSUES
• Four fundamental tissues are recognized:
ECMN
• Epithelial tissue
• Connective tissue
• Muscular tissue
• And nervous tissue.
 COMPOSITION OF TISSUES
• Tissues are made of cells and extracellular
matrix
• Cells produce extracellular matrix and
influence them
• The extracellular matrix
• Environment around the cell
• Consists of;
– many kinds of molecules,
– highly organized
– Form complex structures,
• collagen fibrils
• basement membranes.
– Attach to cell receptors
– Functions
• mechanical support for the cells
• transport nutrients to the cells
• carry away catabolites and secretory products.
Tissues
• Several types of cells
• Specific associations of cells and extracellular matrix.
• types of tissues.
Organs
• formed by an orderly combination of several tissues
• Except the central nervous system
– almost solely by nervous tissue.
• 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.
• Advances in chemistry, physiology, immunology, and
pathology—and the interactions among these fields
—are essential for a better knowledge of tissue
biology.
• Familiarity with the tools and methods of any
branch of science is essential for a proper
understanding of the subject.
 Preparation of Tissues for Microscopic
Examination
• Preparation of histological
• studied with the aid of the light microscope.
• light beam is transmitted through the tissue.
• sectioned to obtain thin, translucent sections.
• living cells, very thin layers of tissues, or
transparent membranes of living animals (eg, the
mesentery, the tail of a tadpole, the wall of a
hamster's cheek pouch) can be observed directly
in the microscope without first being sectioned.
• It is then possible to study these structures for
long periods and under varying physiological or
experimental conditions.
• In most cases, however, tissues must be sliced
into thin sections and attached on glass slides
before they can be examined.
• These sections are precisely cut from tissues
previously prepared for sectioning using fine
cutting instruments called microtomes.
• The ideal microscope tissue preparation should
be preserved so that the tissue on the slide has
the same structure and molecular composition
as it had in the body.
• This is sometimes possible but—as a practical
matter—seldom feasible, and artifacts,
distortions, and loss of components due to the
preparation process are almost always present.
 FIXATION
• Treatment to
– avoid tissue digestion by enzymes present within
the cells (autolysis) or by bacteria
– preserve the structure and molecular composition
• Promptly and adequately
• by chemical
• less frequently, physical methods.
• In chemical fixation
• the tissues are immersed in solutions called
Fixatives
– stabilizing or cross-linking agents
• time to fully diffuse into the tissues
• cut into small fragments
• facilitate the penetration
• to guarantee preservation of the tissue.
• Intravascular perfusion of fixatives can be used.
– Fixative rapidly reaches the tissues through the blood vessels.
• One of the best fixatives for routine light microscopy
is a buffered isotonic solution of 4% formaldehyde.
• Formaldehyde and glutaraldehyde,
• react with the amine groups (NH2) of tissue proteins.
• Glutaraldehyde, a dialdehyde,
– cross-link proteins.
• Fixation very important in preserving ultra structure
in EM.
• Therefore a double fixation procedure
– buffered glutaraldehyde solution
– followed by a second fixation in buffered osmium tetroxid
• to preserve and stain lipids and proteins.
 EMBEDDING/impregnation
• Solid medium to facilitate sectioning.
• Infiltrate embedding substances that impart a
rigid consistency to the tissue.
• Include paraffin and plastic resins.
• Paraffin is used routinely for light microscopy;
• resins are used for both light and electron
microscopy.
• 1st : dehydration and clearing.
• water is first extracted
• Tissue is placed successively in a graded series of mixtures of
ethanol and water (usually from 70% to 100% ethanol).
• The ethanol is then replaced with a solvent miscible with the
embedding medium.
• In paraffin embedding, the solvent used is usually xylene.
• Tissues infiltrated with the solvent become transparent
(clearing).
• Impregnated tissue is placed in melted paraffin in the oven,
typically at 58–60°C.
• Causes the solvent to evaporate, and the spaces within the
tissues become filled with paraffin.
• The tissue together with its impregnating paraffin hardens
after being taken out of the oven.
• Tissues to be embedded with plastic resin are also
dehydrated in ethanol
• Then infiltrated with plastic solvents.
• The ethanol or the solvents are later replaced by plastic
solutions that are hardened by means of cross-linking
polymerizers.
• Plastic embedding prevents the shrinking caused by the
high temperatures needed for paraffin embedding
– and gives much better results.
• The hard blocks containing the tissues are then taken to a
microtome
• Sectioned by the microtome's steel or glass blade to a
thickness of 1–10 microns.
• 1km = 1000 metres
• 1m = 100cm
• 1cm = 10mm
• 1000mm= 1m
• 1,000,000micron(μm )= 1m
• 1000,000,000(nm)= 1m
• 10,000,000,000. Angstroms(Ǻ) = 1m
• 1 micrometer (1 μm) = 0.001 mm = 10–6 m;
• 1 nanometer (1 nm) = 0.001 m = 10–6 mm = 10–9 m.
• The sections are floated on water and transferred to
glass slides to be stained.
• Physical Fixation
• rapid freezing.
• become hard and thus ready to be sectioned.
• A freezing microtome—the cryostat (Gr. kryos, cold, + statos,
standing)
– To section the frozen tissues.
– Rapid
– routinely used in hospitals to study specimens during surgical
procedures.
– in the histochemical study of very sensitive enzymes or small
molecules, since freezing does not inactivate most enzymes.
– Preserve tissue lipids, which zylene dissolves out.
 STAINNING
• Give colour to colourless tissues
• Make various tissue components conspicuous
• permit distinctions to be made between them.
• dyes must be selectively.
• Most of these dyes behave like acidic or basic compounds
and have a tendency to form electrostatic (salt) linkages
with ionizable radicals of the tissues.
• Tissue components that stain more readily with basic dyes
are termed basophilic (Gr. basis, base, + phileo, to love);
• those with an affinity for acid dyes are termed acidophilic.
• Examples of basic dyes are
– toluidine blue
– methylene blue.
– Hematoxylin behaves like a basic dye
– The main tissue components that ionize and react
with basic dyes do so because of acids in their
composition (nucleic acids, glycosaminoglycans,
and acid glycoproteins).
• Acid dyes (eg, orange G, eosin, acid fuchsin) stain the
acidophilic components of tissues such as
mitochondria, secretory granules, and collagen.
• Of all dyes, the combination of hematoxylin and
eosin (H&E) is the most commonly used.
• Hematoxylin stains the cell nucleus and other acidic
structures (such as RNA-rich portions of the
cytoplasm and the matrix of hyaline cartilage) blue.
• Eosin stains the cytoplasm and collagen pink.
• Many other dyes, such as the trichromes (eg,
Mallory's stain, Masson's stain), are used in different
histological procedures.
• The trichromes, in addition to showing the nuclei and
cytoplasm very well, help to differentiate collagen
from smooth muscle.
• A good technique for differentiating collagen is the use
of picrosirius, especially when associated with
polarized light.
• In many procedures (see
Immunocytochemistry), the sections become
labeled by a precipitate, but cells and cell
limits are often not visible.
• In this case a counterstain, usually a single
stain that is applied to a section to allow the
recognition of nuclei or cytoplasm, is used.
• Although most stains are useful in visualizing the various
tissue components,
• they usually provide no insight into the chemical nature of
the tissue being studied.
• In addition to tissue staining with dyes, impregnation with
metals such as silver and gold is a common method,
especially in studies of the nervous system.
• The whole procedure, from fixation to observing a tissue in
a light microscope, may take from 12 h to 21⁄2 days,
depending on the size of the tissue, the fixative, and the
embedding medium.
 Microscope
• mechanical and optical parts.
• The optical components consist of three systems of lenses:
condenser, objective, and eyepiece.
• The condenser collects and focuses light, producing a cone of light
that illuminates the object to be observed.
• The objective lenses enlarge and project the illuminated image of
the object in the direction of the eyepiece.
• The eyepiece further magnifies this image and projects it onto the
viewer's retina, a photographic plate, or (to obtain a digital image) a
detector such as a charged coupled device camera.
• The total magnification is obtained by multiplying the magnifying
power of the objective and eyepiece.
 The optical microscope
• Light microscope
• uses visible light and a system of lenses to magnify images of
small samples.
• Optical microscopes are the oldest and simplest of the
microscopes.
• New designs of digital microscopes are now available
– use a CCD camera to examine a sample and the image is shown
directly on a computer screen without the need for expensive optics
such as eye-pieces.
– Other microscopic methods which do not use visible light include
scanning electron microscopy and transmission electron microscopy.
• Two types:
• simple (one lens) and compound (many lenses).
• Digital microscopes are based on an entirely different system of
collecting the reflected light from a sample.
• Light microscope
• A simple microscope
• uses only one convex lens for magnification, and is the original light
microscope.
• now considered primitive
• Seen in magnifying glass
• View samples in colour against EM
– for forensic analysis, where blood traces may be important, for example.
 COMPOUND MICROSCOPE
• Uses many lenses
1. ocular lens, or eyepiece
2. objective turret
3. objective lenses
4. coarse adjustment knob
5. fine adjustment knob
6. object holder or stage
7. mirror or light
(illuminator)
8. diaphragm and condenser
• The eyepiece –
• a cylinder containing two or more lenses to bring the
image to focus for the eye.
• The eyepiece is inserted into the top end of the body
tube.
• Typical magnification values for eyepieces include 5x,
10x and 2x.
• the objective lens and eyepiece are matched to give
the best possible optical performance.
– most commonly with apochromatic objectives.
• The objective lens
– a cylinder containing one or more glass lenses,
– collect light from the sample.
– At the lower end of the microscope tube one or more
objective lenses are screwed into a circular nose piece which
may be rotated to select the required objective lens.
– Typical magnification values of objective lenses are 4x, 5x,
10x, 20x, 40x, 50x and 100x.
– Some high performance objective lenses may require
matched eyepieces to deliver the best optical performance.
• The stage - a platform below the objective
which supports the specimen being viewed.
• In the center of the stage is a hole through
which light passes to illuminate the specimen.
• The stage usually has arms to hold slides
(rectangular glass plates with typical
dimensions of 25 mm by 75 mm, on which the
specimen is mounted).
• The illumination source - below the stage.
• light is provided and controlled in a variety of
ways.
• At its simplest, daylight is directed via a mirror.
• Most microscopes, now have their own
controllable light source that is focused through
an optical device called a condenser, with
diaphragms and filters available to manage the
quality and intensity of the light.
• Typical compound optical microscope has three objective lenses:
– a scanning lens (4×),
– low power lens (10x)
– high power lens (ranging from 20 to 100×).
– Some microscopes have a fourth objective lens, called an oil immersion lens.
– To use this lens, a drop of immersion oil is placed on top of the cover slip,
– the lens is very carefully lowered until the front objective element is
immersed in the oil film.
– Such immersion lenses are designed so that the refractive index of the oil
and of the cover slip are closely matched
– so that the light is transmitted from the specimen to the outer face of the
objective lens with minimal refraction.
– An oil immersion lens usually has a magnification of 50 to 100×.
• The actual power or magnification of an
optical microscope is the product of the
powers of the ocular (eyepiece), usually about
10×, and the objective lens being used.
• Compound optical microscopes can produce a
magnified image of a specimen up to 1000×
• used to study thin specimens as they have a
very limited depth of field.
 Assignment
• Types of microscopes and their uses.