Tools for studying

cell and tissue structures

Histology:
-morphology of plant and animal tissues
-the cells that compose them both at a morphological and functional level

Modern histology:
-descriptive science
-aspects of molecular and cell biology, which help describe cell organization and function

➢Morphological methods

➢Biochemical/Cell and Molecular Biology methods

Data provided by these methods can help the student
acquire a better understanding of cells, tissues, and
organ functions
 ➢Morphological Methods:

Microscopes and Image diagnostic technology (light

microscopy, TEM and SEM, AFM)
➢Biochemical /Molecular Biology Methods
Gene structure and regulation
DNA sequencing
Polymerase chain reaction
Northern blotting
Southern blotting
DNA microarray/RNA-seq
Bioinformatics

Protein structure and functions

Western blotting
Chromatography
ELISA
X-ray crystallography

Interactions between Biomolecules

Coimmunoprecipitation (protein-protein)
ChiP (protein-DNA)
Electrophoretic mobility shift assay (protein–DNA
or protein–RNA)
Southwestern blotting (DNA-protein)
 ➢Cells and tissues can be studied in vivo and in vitro

(use of animal models, use of cell cultures)

The remarkable anatomical and physiological similarities Cell culture is defined as the removal of animal cells and its
between humans and animals have prompted propagation and cultivation in vitro in an artificial
researchers to investigate a large range of mechanisms environment that is suitable for its growth.
and assess novel therapies in animal models before Cell cultures: short term or established cell lines which are
applying their discoveries to humans widely used for research and diagnosis.
In vitro cell culture
to study every cell
types isolated from -adherent cell cultures
-suspension cultures
the body
-short term cultures
-established cell lines

But how to obtain a cell

culture starting from a
tissue?
 Differential centrifugation
The subcellular fractionation protocol is a procedure for
separating nuclear, membrane and cytoplasmic cell
fractions using centrifugation methods:
1. Put suspension of broken cells in fractionation
buffer and centrifuge at 800 g x 10 min (pellet
contains nuclei and supernatant cytoplasm,
membrane and mitochondria)
2. Centrifuge the supernatant at 10,000 x g for 5 min
(pellet contains mitochondria and supernatant
cytoplasm and membrane fraction)
3. For a membrane fraction, centrifuge the
supernatant in an ultracentrifuge at 100,000 x g for
1 h. Wash and resuspend pellet by adding 400 μL of
fractionation buffer
4. Re-centrifuge for 45 min. Resuspend the membrane
pellet in the same buffer as used for the nuclei
 ➢Morphological methods - Microscopes

Essential instrument of the histologist is

the optical microscope, which allows the
direct observation of the tissues to study.

Why do you need a

microscope?

Because a microscope is an instrument that

magnifies an image and allows visualization of
greater detail than is possible with the eye.
 Resolution limit
(distance by which two objects must be separated to be seen as two objects)

0.2 mm

Resolution limit of the human eye 0.2 mm

Resolution limit of the optical microscope 0.2 m
Resolution limit of the electron microscope 0.25 nm
• Measurement Units in Cytology and Histology
mm (10-3 m)

m (10-3 mm)

nm (10-3 m)

A (10-4 m)
 Optical and electron microscopes

The resolution limit of the human eye is determined by the spacing of

the photoreceptor cells in the retina

Optical tools are needed for improving the resolution limit of the
human eye
 Optical and electron microscopes
• The most important parameter for a microscope is its RESOLUTION and not its magnification
• RESOLVING POWER: minimal distance (d) at which 2 dots are resolved as separated

0.2 mm

0.0002 (2x10-4) mm

=0.2 µm
 Magnification

Telescope: magnification of distance

objects

Microscope: magnification of small,

close obsjects

Process of enlarging the apparent size, not physical size, of something

 Resolution power

We obtain this by
Increasing the resolution
power
Typically, magnification is related to scaling up visuals or images to be
able to see more detail, but this is due to an increase of resolution
 RESOLUTION LIMIT OF A MICROSCOPE
The resolution limit of a microscope can be
calculated by the Abbe equation:
wave lenght of
Minimal distance the light source
i lens aperture

N=refractive index of the medium between

the objective front lens and the specimen
(ranges from 1.00 for air to 1.51 for
immersion oils)
 • Theoretical resolving power of the optical microscope :
0.4m = 0.2m ( m = 10-3mm)
d=
2x1x1
The magnification of the optical microscope is the product of the magnification of
the eyepiece (say 10x) and the objective lens (say 100x), to give a total magnification
Maximum
of 1000×. Modified useful such
environments as the use of oil1.500
magnification times
or ultraviolet light can
increase the magnification, arriving to a maximum magnification of 1500x

• Theoretical resolving power of the electron microscope :

5x10-4m = 2.5x10-4m = 0.25 nm (1nm=10-3m)
d=
2x1x1
The primary improvement in the EM versus the light microscope is that the
wavelength of the EM beam is approximately 1/2,000 that of the light microscope
Maximum
beam, thereby increasinguseful
resolution by a factor of 103250.000
magnification times
. Objects can be magnified to
250000 times their actual size.
 OPTICAL MICROSCOPES
1. Brightfield microscope: commonly used for routine histological examination
2. Stereomicroscope: to study the surfaces of solid specimens or to carry out close
work such as dissection and microsurgery
3. Phase contrast microscope: unstained cells/tissues, especially useful for living cells
4. Dark field microscope: to detect smaller individual particles, useful in examining
autoradiographs
5. Interference microscope: allows quantification of tissue mass
6. Polarized microscope: allows the observation of tissues, as striated muscle,
exhibiting birefrangence
7. Fluorescence microscope: makes use of the ability of certain molecules to fluoresce
under ultraviolet light
8. Confocal microscope: light microscope+scanning system; allowes visualization of a
biologic specimen in three dimensions
 Brightfield microscope

Commonly used for routine histological examination,

consists of:
• a light source for illumination of the specimen
• a condenser lens to focus the beam of light at the level
of the specimen
• a stage on which the slide or other specimen is placed
• an objective lens to gather the light that has passed
through the specimen
• a pair of ocular lenses through which the image
formed by the objective lens may be examined directly
Brightfield microscope

By examining a number of such two-dimensional sections, it is possible to

create the three-dimensional configuration of the examined structure
 Stereo or dissecting microscope

-optical microscope variant

-low magnification observation of a sample,
-typically using light reflected from the surface of an
object rather than transmitted through it.

Two separate optical paths with two objectives and

eyepieces to provide slightly different viewing angles to
the left and right eyes. This arrangement produces a
three-dimensional visualization of the sample being
examined.

Used to study the surfaces of solid specimens or to carry

out close work such as dissection, microsurgery.
Stereo or dissecting microscope
 Phase contrast microscope
-Takes advantage of small differences in the refractive index in
different parts of a cell or tissue sample.
-Light passing through areas of relatively high refractive index (denser
areas) is deflected and becomes out of phase with the rest of the
beam of light that has passed through the specimen.
-Dark portions → dense portions of the specimen;
-Light portions → less dense portions of the specimen.

Enables examination of unstained cells and tissues and is especially

useful for living cells.
 Differential interference contrast (DIC) microscope
Modification of the phase contrast microscope with:
• Polarizer: polarizing filter located between the light source and
the specimen, it produces the necessary plane-polarized light for
interference imaging
• Condenser Nomarski Prism: specialized beamsplitting prism that
separate the polarized light emanating from the polarizer into
two components
• Objective Nomarski Prism: second beamsplitting prism that
recombine the sheared wavefronts in the conjugate plane of the
objective rear aperture
• Analyzer: second polarizer located between the objective lens
and the viewer, components of circular and elliptically polarized
light arriving from the objective prism pass through the analyzer
and subsequently undergo interference to generate the DIC
image at the microscope.

This microscope is especially useful for assessing surface properties

of cells.
 Differential interference contrast microscope
PC-OM IN-OM

Dense subcellular particles (nuclei, nucleoli, mitochondria, filaments, metaphase chromosomes, and lysosomes)
usually display the appearance of raised elevations (hilltops), while lower refractive index inclusions (pinocytotic
vesicles, aqueous vacuoles, and lipid droplets) appear to be sunken depressions (craters)
Phase contrast vs DIC microscope
A fibroblast under brightfield, phase contrast, differential
interference and dark-field microscope: a comparison
 Fluorescence microscope
A molecule that fluoresces emits light of wavelengths in
the visible range when exposed to an UV source

Display autofluorescent molecules such as vitamin A and

some neurotransmitters

Display introduced fluorescence, as in the detection of

antigens or antibodies in immunocytochemical staining
procedures

The specimen is illuminated with light of a specific

wavelength which is absorbed by the fluorophores, causing
them to emit light of longer wavelengths (i.e., of a different
color than the absorbed light)
 Fluorescence microscope

Components:
1. light source: xenon or mercury-vapor
lamp are common; more advanced
forms are high-power LEDs and lasers
2. excitation filter: produce single-
wavelength or narrow-band–wavelength
light
3. dichroic mirror
4. emission or barrier filter: allows only the
narrow band of wavelength of the
fluorescence to reach the eye or to
reach a photographic emulsion or other
analytic processor
 Fluorescence microscope
A fluorophore is a fluorescent chemical compound that can re-emit light
upon light excitation
Fluorophores typically contain several combined aromatic groups, or plane
or cyclic molecules with several π bonds
Fluorescein, via its amine-reactive isothiocyanate derivative fluorescein
isothiocyanate (FITC), has been one of the most popular fluorophores
Other historically common fluorophores are derivatives of rhodamine
(TRITC), coumarin, and cyanine
DAPI and Hoechst are small molecules intrinsically fluorescent that bind
the minor groove of DNA, thus labeling the nuclei of cells
 Fluorescence microscope
Immunofluorescence: technique which uses the highly specific binding of an antibody to its antigen in order to
label specific proteins or other molecules within the cell.
DIRECT IF: primary antibody specific for the molecule of interest can be directly conjugated to a fluorophore;
INDERECT IF: a secondary antibody, which binds specifically to the first antibody, is conjugated to a fluorophore
DAPI Actin
The techniques available for modifying
DNA allow scientists to genetically
modify proteins to also carry a
fluorescent protein reporter (e.g. Tubulin
green fluorescent protein, GFP). In
biological samples this allows a
scientist to directly make a protein of Tubulin Merge
interest fluorescent. The protein
location can then be directly tracked,
including in live cells
Fluorescence microscope
IMMUNOCYTOCHEMISTRY/IMMUNOHISTOCHEMISTRY
to identify specific cell types, organelles and molecules

Underlying basis: specificity of a reaction between an antigen and an antibody

Immunofluorescence: use of antibodies conjugated

with fluorescent dyes (fluorochromes)

Immunohistochemistry: antibodies conjugated with

enzymes (e.g., peroxidase with DAB substrate)
 IMMUNOCYTOCHEMISTRY/IMMUNOHISTOCHEMISTRY
Antibodies are glycoproteins that are produced by specific cells of the immune system in response to a foreign protein, or
antigen. In the laboratory, antibodies can be purified from the blood of animals and conjugated to a fluorochrome (e.g.
fluorescein) or an electron-dense material (e.g. gold)
OM
Antigenic site
Antibody

OM
TEM

E
 Immunofluorescence
Direct IF: the oldest immunocytochemistry
technique used for identifying the distribution of an
antigen within cells and tissues, it uses a
fluorochrome-labeled primary antibody that reacts
with the antigen within the sample. Low intensity of
the signal emission

Indirect IF: the fluorochrome is conjugated with a

secondary antibody directed against primary
antibody. Advantages: It considerably enhances the
fluorescence signal emission from the tissue, a
single secondary antibody can be used to
localize the tissue-specific binding of several
different primary antibodies and the secondary
antibody can be conjugated with different
fluorescent dyes so that multiple labels can be
shown in the same tissue section
Immunofluorescence
-catenin
actin

Epithelial cells
Immunofluorescence
chromatids
kinetochores
microtubules

Mitotic spindle
 Confocal microscope

The two lenses (objective and phototube lens) are

perfectly aligned to focus light from the focal point of
one lens to the focal point of the other lens

The major difference between a conventional and a

confocal microscope is the addition of a pinhole
aperture that is conjugate with the focal point of the
lens; therefore, it is confocal

This precisely positioned pinhole allows only “in-

focus” light to pass into a detector, whereas the “out-
of-focus” light is blocked from entering the detector
 Confocal microscope
Several advantages over conventional widefield
optical microscopy:

• ability to control depth of field

• elimination or reduction of background

information away from the focal plane:
exceptional resolution and clarity from a thin
section of a biologic sample simply by
rejecting out-of-focus light

• capability to collect serial optical

sections from thick specimens: use the
computer to make three-dimensional
reconstructions of a series of these images,
that can be animated for viewing on the
computer or over the Internet from any
orientation desired
 TEM Theoretical resolving
Components: power:
• An electron source, such as a heated tungsten 5x10-4m = 2.5x10-4m =
fi lament, emits electrons. 0.25 nm
• The electrons are attracted toward an anode.
• An electrical difference between the cathode
and the anode creates the electron beam.
•The
Thewavelength of an electron
beam passes through a series ofcan be up to 100,000 times shorter than that of visible light
photons, so the
electromagnetic electron
lenses microscope
to control has a higher resolving power than a light microscope and
the electron
can reveal
beam theitstructure
and focus of smaller objects.
to form an image
• The condenser lens shapes and changes the electromagnetic lenses
diameter of the electron beam that reaches the
specimen plane.
• The beam that has passed through the
specimen is then focused and magnified by an
objective lens and then further magnified by
one or more projector lenses.
• The final image is viewed on a phosphor-
coated fluorescent screen or captured on a
photographic plate.
 TEM (EXAMPLES OF TEM IMAGES)
Images obtained with TEM are black
(electron-dense areas, that have scattered
electrons because of their inherent density
or because of heavy metals added during
specimen preparation) and white (portions
of the specimen through which electrons
have passed)

Using TEM subcellular components can be

visualized

Lymphocyte
TEM (EXAMPLES OF TEM IMAGES)

Secretory cell
 TEM (EXAMPLES OF TEM IMAGES)
Proteoglycan
Development of quantitative electron microscopic
methods has made it possible to define the age-
related structural changes in aggregating
proteoglycans and to correlate the alterations in
their structure with changes in tissue composition
and morphology.

With increasing age the length of the chondroitin

sulfate (GAG)-rich region of aggregating
proteoglycan monomers decreases, the variability in
proteoglycan length increases, the density of keratan
sulfate (GAG) chains increases, the number of
monomers per aggregate decreases, and the
proportion of monomers that aggregate declines.
 SEM Mitochondrion

The principle of SEM is similar to that of TEM, with some

differences:

➢ SEM is based on scattered electrons while TEM is based on

transmitted electrons: Electrons reflected from the surface
(backscattered electrons) and electrons forced out of the
surface (secondary electrons) are collected by one or more
detectors and reprocessed to form a high-resolution three-
dimensional image of a sample surface

➢ SEM focuses on the sample’s surface and its composition,

whereas TEM provides the details about internal
composition (it is like taking a picture with a camera and
having an Xray)
 SEM

Electrons reflected from the

surface (backscattered electrons)
and electrons forced out of the
surface (secondary electrons) are
collected by one or more
detectors and reprocessed to
form a high-resolution three-
dimensional image of a sample
surface
 SEM (EXAMPLES OF SEM IMAGES)
Macrophage: SEM TEM

Lysosome

phagosomes residual
bodies
 SEM (EXAMPLES OF SEM IMAGES)

hair
The outer layer of hair (the cuticle) has overlapping scales of keratin. These scales are thought to
prevent hairs from matting together.
 SEM (EXAMPLES OF SEM IMAGES)

Follicular cells surround the

oocyte and project cytoplasmic
processes in the perioocytic
space. oocyte
The oolemma mostly appears
smooth with short microvilli.

follicular
cells
 SEM (EXAMPLES OF SEM IMAGES)

ant
 Immunoelectron microscopy
Combines the ability of an antibody to
specifically bind a protein with the
high spatial resolution of an electron gold
microscope in order to determine the
protein’s location at subcellular level

The visualization is performed by TEM

aquaporin
Detection of the antibody’s
localization in the sample is possible
by conjugating it with colloidal gold
particles of known diameter, that
increases electron scatter to reveal
high contrast “spots” which allow
analytes to be visualized a protein called
aquaporin is
internalized by
endocytosis
 How to obtain a section for the
microscope starting from
a biological sample
The basic problems are similar for OM and TEM

The principles used in the preparation of sections for viewing with the TEM are
essentially the same as those used in OM, with the added constraint that at every step
one must work with specimens three to four orders of magnitude smaller or thinner than
those used for light microscopy
 Sample preparation
Problem 1

Tissues decompose after collection

Most parts of our tissues are soft

Fixation, usually by a (needs to fix and harden tissue ) The specimen has to be
chemical or mixture of infiltrated with an
Chemical fixation (formalin) and wax paraffin or freezing for OM
chemicals, permanently embedding medium to
preserves the tissue Chemical fixation (glutaraldehyde and osmium tetroxide) and permit sectioning
structure for plastic resins for TEM
subsequent treatments
 Sample preparation
Problem 2

Light and electrons can pass

only through thin or very thin
materials

(needs tissue sectioning)

5-15 m for OM
50-100 nm for TEM
 Sample preparation
Problem 3

Cell and tissue structures are optical

empty (do not make contrasts)
(needs to stain tissue sections)

Dye staining (H&E or other stainings) for OM

Heavy-metal ions (osmium tetroxide or others) for TEM
 Eosin
A typical
protocol for the
preparation of
histological Hematoxylin

samples in
paraffin

*
 Histological slide preparation

Nostra Signora
del Buon consiglio
 STAININGS

➢Cyto-histomorphological stainings: cell and tissue structural features (H&E and

others)

➢Cyto-histochemistry stainings: function of cells and the extracellular

components of tissues (may be based on specific binding of a dye, use of a
fluorescent dye–labeled antibody with a particular cell component, or the inherent
enzymatic activity of a cell component)
CYTO-HISTOMORPHOLOGICAL
STAININGS
Hematoxylin and Eosin (H&E)
 Hematoxylin and Eosin (H&E)
Hematoxylin behaves as a basic dye (positive
charge), it reacts with anionic components of
cells and tissue (phosphate groups of nucleic
acids, the sulfate groups of
glycosaminoglycans, and the carboxyl groups
of proteins), staining them purple. The ability
of such anionic groups to react with a basic
dye, as hematoxylin, is called basophilia [Gr.,
base-loving].

Eosin is an acidic dye (negative charge), it

reacts with cationic groups in cells and
tissues (ionized amino groups of proteins),
staining them pink. The reaction of cationic
groups with an acidic dye, as eosin, is called
acidophilia [Gr., acid-loving].
 Azan-Mallory (AM)
The AZAN staining (acronym of AZocarmine.-ANilin blue) modified
by Mallory is a technique that stains the collagen fibers of
connective tissue.
The acidic azocarmine to stain the cell in red is associated to a
contrast staining obtained by the anilin blue, which colors the
collagen fibers very strong blue.
 Azan-Mallory (AM)

Loose connective tissue typically

contains many cells, stained red by
azocarmine. Because dense
connective tissue contains thick
collagen bundles, it stains more
intensely with the blue dye (anilin
blue)

Epiglottis
 H&E vs Mallory’s trichrome vs Azan-Mallory
Azan-Mallory however is not a stain. It is a
colloquial term used to describe the ‘Azan’
modification of ‘Mallory’s’ trichrome connective
tissue stain. Frank Mallory used acid fuchsin,
orange G and analine blue to stain nuclei and
muscle red, collagen blue and red blood cells
orange. M. Heidenhain came along and tweaked
the recipe by adding azocarmine in place of the
acid fuchsin to make the nuclei dark red, muscle
orange and collagen blue. The differences between
the two are not particularly huge. But they are
dramatically different from H&E!!
 May-Grunwald-Giemsa
This method is used for staining blood smears.
May-Grünwald staining combines the effect of
acidic eosin and alkaline methylene blue.

This allows to stain all cellular components of the blood

pH (6.5 and 6.8) is very important for a correct staining

1. Methylene blue stains blue the acidic components of the cell

2. Eosin stains orange-red the basic components of the cell
3. Giemsa (eosin & azurII) stains red and purple the basic cellular
components
 May-Grunwald-Giemsa
Eosinophil
Platelets
Neutrophil

Lymphocyte

Basophil
Monocyte
 Alkaline phosphatase staining

➢ Method used to identify and monitor stem cells by staining for alkaline phosphatase activity
➢ Pluripotent stem cells typically have higher levels of alkaline phosphatase activity than differentiated cell types,
so alkaline phosphatase staining / activity is often used as a biomarker of stem cells
➢ Add in the cell culture a substrate of AP, so the enzyme will act on this substrate, producing a red colour in stem
cells that is imaged with light microscopy
 TUNEL staining

Method for detecting DNA fragmentation,

widely used to identify and quantify dying
cells, or to detect excessive DNA breakage
in individual cells.

The assay relies on the use of terminal

deoxynucleotidyl transferase (TdT), an
enzyme that catalyzes attachment of
deoxynucleotides, tagged with a
fluorochrome or another marker, to 3'-
hydroxyl termini of DNA double strand
breaks, usually generated during cell death.
 Silver staining (to identify the Golgi apparatus)
Technique to produce yellow to orange or brown shades, by adding a mixture containing silver compounds (notably
silver nitrate)

Wall of ductus epididymis showing the apically oriented Golgi apparatus stained
black with silver. Note basal location of nuclei (hematoxylin)
Nucleic acids staining
 DNA IS DEMONSTRATED BY

FEULGEN technique demonstrates SUGAR (Deoxyribose)

It stains DNA only

METHYL GREEN PYRONIN (MGP) technique demonstrates

PHOSPHATE
It stains both DNA and RNA

Acridine Orange is a cell-permeant nucleic acid binding

dye that emits green fluorescence when bound to dsDNA
and red fluorescence when bound to ssDNA or RNA.
 Nucleic acids staining
DNA green
RNAs red
Nucleolus
(nucleolar RNA)

Methyl green-pyronin (MGP) staining

 PAS staining

The periodic acid–Schiff (PAS)

reaction stains carbohydrates and
carbohydrate-rich macromolecules

It is used to demonstrate glycogen

in cells, mucus in various cells and
tissues, the basement membrane
that underlies epithelia, and
reticular fibers in connective tissue

Goblet cell