HISTOLOGY

a subdiscipline of anatomy
the study of tissues
largely a visual science that relies on microscopy and other
imaging

modalities

substructure
Goals of Histology
Make small

and

to

reveal

complex

cell,

tissue,

structures

and

and

vessel to vessel (e.g. short term culture of WBC)

b. Tissue Culture
A.K.A. explant
immature tissue culture, incubated
embedded in coagulum of blood plasma + embryonic

organ

processes

observable
Understand the relationship between tissue structure and

function
Establish a basis for learning histopathology, which involves

juice
cells proliferate in the zone of outgrowth
c. Organ Culture
maintenance of mature tissue or organ fragments
study direct effects of drugs or hormones on various

the relationship between abnormal tissue structure and

functional defects
Provide a basis for treating diseased and injured tissues

HISTOLOGICAL TECHNIQUES
Direct Observation of Living Cells and Tissues

cells are studied while still alive

colorless
phase contrast microscope
amoeboid movement
phagocytic activity of blood cells
a. Exteriorization and Transillumination
organs with long pedicles can be brought outside the
body and placed in a suitable moist container permitting
their transillumination and direct microscopic analysis
b. Transparent Chamber Method
installation of chambers of metal glass in the flexible ear
of rabbits

treat with enzymes or use spatula (to harvest)

cultivated in suspension or petridish
a. Cell Culture
non adherent, dividing cells are transferred from

tissue
Uses of Cell/Tissue/Organ Culture

study of normal and cancerous cells

development of new drugs
study intracellular parasites/viruses/mycoplasma/protozoa
determination of human karyotypes
detect genetic disorders
molecular biology and recombinant DNA technique

Mechanical Micromanipulation and Microdissection

INSTRUMENT: Micromanipulator
moves glass needles or pipets so a single cell can be

manipulated or directed
moves fixed cells while studied under SEM
e.g. granules, vacuoles, mitochondria can be moved

Use of Radiation Probes

selective staining and probing of organelle with colored

radioactive dye combined with high intensities of light

Cell/Tissue/Organ Culture

in vitro in glass cultivation

isolate the cells/tissues/organ fragments
bathed in plasma, salts, amino acids, vitamins

available from laser sources

bombarding with beams of radioisotope & UV light
Application: removal of nucleus from a cell & selective

destruction of specific cell organelles

provides the opportunity for a new form of cellular
microsurgery

Cinematography

motion pictures taken through the objectives of microscope

to record cell activity (e.g. movement of cells and

material is placed in a shell freezer (w/ dry ice, methanol, or

liquid nitrogen)
reagents are added to make protein content of tissue

organelles, mitosis, phagocytosis, muscle contraction, cilia

movement)
Differential Centrifugation

cell fractionation
separation of homogeneous

heterogeneous population of cells

uses repeated centrifugation at progressively higher speeds
smaller components need greater centrifugal force to

sediment it
STEPS: cells (disrupted) homogenate layered in sucrose

cell

organelles

from

centrifuge at high speed

Microincineration

tissue slices leave ash which retain fine structural details

minerals can be identified within the cells

Frozen Section Method

a piece of tissue is placed directly on a stage of special

microtome with an outlet for carbon dioxide

for sectioning biopsy material
during operation for examination of cancer cell

Freeze Drying Technique

A.K.A. lyophilization/cryodessication
freezing uses lyoprotectant (polyhydroxycompounds)
STEPS: freezing primary drying secondary drying
tissue is frozen and dehydrated at low temperature in high

vacuum
dehydration process typically used to preserve a perishable
material or to make a material more convenient for
transport

insoluble
should be done below the materials eutectic pt.
a. Primary Drying
pressure is lowered and enough heat is applied to the
material for the water to sublimate
about 95% of water is sublimated
pressure is controlled by partial vacuum
heat is brought by conduction or radiation
b. Secondary Drying
higher temperature than primary drying
removes unfrozen water molecules
after this step, only 1 -4% water is retained
Properties of a Freeze-Dried Product

sealed
lesser damage to substance than simple

dehydration
material not shrunk or toughed
flavors and odor remain unchanged
can be reconstituted (rehydrated)

Use of Stain

most commonly used techniques are histochemistry (and

to differentiate tissue elements as certain cellular elements

to produce a contrast
most stains differentiate between the acid and basic
components of cells
other stains differentiate the fibrous components of the

physicochemical processes that not only stain the tissue

extracellular matrix
some tissues can be stained by forming metal deposits on

intracellular molecules of interest

tissue (e.g. nerve cells and extracellular fibers)

ROUTINE STAIN: H&E
a. Basophilic Stain
stain the acidic components of cell (e.g. DNA, RNA)
hematoxylin (blue color)
b. Acidophilic Stain
stain the basic components of cell (e.g. cytoplasmic

immunocytochemistry,

autoradiography,

column chromatography, and gene analysis

use chemical reactions, enzymatic processes,

and

but also permit the localization of extracellular and

Immunohistochemistry

uses labeled antibodies to localize specific cell and tissue

antigens and is the most sensitive, specific, and widely

used histochemical method

can be used with LM or TEM
because many targeted antigens

are

proteins

whose

structure may be altered by fixation and clearing, frozen

elements)
eosin (pink color)

sections are often used

Methods of Staining
1. Vital Staining - staining fresh on living, unfixed tissue cells
a. Intra Vital Staining - using intravenous injection of
dyes
b. Supra Vital Staining - adding dyes to the medium of
the cells already removed from the organism
2. Staining Of Fixed Dead Tissues - tissues

cytochemistry),

Labels Used in Immunohistochemistry

LM: fluorescent molecule or enzyme (e.g. peroxidase,
alkaline phosphatase bound to antibody)
TEM: colloidal gold or ferritin iron bound to antibody

killed,

embedded, sectioned, stained and mounted on slides

may use polyclonal or monoclonal antibodies

Polyclonal - more sensitivity by being directed against

many antigenic determinants

Monoclonal - specific for one antigen, lower sensitivity
may be direct or indirect, with label directly attached
to the primary antibody (which binds the molecule to
be localized), or with the primary antibody being
unlabeled and visualized by a labeled secondary

Advanced Visualization Procedures

in elucidating functional aspects of the cells, tissues, and

organs being studied

antibody
antibody

that

binds

specifically

to

the

primary

radioactive

isotopes

are

incorporated

into

components specifically
because fixation and clearing inactivate enzymes, frozen

sections are commonly used

sections are incubated in solutions containing substrates for
the enzymes of interest and reagents that yield insoluble

localizes in a tissue section a radioactive substance (drug,

enzyme etc.) that the living cells metabolized

uses a radioactive isotopes which is integrated into the

molecule that is being investigated

isotopes that are low energy B-emitters are usually best
H3 and C14 are commonly used

Procedures:

radioactive particles emitted expose the emulsion

emulsion if developed like film and then cover slipped

and viewed by light microscopy

Result: Microscopic exam displays the presence of silver

involves packing a hollow column with a semipermeable

material (often tiny resin or agarose beads) and applying a
cell or tissue homogenate, or a centrifugal fraction of such a

sample, to the top of the column

after the sample percolates into the column, solvent is

added and allowed to flow through

timed fractions of the flow-through material (eluate),
containing different molecules from the homogenate, are

Autoradiography

detected by thin layer of photographic emulsion

the slide is placed in the dark for several weeks and the

Column Chromatography

colored or electron-dense precipitates at sites of enzyme

activity

molecule was located

uses enzymes (such as acid phosphatase, dehydrogenases,

and peroxidases) in specific cell organelles to localize those

macromolecules (commonly tritium H)

the presence of the isotopes and the macromolecule is

grains over the regions where the isotope labeled

Enzyme Histochemistry

collected
release (elution) is retarded by interactions with the packing
material and results in separation
Column Chromatography Types
1. Ion-exchange - charge
2. Gel-filtration - size
3. Affinity - binding affinity

GENETIC TECHNOLOGY

sequences. It is highly individualized, and is one of the

different technologies for understanding the genes which

best ways to get an unequivocal identification of a

have become tools of unlimited diagnostic and therapeutic

person. A single-locus probe searches for repetitions of

power
A. Gene Splicing
Genes from one organism can be spliced into another

only one specific sequence. It works with 50 times less

using a technique called gene splicing. If a scientist

wanted to splice human genes into a bacterial plasmid,
he would first cut both DNA fragments with the same

genetic material, but is less definitive in its results.

Scientists often use three or four single-locus probes to
identify individuals, whereas only one multi-locus probe
would work.

restriction enzyme (an enzyme that breaks DNA at

certain base sequences, leaving "sticky ends"). He would
then take the human gene he wanted to splice into the
plasmid and connect them using the "sticky ends" left by
the enzymes. He would probably then add strengthening
enzymes to strengthen the bond between the DNA
fragments. The transgenic bacterium would most likely
then be allowed to divide repeatedly, and the resulting
bacterial colony would then express the gene.

C. In Situ Hybridization
is a method of analyzing the tissue distribution of
particular nucleotide sequences in DNA (e.g. specific

B. Genetic Fingerprinting
Genetic fingerprinting is a powerful forensic tool used to

genes) and RNA (e.g. specific mRNAs)

hybridization refers to the binding of complementary

nucleotide sequences to one another with specificity

recombinant DNA technology permits copies of

identify the perpetrator of a crime through traces of

selected single-strand nucleotide sequences to be

genetic material left at the scene. It utilizes repetitions of

DNA sequences, which differ from person to person, to
uniquely identify an individual. A multi-locus probe
searches for multiple repetitions of several different DNA

synthesized in large numbers

synthetic sequences complementary to the RNA or
DNA sequence, an investigator wishes to localize, are

termed probes and can be labeled with radioisotopes

Electrophoresis

(e.g. 32P), biotin, or digoxigenin

radiolabeled
probes
are
demonstrated

fragments of different lengths. The DNA samples are

autoradiography
biotin-labeled probes
enzymes

(e.g.

are

demonstrated

peroxidase)

or

by
with

fluorochromes

covalently linked to avidin, a molecule with high

is

method

of

separating

DNA

placed in tiny "wells" at one end of an agarose gel. An

electric current is then passed over the gel, separating
the fragments. The DNA bands are then revealed with a
radioactive probe.

affinity for biotin

digoxigenin-labelled probes are demonstrated by
indirect immunohistochemistry using antidigoxigenin

primary antibodies
labeled probes were first used to analyze nucleic
acids

isolated

cell

or

tissue

or

tissue

homogenates
the term in situ refers to the application of this
technique

from

to

tissue

sections,

smears

of

cells,

cultures, or even whole embryos

when such specimens are incubated with labeled
probes, the probes bind to and reveal the distribution
of their complementary sequences

E. Blotting and Electron Transfer

used to analyze molecules

first

separated

by

electrophoresis
gels used for electrophoresis, restrict the access of
large native molecule, such as antibodies and large

FISH: Fluorescence In Situ Hybridization

nucleic acids, to their target molecules

blotting and electron transfer remove the separated
molecules from the gels and immobilize them on

membranes
transfer from the gels to the membrane may be

accomplished by blotting or by electric charge

in blotting techniques, molecules in the gel are
carried by the flow of a buffer across the gel and

D. Electrophoresis

through the membrane

the membranes carrying the more accessible target
molecules are incubated with labeled antibodies or
complementary nucleic acid sequences (probes) to

reveal the positions and relative amounts of the

molecules of interest
Types ff Blotting Techniques:
Western blotting - labeled antibodies to reveal
the presence and amount of a specific protein on

the membrane
Northern blotting

complementary RNA sequences

Southern blotting - labeled probes localize

labeled

probes

reveal

specific DNA sequences

Southern Blotting

F. PCR Amplification
PCR (polymerase chain reaction) amplification is an
extremely

powerful

technique

by

which

single

molecule of DNA can be amplified millions of times in a

single

afternoon.

The

technique

has

enormous

applications fields from forensic science, where it can

amplify trace DNA samples left at the scene of a crime;
to archaeology, where it can show some of the genome
of ancient organisms; to modern hospital testing, where
the DNA in a tiny blood sample can be used for literally
hundreds of genetic tests.

TYPES OF MICROSCOPE
1. Light or Optical Microscope
a. polarizing
b. differential interference
c. phase-contrast
d. fluorescence
e. dark-field
f. bright-field
2. UV Microscope
3. Electron Microscope
a. TEM
b. SEM
LIGHT MICROSCOPY
Mechanism of Light Microscope

resolves the specimens image and projects it to the ocular

Magnification

the wave peak of light rays from another source, the rays

interact to produce reinforcement (relative brightness)

However, if the wave peak from one light source coincides
with the wave through from another light source, the rays
interact to produce interference (relative darkness)

Light Source

usually lit by bulbs that emit white light (average 550 nm) of

varying intensity
halogen bulbs with tungsten filaments emit intense white
light and are commonly used in compound bright-field
microscopes

Microscope Lenses (Light microscopes have glass lenses)

condenser lens collects light from the source and projects

it as a cone through the specimen

increases the specimens apparent size

objective magnification X ocular magnification
ratio of image size to the actual size

Numerical Aperture

The principle is based on the wave nature of light rays, and

valleys match) or out of phase
If the wave peak of light rays from one source coincides with

lens
ocular lens further enlarges the image and projects it into
the observers retina, a screen, or photographic emulsion

light-gathering capacity of the microscope

NA resolving power
measure of the size/angle of the cone of light delivered by
the illuminating condenser lens to the object plane and of

the fact that light rays can be in phase (their peaks and

objective lens mounted on rotating turret, enlarges and

the cone of light emerging from the object

related to the width of the lens opening (aperture)

Resolution

measure of the capacity of the microscope to distinguish 2

close but distinct points

human eye : 200 m
light microscope : 0.2 m
electron microscope : 0.002 m
independent of magnification
calculated from the NA of the objective and the wavelength
of illumination:

R=

0.61
NA

Refractive Index

measures the comparative velocity of light in different

TYPES OF LIGHT MICROSCOPES

media
measure of the optical density of an object or the speed with

Bright-Field Microscope

which it is traversed by a light wave

the air between the lens and the coverslip bends some of

the light projected through the specimen

using immersion oil between the coverslip and an oil

most common tool of histology and histopathology

bright-field: entire field is illuminated by an ordinary

condenser
specimens must be translucent and stained to provide
contrast

immersion objective lens maintains the refractive index,

thus improving resolution

Dark-Field Microscope

Working Distance

distance between the surface of the lens and the surface of

the cover glass or the specimen when in sharp focus

NA resolving power working distance

The Properties of the Microscope Objectives

examines living microorganisms that are:

* invisible in brightfield microscopy
* do not stain easily
* distorted by staining
uses a special condenser with an opaque disc that
blocks light from entering the objective lens directly
specimen appears light against a bright background
Application:
* detecting T. pallidum in the diagnosis of syphilis

Polarizing Microscope

detects orderly arrangement of fibrous proteins or

stained linearly oriented structures of living cells in

Objecti
ve
Scanne
r
LPO

Ring

Size

Lens

Mag

short

large

lowes

st

yello

est
shorte

larger

lower

red

HPO

blue

OIO

white

longer
longe
st

smalle
r
small

Function
locate structures
initial focusing,
general outline

higher

shows details

highe

examines

st

microorganism

tissue culture or fixed stained preparations (e.g.)

provides information about structural arrangement at the
molecular level
Modification: two filters
* POLAROID: between the light source and condenser
* ANALYZER: at the draw tube
Application: spindle fibers of dividing cells, banding
patterns of striated muscle, mineral elements, ash residues

Phase-Contrast Microscope

visualizes differences of refractive index within cells

fluorescing compounds (fluorochromes: fluorescein or

and tissues using a condenser lens system containing an

annular (ring-shaped) diaphragm

2 sets of light rays brought together form an image on the

phase) shades of gray black (out of phase)

basic tool for tissue culture
different protoplasmic constituents produce phase variations

into intensity variations and thereby enables the eye to

fixed
teaching films of mitosis usually employ dark

stimulate the fluorochrome

barrier filter between the objective and ocular lenses
protects the eyes from UV rays and projects only the

detect mere contrast between different structures

Application:
* useful for the study of unstained cells, living or
*

rhodamine)
excitation filter between the light source and the
specimen filters out all wavelength except that needed to

ocular lens containing areas that are relatively light (in

allows localization of substances labeled with

emitted light
fluorochromes stimulated by UV light emit visible light
* fluorochrome auramine for M. tuberculosis (glows
*

medium phase contrast microscopy to render

yellow)
fluorescein isothiocyanate (FITC) for B. anthracis

(apple green)
Application: most precise method of localizing specific
proteins within tissues

chromosomes and other cell organelles darker than

the surrounding cytoplasm

Scanned-Probe Microscope

Interference Microscope

combines optical features of phase contrast and

polarizing microscopes to provide contrast in

unstained material
provides a colored 3D-image
measures phase retardation induced by specimen

components by relying on differences in refractive index

can be used to calculate mass of cellular components
compares the refracted light with an unimpeded

uses probes to closely examine the specimen surface

without causing damage or modification
Application:
* maps atomic and molecular shapes
* characterizes magnetic and chemical properties
* determines temperature
Types of Scanned Probe Microscopes:
Type

Fluorescence Microscope

M.)

s
Uses

reference beam and provide an electronic readout of the

data
2 light beams separated by beam splitting prisms

STM (Scanning Tunneling

View

thin metal probe

(tungsten)
detailed view of molecule

Perk

(DNA)
greater resolution than

EM

AFM (Atomic Force

M.)
metal and diamond
probe
3D image
no special preparations

Confocal Microscope

allows visualization of 3-D structures without cutting

sections
uses a scanning laser beam to make a series of sharp

Electron Microscope

permits the visualization of ultrastructures, subcellular

and then display these as a combined high resolution image

Application: 2- or 3-D images of cells for biomedical

structures, and single macromolecules such as myosin

uses electromagnetic field, fluorescent screen/TV monitor,

application

and electron beams (W filaments) instead of glass lenses

electromagnets spread and focus the electron beam
e-beam wavelength is far shorter than that of visible

light
e-beam resolution is about 1000X greater than visible

light
resolving power is about 200 nm
TEM resolving power is 0.2 nm providing a

magnification of 150,000X
Advantages: high resolution, high magnification
Disadvantages: requires vacuum enclosed system, high

images on a photomultiplier tube, computers to record,

UV Microscope

sees beyond what a standard optical microscope can

image
some materials that are transparent or clear in normal

microscopes can be imaged with UV microscopes

can image microscopic samples in the visible and the UV

region
have features that make them superior to normal visible
range microscopes (special UV optics, light sources and

cameras)
by using shorter wavelengths of UV light rather than
longer wavelengths of visible light, higher image

resolution can be obtained

Application: imaging protein crystals that are transparent
in the visible range but can be easily seen at 280 nm due to
the strong absorbance of certain amino acids

voltage, mechanical stability, living tissue cannot be used,

different way of specimen preparation, well-trained staff

Tissue Preparation for Light Microscopy

1. Fixation
preservation of tissue for study
kills the tissue and bacteria
coagulate or cross-link proteins, making them insoluble
COMMON FIXATIVES:
buffered formalin (4% formaldehyde in buffered
isotonic saline)
Bouins fluid (picric acid)
Carnoys fixative
2. Dehydration and Clearing
DEHYDRATION: removal of water from the tissue and

replacement with ethanol (50-70-100%)

fixative is also removed in early steps of dehydration by

several washes of 50% ethanol for 2 hours each

CLEARING: 100% ethanol is replaced by solvent miscible

with the embedding medium (xylene)

as the tissues become infiltrated, they become more

transparent
typically, first mixture of 50% ethanol and 50% xylene

followed by 100% xylene for an hour each

3. Infiltration (Interpenetration)
xylene is replaced by paraffin in an oven at 58-60C
tissues are infiltrated/saturated by immersion in a

medium in which they are finally embedded (e.g. wax)

50:50 mixture of xylene and paraffin (30 minutes) two

changes of 100% paraffin

first paraffin bath lasts for 2 hours, second bath is 3
hours to overnight; best not to exceed 5-6 hours since

tissue tends to shrink in the heat

4. Embedding
tissue is oriented and embedded in a paraffin block
block is placed in ice water to solidify
5. Sectioning
small block of paraffin containing the tissue is mounted

in microtome
MICROTOME: designed to cut thin slices (thickness
between 2 and 10 microns or .002 to 0.010 m)

paraffin affixed to a slide dissolve remove dissolved

paraffin
6. Mounting and Staining
most tissues are colorless (need staining)
(e.g. dyeing or metallic coating of tissue components,

H&E)
PURPOSE OF MOUNTING: for protection and to make the

preparation permanent
coverslip is placed over the section

PROCESSING TISSUES FOR LM AND TEM

PROCEDUR
PURPOSE
LM
E
Preserves tissue
morphology by
Formaldehy
1. Fixation
coagulating
de solution
proteins; stops
autolysis
Pass thru
2.
Removes water
graded
Dehydratio
from cells and
ethanol
n
tissue
series (35
100%)
Enables cells and
tissues to be
Benzene
3. Clearing
penetrated with
(organic
paraffin (LM) or
solvent)
plastic (TEM)
Penetrates cells
and intracellular
4.
spaces giving
paraffin
Embedding
tissue rigidity for
sectioning
Provides thin
5.
5-10 um on
sections of cells
Sectioning
microtome
and tissues
Provides
supporting
6.
medium for
Glass slide
Mounting
viewing and
handling
Removes paraffin
Pass from
7.
so that tissue can
benzene
Rehydratio
be stained with
100% EtOH
n
aqueous solution
35% EtOH
Helps visualize
Hematoxylin
8. Staining
tissue and cell
and Eosin
components
9.
Pass from
Makes permanent
Dehydratio
35%EtOH

TM
Glutaraldehy
de and
osmium
tetroxide

Propylene
oxide
(organic
solvent)

Plastic (Epon)

10-20 nm on
ultramicroto
me

Fine wire grid

Pass down
100% EtOH
35% EtOH
Uranyl
acetate
Pass thru
100% EtOH

100%EtOH
and air dry.
benzene
Store in
and mount
dessicant.
COMMON STAINS AND THEIR AFFINITIES
APP.
TYPES
STAINS
Basophylic tissue
Hematoxylin
components(DNA,
Toluidine Blue
Basic dyes
RNA, polyanions
MB
such as sulfated
Alcian Blue
glycosaminoglycans)
Acidophilic tissue
Eosin
components (basic
Acidic dyes
Orange G
proteins in
Acid Fuschin
cytoplasm)
LM
Long chain
Lipid-soluble
Oil red O
hydrocarbons (fats,
dyes
Sudan black
oils, waxes)
Complex
Periodic Acidcarbohydrates
Schiff (PAS)
Multicomponen
(glycogen,
Reaction
t histochemical
glycosaminoglycan)
Nuclear chromatin
reaction
Feulgens
(DNA and associated
Reaction
proteins)
Uranyl
Nonspecific; adsorb
Acetate
to surfaces and
Lead Citrate
enhance contrast
Actually a fixative,
but binds to
Osmium
phosphate groups of
Tetroxide
membrane
Heavy metal
phospholipids,
TEM
(electron
enhancing contrast
dense)
Polyanions; complex
carbohydrates
e.g. Oligosaccharides
Ruthenium
of glycocalyx and
Red
glycosaminoglycans
of the extracellular
matrix
n