Lecture No.

1
Romena Liza F. Restua, RMT, MSMT, DrPH©
 HISTOLOGY
◦ the study of the microanatomy of cells, tissues, and
organs as seen through a microscope
◦ examines the correlation between structure and
function.
 HISTOPATHOLOGY
◦ is the examination of tissues from the body under a
microscope to spot the signs and characteristics of
disease
 CYTOLOGY
◦ the study of cell as fundamental units of living
things.
 TISSUE PROCESSING
◦ Procedure/process of preparing the tissue by
embedding it in a solid medium that is firm enough
to support it and give sufficient rigidity to enable
thin sections to be cut and yet soft enough to
enable the knife to cut the sections with little
damage to the knife or the tissue

 PATHOLOGIST
◦ a scientist who studies the causes and effects of
diseases, especially one who examines laboratory
samples of body tissue for diagnostic or forensic
purposes.
 HISTOTECHNICIAN
◦ is a clinical laboratory technician who prepares very
thin samples of body tissue to be examined under a
microscope by a pathologist.
LIGHT MICROSCOPE
 “COMPOUND microscope” or “OPTICAL
microscope”
 Principle: Refraction of lights

ELECTRON MICROSCOPE
 Principle: Deflection of beam of electrons
 All types of Light Microscope are based on
the interaction of light and tissue
components and can be used to reveal and
study tissue features.
1. Bright-field Microscope
2. Dark-field Microscope
3. Fluorescence Microscope
4. Phase-Contrast & Differential Interference
Microscope
5. Confocal Microscope
6. Polarizing Microscope
 Most widely used by students of histology.
 Stained preparations are examined by means of
ordinary light that passes through the specimen.
 It is composed of MECHANICAL and OPTICAL
PARTS.
1. Condenser= collects and focuses light,
producing a cone light that illuminates the
object to be observed.
2. Objective lenses= enlarge and project the
illuminated image of the object in the
direction of the eyepiece.
3. Eyepiece/ Ocular lens= magnifies the image
and projects it onto the viewer’s retina.
 light seems to radiate from the specimen
while all the rest of the field is black.

 Resolution is as good as that in bright

field while contrast is enhanced.

 Syphilis is what made dark field popular.

Schaudinn discovered Treponema
pallidum – the syphilis spirochete – in 1905
using dark field microscopy.
 Fluorescence= when certain substances are
irradiated by light of a proper wavelength,
they emit light with a longer wavelength.
 Tissue sections are irradiated by UV light and
the emission is in the visible portion of the
spectrum.
 The fluorescent substance appear BRILLIANT
on a DARK BACKGROUND.
 ACRIDINE ORANGE= fluorescent stain which
binds both in DNA and RNA.
 Uses a lens system that produces visible images
for transparent objects.

 PRINCIPLE: Light changes its speed when passing

through cellular and extracellular structures with
different refractive indices.

 It allows observation of living cells and tissue

cultures without staining.
 Another method of observing unstained cells or
tissue sections.

 It produces an image with more apparent Three

Dimensional(3D) aspect as compared to routine
phase-contrast microscopy.
  The point light source, focal point of the lens and
detector’s pinpoint aperture are all OPTICALLY
CONJUGATED or ALIGNED to each other in the focal
plane and unfocused light does not pass through
the pinhole.

 It avoids STRAY LIGHT and achieves GREATER

RESOLUTION by using:

1. small point of high intensity light provided by a

laser.
2. a plate with a pinhole aperture in front of the
image detector.
 allows the recognition of structures made
of highly organized molecules like collagen,
cellulose, microtubules and microfilaments.

 Polarized structures appears BRIGHT

against a DARK BACKGROUND.

 BIREFRINGENCE= ability to rotate the

direction of the vibration of the polarized
light.
 All are based on the interaction of electrons
and tissue components.

 The wavelength in the electron beam is much

shorter than of light, allowing a thousand-
fold increase in resolution.
1. Transmission Electron
Microscope (TEM)

2. Scanning Electron
Microscope (SEM)
 It requires very thin sections about 40-90 nm.
 Allows magnifications of up to 400,000X to be
viewed with details only applied to isolated
molecules and particles.
 Very thin sections can be observed with
details at magnifications of up to about
120,000X.
 If electrons can readily pass in the specimen
it will appear BRIGHTER or ELECTRON LUCENT
showing that it is coming from a THIN TISSUE
SECTION.
 If electrons were absorbed and deflected it
will appear DARKER or MORE ELECTRON
DENSE showing that it is coming from a
THICK TISSUE SECTION.
 permits PSEUDO Three-Dimensional views
of the surfaces of the cells, tissues and
organs.
Light Electron beams emitted by tungsten filaments or
electron particles
Glass lens Electron magnetic fields or electric field
Low resolving power Higher resolution
Open system Vacuum enclosed system
Direct viewing of objects Fluorescent screen
Less expensive Highly expensive
Do not require high voltage Require high voltage
 Eyepiece: The lens the viewer looks through
to see the specimen. The eyepiece usually
contains a 10X or 15X power lens.

 Diopter Adjustment Knob: Useful as a means

to change focus on one eyepiece so as to
correct for any difference in vision between
your two eyes.
 Diopter Adjustment Scale: Useful in changing
the distance of the eyepiece with one
another.
 Body tube (Head): The body tube connects
the eyepiece to the objective lenses.

 Arm: The arm connects the body tube to the

base of the microscope.

 Coarse adjustment: Brings the specimen into

general focus. (SCANNER and LPO)

 Fine adjustment: Fine tunes the focus and

increases the detail of the specimen. (HPO
and OIO)
 Nosepiece: A rotating turret that houses the
objective lenses. The viewer spins the nosepiece
to select different objective lenses.

 Objective lenses: One of the most important

parts of a compound microscope, as they are the
lenses closest to the specimen.

 Stage: The flat platform where the slide is placed.

 Stage clips: Metal clips that hold the slide in

place.

 Stage height adjustment (Stage Control): These

knobs move the stage left and right or up and
down.
 Aperture: The hole in the middle of the stage that allows
light from the illuminator to reach the specimen.

 On/off switch: This switch on the base of the microscope

turns the illuminator off and on.

 Illumination: The light source for a microscope. Older

microscopes used mirrors to reflect light from an external
source up through the bottom of the stage; however, most
microscopes now use a low-voltage bulb.

 Iris diaphragm: Adjusts the amount of light that reaches

the specimen.

 Condenser: Gathers and focuses light from the illuminator

onto the specimen being viewed. Base: The base supports
the microscope and it’s where illuminator is located.
 Method of preparation of tissue for
microscopic examination.
 Paraffin wax method – uses paraffin for
impregnation of tissue at room temperature.
The most common procedure used in the
study of tissue in the laboratory and hospital
set-up.
 Frozen section method – quick method in
which the tissue are hardened at low
temperature by the use of a cryostat or
freezing microtome.
 Celloidin method- uses celloidin as
embedding medium useful for large objects
(whole brain), and for hard brittle specimen
(cartilages)
 F-IXATION
D-EHYDRATION
C-LEARING
I-MPREGNATION
E-MBEDDING
T –RIMMING
S-ECTIONING
S-TAINING
M-OUNTING
L-ABELING
Procurement of tissue specimen.
Fixation- MOST CRITICAL STEP
 submerging the specimen in a chemical substance in order to
preserve the fresh tissue for examination.

Purpose:
 To preserve tissue morphology and chemical composition(Primary
aim)
 To act as disinfectant
 To kill microorganisms
 To harden the tissue(Secondary aim)
 To permit better staining reaction

Fixative used:
◦ 10 % Neutral formalin (most commonly used)
◦ 10 % Formol saline
◦ Bouin’s solution
◦ Zenker’s solution
◦ Potassium Bichromate
◦ Osmic acid
◦ Glutaraldehyde
Dehydration
- immersing the tissue in increasing
concentration of alcohol
 Purpose: To remove water from the tissue
 Dehydrating Agent: Graded concentration of
ethyl alcohol (65% up to 100 % alcohol)
Clearing (dealcoholization)
- replacing the alcohol with clearing agents.
 Purpose: to impregnate tissue with a paraffin
solvent, since alcohol is insoluble with paraffin, it
must be replaced by a clearing agent so that it
can be impregnated with paraffin wax.
 Clearing agents:
◦ Xylene (Xylol)- most commonly used
◦ Chloroform
◦ Benzene
◦ Cedarwood Oil
◦ Ether
◦ Carbon tetrachloride
Impregnation
-Infiltration of tissue with melted
paraffin wax.
Embedding
- placing the tissue in paraffin blocks.
 Purpose: Paraffin penetrates all intercellular
spaces and even into the cells making the
tissues more resistant to sectioning.
Trimming
Process of removing EXCESS WAX after
embedding.
Sectioning
- cutting of tissue by use of microtome. The
cut tissue, paraffin ribonettes are placed in
basin of water.
- 4-6 u= routine histologic procedure.
- 10-15 u=frozen section.
- 0.5 u= electron microscopy.
 Placing the cut sections in clean glass
slides. Egg albumin is used as adhesive.

 Dissolving or melting the embedding

medium (Paraffin wax) by passing the
slide over a flame (alcohol lamp or
Bunsen burner)

 Rehydration- immersing the tissue in

decreasing concentration of alcohol.
Staining
- with appropriate stain.
 Hematoxylin and Eosin stain (H and E stain) is
usually used.
 Hematoxylin (basic dye)- gives a bluish or
purple color to the nucleus.
 Eosin (acid dye)- imparts reddish or pinkish
color to the cytoplasm.
 Dehydration- immersing again the tissue in
increasing concentration of alcohol.

 Clearing
Mounting
- Placing cover slip with a few drops of Canada
balsam as a mounting medium on processed
tissue.
Labeling
- Label the organ on the prepared slides.