HISTOLOGY

MT/MLS 3rd year first semester s. y. 2019-2020

EFINITIONS… • histos - web or tissue
• logia - study or learning

The Greek root histos can be

translated as either "tissue" or
"web," both of which are appropriate
because tissues are usually webs of
interwoven filaments and fibers,
both cellular and non cellular, with
membranous linings.
 HISTOLOGY
• Histology is the study of the
tissues of the body and how
these tissues are arranged to
constitute organs.
• This subject involves all
aspects of tissue biology,
with the focus on how cells’
structure and arrangement
optimize functions specific to
each organ.
• Histology is the study of
tissues and organs under the
microscope.
HE HIERARCHY OF STRUCTURAL ORGANIZATIO
IGNIFICANCE…
t is one of the tools for the diagnosis of diseas

great many diseases reveal themselves at the

ellular level: many cancers, bone and connective

issue disorders, vascular diseases, liver disease

idney malfunctions, and others can be definitivel

iagnosed using histological techniques.

 WHAT TO LEARN IN HISTOLOGY?

rn the (microscopic) structure while

relating the structure with functions

Physiologic
Biochemical

Disease(pathology)
Treatment
Prevention
 TISSUES AND CELLS

h century
0 - Robert Hooke examined cork with a microscope and
found it was composed of tiny “chambers”.

ook called these chambers cells because they

eminded him of the small rooms or chambers
ound in monasteries that, at that time, were
escribed by the the latin word “cella”.
oke published this information, as 17TH
ll as the results of other CENTURY
croscopic research he had performed
his Micrographia.

milar compartments were found to be

esent in animal tissue.

ditional study revealed that, in

ving tissues, these compartments
re filled with a fluid substance
ich is, of course, the cell
toplasm.
2 - Schleiden and Schwann THE CELL THEORY
dently hypothesized that all
nd animal tissues are
d of cells.

lieved that cells were the

te” potentially independent
MATTHIAS JAKOB SCHLEIDEN
a living organism. 1804 - 1881

, further studies revealed

ese small cells contained
aller structures in their
sm.
THEODOR SCHWANN
1810 - 1882
tains were not used to examine cells in these early
tudies. Scientists using the microscope relied entire
n differences in refractive index to make structures
issues visible.

his didn’t work very well - not enough contrast

nitially only the nucleus (nut) of the cell was noted

ut it soon became obvious that there was an even smal
tructure within the nucleus that was given the name
nucleolus” which means “smaller nut”.
MICROSCOPES
istology requires the use of “Microscopes” t
iew the structures under increasing
agnifications.

his requires preparation of slides that will

hen be viewed under microscopes.

arious stains are introduced to increase

ontrast.
nciple

Microscopy is to get a magnified image, in which

uctures may be resolved that which could not be
n without the help of an unaided eye.
 TERMS AND DEFINITIONS

gnification

It is the ratio of the size of an object seen under

microscope to the actual size observed with unaided eye

The total magnification of microscope is calculated by

multiplying the magnifying power of the objective lens

that of eye piece.

 TERMS AND DEFINITIONS

lving power

It is the ability to differentiate two close points as

separate.

The resolving power of human eye is 0.25 mm

→ The light microscope can separate dots
that are 0.25μm apart.
→ The electron microscope can separate dots
that are 0.5nm apart.
 LIGHT MICROSCOPY

Conventional bright-field microscopy, as well as m

specialized applications like fluorescence, phas

contrast, confocal, and polarizing microscopy, are

based on the interaction of light with tissue

components and are used to reveal and study tiss

features.
 BRIGHT-FIEL
pes of Light Microscopes
MICROSCOP
ight-field microscopy:

Widely used, involves use

fixed and stained slides
ew under an ordinary
.

udes an optical system and Light microscope has a maxi

echanisms to move and focus the
specimen.
resolving power of approxim
0.2 μm.
This power is sufficient to
the image 1000-1500 times.
luorescence microscopy:
sed to view inherently fluorescent substance
or those tissues that have been labeled by
uorescent stains.
es a light of a different wavelength
V light) is focused onto the cells which i
emit light in the visible spectrum that ca
iewed.
escent compounds with affinity for specific cell
molecules may be used as fluorescent stains.

ine orange, which binds both DNA and RNA, is an exa

observed in the fluorescence microscope, these nucl

emit slightly different fluorescence, allowing the
calized separately in cells.

odies labeled with fluorescent compounds are extrem

tant in immunohistologic staining.
Components of cells
often stained with
compounds visible by
fluorescence microsc

(a) Acridine orange

nucleic acids and ca
DNA in cell nuclei (
emit yellow light an
RNA-rich cytoplasm (
appear orange in the
cells of a kidney tu
(b)Cultured cells stain
DAPI (4′,6-diamino-
phenylindole) that
DNA and with fluoresc
phalloidin that binds
filaments show nuclei
blue fluorescence and
filaments stained gre

(c) Important informati

as the greater densit
microfilaments at the
periphery is readily
apparent.(Both X500)

(Figure 1–4b, used with permis

Drs. Claire E. Walczak and Ran
Indiana University School of M
Bloomington.)
hase-Contrast Microscopy
ined cells and tissue sections, which
sually transparent and colorless, can be
ed with these modified light  Uses modifi
scopes. objective l
and condens
lar detail is normally difficult to see allow the
stained tissues because all parts of the viewing of
men have roughly similar optical living tiss
ties. without pri
fixing or
staining.
-contrast microscopy, however, uses a
system that produces visible images from
parent objects and, importantly, can be
with living, cultured cells
re 1–5).
-contrast microscopy is based on the principle that
changes its speed when passing through cellular
xtracellular structures with different refractive
es.

changes are used by the phase-contrast system to c

tructures to appear lighter or darker in relation t
other. Because they allow the examination of cells
ut fixation or staining, phase-contrast microscopes
nent tools in all cell culture laboratories.

ification of phase-contrast microscopy is different

ference microscopy with Nomarski optics, which prod
age of living cells with a more apparent three-
sional (3D)aspect (Figure 1–5c).
Confocal Microscopy
bright-field microscope uses large light source to
oject light thus reducing the contrast in the image
d hence poor resolution.

e confocal microscope therefore uses a small yet

tense source of light- the laser and also a plate
aring a pin-hole aperture to reduce scattering of
ght.
ocal microscopes include a computer-driven mirror

em (the beam splitter) to move the point of illuminatio

ss the specimen automatically and rapidly.

tal images captured at many individual spots in a very

e of focus are used to produce an “optical section” of

e
 utility of all light microscopic methods is greatly

ended through the use of digital cameras.

y features of digitized histological images can be anal

ntitatively using appropriate software.

h images can also be enhanced to allow objects not dire

ible through the eyepieces to beexamined on a monitor.

ELECTRON MICROSCOPY
What is the smallest thing yo
have ever seen?
EEING WITH ELECTRONS

rons are the minute charged particles that

py the outer regions of atoms.
y're also the particles that carry electricity
nd circuits.)

electron microscope, a stream of electrons

s the place of a beam of light.

ectron has an equivalent wavelength of just

1 nanometer, which allows us to see things
ler even than light itself (smaller than the
length of light's photons).
 HOW ELECTRON MICROSCOPES WORK
ever we used an ordinary microscope, the
idea is simple. There's a light at the bottom
hines upward through a thin slice of the
men.
ok through an eyepiece and a powerful lens to
considerably magnified image of the specimen
ally 10–200 times bigger).
ere are essentially four important parts to an
ary microscope:
source of light.
specimen.
lenses that makes the specimen seem bigger.
magnified image of the specimen.
 HOW ELECTRON MICROSCOPES WORK
ctron microscope, these four things are slightly

ght source is replaced by a beam of very fast moving

ons.

pecimen usually has to be specially prepared and held

a vacuum chamber from which the air has been
ed out (because electrons do not travel very far in air).

nses are replaced by a series of coil-shaped

omagnets through which the electron beam travels.
ordinary microscope, the glass lenses bend (or refract)
ht beams passing through them to produce
fication. In an electron microscope, the coils bend the
on beams the same way.

mage is formed as a photograph (called an electron

graph) or as an image on a screen.
 HOW ELECTRON MICROSCOPES WORK

e are a few different types of electron microscopes and they all work in
rent ways.

wo most familiar types are called:

Transmission Electron Microscopes (TEMs)

canning Electron Microscopes (SEMs),

 HOW A TRANSMISSION ELECTRON MICROSCOPE (TEM) WORKS
ission electron microscope fires a beam of electrons through a specimen to
magnified image of an object.

age electricity supply powers the cathode.

de is a heated filament, a bit like the electron gun in an old-fashioned cathode-ray

) TV. It generates a beam of electrons that works in an analogous way to the beam
an optical microscope.

magnetic coil (the first lens) concentrates the electrons into a more powerful beam.

ectromagnetic coil (the second lens) focuses the beam onto a certain part of the

men sits on a copper grid in the middle of the main microscope tube. The beam
ough the specimen and "picks up" an image of it.

ctor lens (the third lens) magnifies the image.

e becomes visible when the electron beam hits a fluorescent screen at the base of
ne. This is analogous to the phosphor screen at the front of an old-fashioned TV .

e can be viewed directly (through a viewing portal), through binoculars at the side, or
onitor attached to an image intensifier (which makes weak images easier to see).
 HOW A SCANNING ELECTRON MICROSCOPE (SEM) WORKS

electron microscope scans a beam of electrons over a specimen to produce a

age of an object. That's completely different from a TEM, where the beam of electrons
ough the specimen.

e fired into the machine.

rt of the machine (where the object is scanned) is contained within a sealed vacuum
ause precise electron beams can't travel effectively through air.

harged electrode (anode) attracts the electrons and accelerates them into an energetic

agnetic coil brings the electron beam to a very precise focus, much like a lens.

lower down, steers the electron beam from side to side.

stematically scans across the object being viewed.

m the beam hit the surface of the object and bounce off it.

gisters these scattered electrons and turns them into a picture.

gnified image of the object is displayed on a TV screen.

ning electron microscopes (SEMs), are designed to make images of the surface
s.
s in a TEM, the top of a SEM is a powerful electron gun that shoots an electron b
specimen.
es of electromagnetic coils pull the beam back and forth, scanning it slowly and
matically across the specimen's surface.
d of traveling through the specimen, the electron beam effectively bounces straigh
ectrons that are reflected off the specimen (known as secondary electrons) are dir
n, similar to a cathode-ray TV screen, where they create a TV-like picture.
are generally about 10 times less powerful than TEMs (so we can use them to s
10 nanometers in size).
e plus side, they produce very sharp, 3D images (compared to the flat images pro
) and their specimens need less preparation.
 1 2
ypical images produced by a SEM.

artificially colored, scanning electron micrograph showing Salmonella typhimurium

ding cultured human cells.
anning electron micrograph of the bacteria Escherichia coli (E. coli).

courtesy of Rocky Mountain Laboratories, US National Institute of Allergy and Infectious Diseases (NIA
stitute of Health.
 AUTORADIOGRAPHY
roscopic autoradiography is a method of localizing newly
nthesized macromolecules in cells or tissue sections.

dioactively labeled metabolites (nucleotides, amino acids,

gars) provided to the living cells are incorporated into specific
cromolecules (DNA, RNA, protein, glycoproteins, and
ysaccharides) and emit weak radiation that is restricted to th
ions where the molecules are located.

autoradiography the specimen itself is the source of the radiation, whic

ginates from radioactive material incorporated into it.
 Autoradiography Method

cells are briefly exposed to a ‘pulse’ of a specific radioactive compound.

ssue is left for a variable time.
les are taken, fixed, and processed for light or electron microscopy.
ns are cut and overlaid with a thin film of photographic emulsion.
the dark for days or weeks (while the radioisotope decays). This exposure
ds on the activity of the isotope, the temperature and the background radia
oduce with time a contaminating increase in ‘background’ silver grains in th
hotographic emulsion is developed (as for conventional photography).
 Autoradiography Method

terstaining e.g. with toluidine blue, shows the histological details of the tissue. The
ust be able to penetrate, but not have an adverse affect on the emulsion.
atively, pre-staining of the entire block of tissue can be done (e.g. with Osmium on
ns coated with stripping film [or dipping emulsion] as in papers by McGeachie and
nds) before exposure to the photographic emulsion. This avoids the need for individ
) staining each slide.
ot necessary to coverslip these slides
osition of the silver grains in the sample is observed by light or electron microscop
ains are in a different plane of focus in the emulsion overlying the tissue section. O
x100 objective is used for detailed observation with the light microscope.
e autoradiographs provide a permanent record. Full details on the batch of emulsio
, exposure time and conditions should be kept for each experiment.
re are autoradiographs from the salivary gland of a mouse injected with 3H-fucose 8 hours
ation. Fucose was incorporated into oligosaccharides, and the free 3H-fucose was removed
nd sectioning of the gland. Autoradiographic processing and microscopy reveal locations of
ed glycoproteins containing that sugar.

grains of silver from the light-sensitive material coating the specimen are visible over cell
etory granules and the duct indicating glycoprotein locations. (X1500)
ame tissue prepared for TEM autoradiography shows silver grains with a coiled or amorpho
ce again localized mainly over the granules (G) and in the gland lumen (L). (X7500)
PLICATIONS
find and investigate the various properties of DNA

find the location and amount of particular substance within a cells includi

l organelle, metabolites etc.

sue localization of radioactive substance

find the site and performance of a targeted drug.

locate the metabolic activity site in the cell.

e small size of cells and matrix components makes histology dependent

e use of microscopes and molecular methods of study.

vances in biochemistry, molecular biology, physiology, immunology, and

thology are essential for a better knowledge of tissue biology.

miliarity with the tools and methods of any branch of science is essentia

a proper understanding of the subject.

PREPARATION OF TISSUES
FOR STUDY
The most common procedure used in histologic research is the preparat
of tissue slices or “sections” that can be examined visually with transmit
ight.
Because most tissues and organs are too thick for light to pass through,
translucent sections are cut from them and placed on glass slides for
microscopic examination of the internal structures.
The ideal microscopic preparation is preserved so that the tissue on the
slide has the same structural features it had in the body.
However, this is often not feasible because the preparation process can
remove cellular lipid, with slight distortions of cell structure.
The basic steps used in tissue preparation for light
microscopy
 issues studied histologically are prepared as shown, with this sequence of st

tion: Small pieces of tissue are placed in solutions of chemicals that cross-l
eins and inactivate degradative enzymes, which preserves cell and tissue
cture.
ydration: The tissue is transferred through a series of increasingly concentr
hol solutions, ending in 100%, which removes all water.
aring: Alcohol is removed in organic solvents in which both alcohol and para
ible.
ltration: The tissue is then placed in melted paraffin until it becomes compl
rated with this substance.
bedding: The paraffin-infiltrated tissue is placed in a small mold with melted
ffin and allowed to harden.
mming: The resulting paraffin block is trimmed to expose the tissue for sectio
rotome is used for sectioning paraffin-
ed tissues for light microscopy.
rimmed tissue specimen is mounted in
ffin block holder, and each turn of the
eel by the histologist advances the holder
led distance, generally from 1 to 10 μm.
each forward move, the tissue block
ver the steel knife edge and a section is
thickness equal to the distance the block
d. The paraffin sections are placed on
des and allowed to adhere, deparaffinized,
ned for light microscope study.
EM, sections less than 1 μm thick are
d from resin embedded cells using and
rotome with a glass or diamond knife.
 STAINING
ost cells and extracellular material are completely colorless,
d to be studied microscopically tissue sections must be stained
dyed).

ll components such as nucleic acids with a net negative charge

nionic) have an affinity for basic dyes and are termed
asophilic; cationic components, such as proteins with many
nized amino groups, stain more readily with acidic dyes and ar
rmed acidophilic.
asic dyes includes: toluidine blue, alcian blue, and methylene blue.
ematoxylin behaves like a basic dye, staining basophilic tissue componen

he main tissue components that ionize and react with basic dyes do so be
of acids in their composition (DNA, RNA, and glycosaminoglycans).

cid dyes (eg, eosin, orange G, and acid fuchsin) stain the acidophilic
omponents of tissues such as mitochondria, secretory granules, and collag
atoxylin and Eosin (H&E) is used most commonly.

atoxylin stains DNA in the cell nucleus, RNA-rich portions of the

lasm, and the matrix of cartilage, producing a dark blue or purple col
ntrast, eosin stains other cytoplasmic structures and collagen pink
re 1–2a).

n is considered a counterstain, which is usually a single dye applied

rately to distinguish additional features of a tissue.

complex procedures, such as trichrome stains (eg, Masson trichrome

greater distinctions among various extracellular tissue components
Periodic Acid-Schiff (PAS) reaction utilizes the hexose rings of
saccharides and other carbohydrate-rich tissue structures and stains su
omolecules distinctly purple or magenta.

DNA of cell nuclei can be specifically stained using a modification of the

edure called the Feulgen reaction.
d-rich structures of cells are revealed by avoiding the processing steps
remove lipids, such as treatment with heat and organic solvents, and
ning with lipid-soluble dyes such as Sudan black, which can be useful in
nosis of metabolic diseases that involve intracellular accumulations of
esterol, phospholipids, or glycolipids.

common methods of staining can employ metal impregnation techniqu

cally using solutions of silver salts to visual certain specific cellular
ments in nervous tissue.
preparation, from tissue fixation to
vation with a light microscope, may
rom 12 hours to 2½ days, depending
e size of the tissue, the embedding
um, and the method of staining.

nal step before microscopic

vation is mounting a protective glass
slip on the slide with clear adhesive.
ISTOLOGY & ITS METHODS OF STUDY (KEY POINTS
aration of Tissues for Study
emical fixatives such as formalin are used to preserve tissue
ucture by cross-linking and denaturing proteins, inactivating
zymes, and preventing cell autolysis or self-digestion.
hydration of the fixed tissue in alcohol and clearing in organic
vents prepare it for embedding and sectioning.
bedding in paraffin wax or epoxy resin allows the tissue to be
o very thin sections (slices) with a microtome.
tions are mounted on glass slides for staining, which is requir
eal specific cellular and tissue components with the microsco
 ISTOLOGY & ITS METHODS OF STUDY (KEY POINTS

paration of Tissues for Study

e most commonly used staining method is a combination of th
ains hematoxylin and eosin (H&E), which act as basic and acid
es, respectively.
ll substances with a net negative (anionic) charge, such as DN
NA, react strongly with hematoxylin and basic stains; such mat
said to be “basophilic.”
tionic substances, such as collagen and many cytoplasmic pro
act with eosin and other acidic stains and are said to be
cidophilic.”
ISTOLOGY & ITS METHODS OF STUDY (KEY POINTS

t Microscopy
ight-field microscopy, the method most commonly used by bo
udents and pathologists, uses ordinary light and the colors are
parted by tissue staining.
uorescence microscopy uses ultraviolet light, under which onl
orescent molecules are visible, allowing localization of fluores
obes which can be much more specific than routine stains.
ase-contrast microscopy uses the differences in refractive ind
rious natural cell and tissue components to produce an image
thout staining, allowing observation of living cells.
ISTOLOGY & IT’S METHODS OF STUDY (KEY POINTS

t Microscopy
ase-contrast microscopy uses the differences in refractive ind
rious natural cell and tissue components to produce an image
thout staining, allowing observation of living cells.
nfocal microscopy involves scanning the specimen at successi
cal planes with a focused light beam, often from a laser, and
oduces a 3D reconstruction from the images.
ISTOLOGY & IT’S METHODS OF STUDY (KEY POINTS
radiography
is process localizes cell components synthesized using radioactive
ecursors by detecting silver grains produced by weakly emitted
diation in a photographic emulsion coating the tissue section or ce
th either light microscopy or TEM, autoradiography permits uniqu
udies of processes such as tissue growth (using radioactive DNA
ecursors) or cellular pathways of macromolecular synthesis.
ISTOLOGY & IT’S METHODS OF STUDY (KEY POINTS
& Tissue Culture
ls can be grown in vitro from newly explanted tissues (primary cultures
long-established cell lines and can be examined in the living state by ph
ntrast light microscopy.

L APPLICATION
ure is very widely used to study molecular changes that occur in cancer; to ana
us viruses, mycoplasma, and some protozoa; and for many routine genetic or
somal analyses. Cervical cancer cells from a patient later identified as Henriett
d from the disease in 1951, were used to establish one of the first cell lines, cal
lls, which are still used in research on cellular structure and function througho
ISTOLOGY & IT’S METHODS OF STUDY (KEY POINTS
yme Histochemistry
istochemical (or cytochemical) techniques use specific enzymatic activi
ghtly fixed or unfixed tissue sections to produce visible products in the s
nzyme locations.
xation and paraffin embedding denatures most enzymes, so histochemi
sually uses frozen tissue sectioned with a cryostat.
nzyme classes for which histochemical study is useful include phosphat
ehydrogenases, and peroxidases, with peroxidase often conjugated to
ntibodies used in immunohistochemistry.
ISTOLOGY & IT’S METHODS OF STUDY (KEY POINTS
DICAL APPLICATION
use cells in some diseases, including many cancer cells, often produce prot
ue to their pathologic condition, immunohistochemistry can be used by
ologists to diagnose many diseases, including certain types of tumors and
s-infected cells.
ISTOLOGY & IT’S METHODS OF STUDY (KEY POINTS

Sections of cells or tissues

are essentially 2D planes
through 3D structures,
and understanding this
fact is important for their
correct interpretation and
study.
 SHORT QUIZ
1.
THE CELL
SHORT QUIZ