Microscopy

Microcope
Microscope: A microscope is an instrument used to
see objects that are too small for the naked eye.

Microscope provides magnification to see

microorganisms with magnifying ranges from X100 to
X400000.

Microscopy: The science of investigating small

objects using microscope is called microscopy.

Microscopic: Microscopic means invisible to the eye

unless aided by a microscope.
 History
Zacharias Janssen - Dutch spectacle makers ---- 1590
Magnification was only
around 9x

In 1665, Robert Hooke stated that life’s smallest structural units

were cells with the help of a crude microscope.
Micrographia, published in 1665, is a historic book written by
Robert Hooke documenting his observations through different
lenses.
He devised a compound microscope and used it to observe
fleas, sponges, bird feathers, plants and molds.
 Antonie van Leeuwenhoek (1632-1723)
a Dutch draper and scientist

Leeuwenhoek observed animal and plant

tissue, human sperm and blood cells,
minerals, fossils, and many other things
that had never been seen before on a
microscopic scale.

Magnification was around 270x

•18th century – Technical innovations improved
microscopes, leading to microscopy becoming popular
among scientists.

•1872 – Ernst Abbe, then research director of the Zeiss

Optical Works, wrote a mathematical formula called the
"Abbe Sine Condition". His formula provided
calculations that allowed for the maximum resolution in
microscopes possible.

• 1903 – Richard Zsigmondy developed the

ultramicroscope that could study objects below the
wavelength of light.
•1931 – Ernst Ruska co-invented the electron microscope
for which he won the Nobel Prize in Physics in 1986.
Later, he developed SEM and TEM.

•1932 – Frits Zernike invented the phase-contrast

microscope that allowed for the study of colorless and
transparent biological materials.

•1981 – Gerd Binnig and Heinrich Rohrer invented the

scanning tunneling microscope (STM) that gives three-
dimensional images of objects down to the atomic level.
 Resolving power

Resolving power----- The ability to

distinguish two adjacent points as distinct
and separate.

The resolving power of a microscope is a

function of wavelength of light used and the
numerical aperture of lens system.
 Numerical aperture

Numerical aperture----- The sine value of half

aperture angle multiplied by refractive index (n) of
the medium filling the space between front lens
and cover slip
Numerical aperture (NA) = n sinθ
•n = Refractive Index
•θ = ½ the maximum cone of light than
can enter the lens

Refractive index = The velocity of light in a vacuum divided by

its velocity in a specified medium
Limit of resolution—- Smallest distance by
which two objects can be separated and still be
distinguishable as two objects.

Resolution, d= λ/2NA , Where λ= wavelength

of light

e.g. NA = 1.3 and λ= 0.55 μm for green light,

then d = 0.55/2x1.3 = 0.21 μm

Smallest objects can be seen details by light

microscope having dimentions of
approximately 0.2 μm
 Magnification

Magnification is the process of enlarging something

only in appearance, not in physical size.

Most microscope having three objectives

a) Oil-immersion
b) High-dry Each objectives provides a different
c) Low-power objectives degree of magnification

Total magnification of system= magnifying power of objective

X
magnifying power of eyepiece
 Microcope
Based on the principle of magnification

1. Light or optical microscopy

Magnification is obtained by a system of
optical lenses using light waves
a) Bright field b) Dark field c) Fluorescence
d) Phase contrast

2. Electron microscopy
Use of electron beam in place of light waves to
produce the image.
 Bright-field Microscopy
Also called compound light microscope
Microscopic area---- Lighted brightly
Microorganisms---- appear dark
Magnification----- X1000 to X2000
The specimen appears darker on a bright background.
 PARTS OF A MICROSCOPE

1. Ocular lens (eyepiece)

The lens of the microscope that you look through.
There are two ocular (binocular) that magnify the
specimen 10X

2. Ocular Adjustment Knob

The width of the ocular can be adjusted by
turning the dial to the left or right.
3. Nosepiece
The part at the bottom of the body tube that
holds the objective lenses and allows
them to be turned
Scanning lens- (red ring) magnifies 4X
Low power lens-(yellow ring) magnifies 10X
High dry lens- (blue lens) magnifies 40X
Oil Immersion Lens-(white ring) magnifies
100X
4. Arm
The part of the microscope supporting the
body tube.
 5. Stage
The flat part below the objectives lens where
the slide is placed
6. Clip
The part that holds the slide in place so it
doesn’t move
7. Mechanical Stage Adjustment
The two knobs located under the stage
and to the left adjust the position of the
slide. They may be used to move the slide
up and down or from side to side view the
entire specimen
8. Condenser
The condenser receives the light from the source in
the base and it focuses it into a cone on the specimen.

9. Diaphragm
The part that controls the amount of light entering
the field of view
10. Course adjustment
The large knob on the microscope that you turn to
bring the object into focus
11. Fine adjustment
The small knob on the microscope that brings the
image into focus
11. Light source
The lamp (or mirror) under the stage that
sends light through the object being
viewed.
12. Base
The bottom part that supports the rest of the
microscope
 Principle of Bright field Microscope
•For a specimen to be the focus and produce an image under
the Bright field Microscope, the specimen must pass through a
uniform beam of the illuminating light. Through differential
absorption and differential refraction, the microscope will
produce a contrasting image.
•The specimens used are prepared initially by staining to
introduce color for easy contracting characterization. The
colored specimens will have a refractive index that will
differentiate it from the surrounding, presenting a combination
of absorption and refractive contrast.
•The functioning of the microscope is based on its ability to
produce a high-resolution image from an adequately provided
light source, focused on the image, producing a high-quality
image.
•The specimen which is placed on a microscopic slide is
viewed under oil immersion or/and covered with a cover slip.
 Applications

Bright field Microscope is used in several fields, from basic

biology to understanding cell structures in cell biology,
microbiology, bacteriology to visualizing parasitic organisms in
parasitology. Most of the specimens to be viewed are stained
using special staining to enable visualization.
Specific applications
•Used to visualize and study the animal cells
•Used to visualize and study plant cells.
•Used to visualize and study the morphologies of bacterial cells.
•Used to identify parasitic protozoans such as Paramecium.
 Advantages of Bright field microscope

•It is simple to use with few adjustments involved while viewing

the image.
•It can be used to view both stained and unstained.
•The optics of the microscope do not alter the color of the
specimen.
•The microscope can be adjusted and modified for better viewing
such as installing a camera, to form a digital microscope or in the
way image illumination is done such as by use of fluorochromes
on the specimen and viewing under a dark environment, forming
a dark field microscope.
Disadvantages of Bright field microscope
•The aperture diaphragm may cause great contrast which may
distort the outcome of the image.
•It can not be used to view live specimens such as bacterial
cells.
•It has low contrast hence most specimens must be stained for
them to be visualized.
•The use of oil immersion may distort the image.
•The use of a cover slip may damage the specimen.
•Staining may introduce extraneously unwanted details into the
specimen or contaminate the specimen.
•It is tedious to stain the specimen before visualizing it under
the bright field microscope.
•The microscope needs a strong light source for magnification
and sometimes the light source may produce a lot of heat
which may damage or kill the specimen.
 Dark-field microscopy

A dark background against which objects

are brilliantly illuminated
A special kind of condenser transmits a
hollow cone of light to the objects.
Some of light rays will be scattered which
enter the objective and reach the eye.
Particularly valuable for examination of
unstained microorganisms suspended in fluid

A dark field microscope is a type of optical microscope that

uses oblique illumination to light the specimen, causing it
to appear bright against a dark background.
 Principle of Dark field microscopy
•Dark field microscopy is a high-resolution microscopy due to the
contrast by light scattering, which allows for a bright sample on a
black background. Basically, the condenser of the light source has a
diaphragm with a central circular opening so that the central light
rays are blocked and a ―cone of light‖ is projected with larger
angles. More light rays are projected from many angles to hit the
sample. Therefore, when one wants to see the transparent sample,
the sample will scatter, refract, or reflect the light rays directed
towards it; only the deflected ray will enter the objective. This
creates the bright sample on the black background. Therefore, this
transparent microscopy works best with transparent samples or
organisms; unstained living samples can be examined as long as
their refractive index is the same as that of the surrounding fluid.
Therefore, it works best with unstained organisms because it makes
them look better; It does not use transmitted light, which allows
living or delicate organisms to be observed.
Uses of Dark field Microscope
•It is useful for the demonstration of very thin bacteria not
visible under ordinary illumination since the reflection of
the light makes them appear larger.
•This is a frequently used method for rapid demonstration of
Treponema pallidum in clinical specimens.
•It is also useful for the demonstration of the motility of
flagellated bacteria and protozoa.
•It is used to study marine organisms such as algae,
plankton, diatoms, insects, hairs, yeast and protozoa as well
as some minerals and crystals, thin polymers and some
ceramics.
•It is used to study mounted cells and tissues.
•It is more useful in examining external details, such as
outlines, edges, grain boundaries and surface defects than
internal structure.
Advantages of Dark field Microscope

•Dark field microscopy is a very simple yet effective

technique.
•It is well suited for uses involving live
and unstained biological samples, such as a smear from a
tissue culture or individual, water-borne, single-celled
organisms.
•Dark field microscopy techniques are almost entirely free
of artifacts, due to the nature of the process.
•A researcher can achieve a dark field by making
modifications to his/her microscope.
Limitations of Dark field Microscope

•The main limitation of dark field microscopy is the low

light levels seen in the final image.

•The sample must be very strongly illuminated, which can

cause damage to the sample.
 Dark-field microscopy

Bright-field microscopy
 Fluorescence microscopy
Fluorescence microscopy is a very powerful
analytical tool that combines the magnifying
properties of light microscopy with visualization of
fluorescence.
Absorb light of a particular wavelength and energy

Emit longer wavelength and a lesser energy content

Particles Phenomenon

Fluorescent Fluorescence
 Fluorescence microscopy
Certain molecules (or atoms) when exposed to light
radiation of short wavelength (high frequency), emit
light of longer wavelength. The process is called
fluorescence.

The substance that exhibits

fluorescence is called
florescent substance or
fluorochromes. e.g
berberine sulfate, acridine
orange, acridine yellow
Fluorescence microscopy
Applications of Fluorescence Microscope

•To identify structures in fixed and live biological

samples.

•Fluorescence microscopy is a common tool for today’s

life science research because it allows the use of
multicolor staining, labeling of structures within cells,
and the measurement of the physiological state of a cell.
Advantages of Fluorescence Microscope

•Fluorescence microscopy is the most popular method for

studying the dynamic behavior exhibited in live-cell
imaging.
•It helps to isolate individual proteins with a high degree
of specificity among non-fluorescing material.
•The sensitivity is high enough to detect as few as 50
molecules per cubic micrometer.
•Different molecules can now be stained with different
colors, allowing multiple types of the molecule to be
tracked simultaneously.
•It gives a clear advantage over other optical imaging
techniques, for both in vitro and in vivo imaging.
Limitations of Fluorescence Microscope

•Fluorophores lose their ability to fluoresce as they are

illuminated in a process called photobleaching.
Photobleaching occurs as the fluorescent molecules
accumulate chemical damage from the electrons excited
during fluorescence.
•Cells are susceptible to phototoxicity, particularly with
short-wavelength light. Furthermore, fluorescent
molecules have a tendency to generate reactive chemical
species when under illumination which enhances the
phototoxic effect.
•Unlike transmitted and reflected light microscopy
techniques fluorescence microscopy only allows
observation of the specific structures which have been
labeled for fluorescence.
 Fluorescent antibody technique
(Immunofluorescence)
Fluorescent dye attached to the antibody
(labeled antibody)

Bacteria combine with labeled antibody

(fluorescent antibody technique)

This technique involves the use of a single fluorescently

labeled primary antibody that is used to bind to a target
antigen.
 Phase-contrast microscopy

Phase-contrast microscopy (PCM) is an optical

microscopy technique that converts phase shifts in
light passing through a transparent specimen to
brightness changes in the image.

Valuable for studying living unstained cell

Widely used in applied and theoretical biological
studies
Light microscope with a phase contrast objective
and phase contrast condenser
Possible to distinguish unstained structures within a cell
of slightly different RI/thickness
 Phase-contrast microscopy
(How does work?)
Light

Materials

Phase change (RI/thickness)

Variation in brightness of structures

Detected by eye
 Use of Phase-contrast microscopy

The visualization of living cells

The visualization of unstained cells
To view various cell organelles (mitochondria, nucleus and
vacuoles
To study cellular events such as cell division, phagocytosis,
cyclosis (cytoplasmic streaming)
To visualize cellular movements such as chromosomal and
flagellar movements
To study the membrane permeability of cells and organelles
To observe living cells in tissue culture monitoring their
growth
 Advantages of Phase contrast Microscope

•Living cells can be observed in their natural state without

previous fixation or labeling.
•It makes a highly transparent object more visible.
•No special preparation of fixation or staining etc. is needed to
study an object.
•Examining intracellular components of living cells at
relatively high resolution.
•To study living cells and how they proliferate through cell
division.
•Phase-contrast optical components can be added to virtually
any bright field microscope, provided the specialized phase
objectives and condenser with annular phase ring of the
correct size.
Limitations of Phase contrast Microscopy

•Phase-contrast condensers and objective lenses add

considerable cost to a microscope, and so phase contrast
is often not used in teaching labs except perhaps in
classes in the health professions.

•To use phase-contrast the light path must be aligned.

•Generally, more light is needed for phase contrast than

for corresponding bright-field viewing, since the
technique is based on the diminishment of the brightness
of most objects.
Bright-field Dark-field

Phase-contrast
 Transmission electron microscopy

Much higher resolution

Production of image using
extremely short waves of
electron beams and magnetic field
Magnification upto x 400000
Specimen prepared as dry thin film
Film placed between magnetic
condenser and magnetic field
Image seen in fluorescent screen
Comparison of optical microscope and TEM
 Various techniques in electron microscopy

Shadow casting
Extremely thin layer metal ( e.g. platinum)
Production of shadow at an oblique angle on the organism
Information of shape of specimen
Negative staining
Staining outside of the object (phosphotungstic acid)
Unable to penetrate the specimen but forming thick deposits
Virus or bacterial flagella can be seen

Ultrathin sectioning
Thick organism cut into thin slices to examine
To see the internal structure

Freeze-etching
Reveal internal structures of cell in a frozen block
Carbon replicas of exposed surfaces are prepared to reveal
Internal structures.
Localization of cell constituents
To determine specific components of cell e.g. Ferritin-
labeled antibody (iron containing)
Ferritin affects passage of electron beam
The combination of Ferritin-labeled antibody with antigen
produce complex that have higher contrast in electron
microscope.

Localization of enzymes in thin sections

To locate the position of a specific enzyme in cell
e.g. isocytrate dehydrogenase by gold colloid
Autoradiography
Examination of chemical constituents by radioactive materials
 Scanning electron microscope

A scanning electron microscope (SEM) is a type

of electron microscope that produces images of a
sample by scanning the surface with a focused
beam of electrons.
 Scanning electron microscope
Narrow electron beam to the specimen

Release of shower of secondary electron and other radiation

Secondary electrons collected by a detector

generating electronic signal

Electronic signal produce images of the specimen

 Limitation of electron microscopy

Cells can not be examined in living state.

Drying process may alter some morphological

characteristics.

Thin section of specimen have to be used due to low

penetration power of electron beam

 Preparation of light microscope examinations
1. The wet-mount and hanging-drop techniques

Drop of fluid containing organisms

Placed onto glass slide

Covered the drop with cover slip

Sealed the cover slip and slide by petroleum jelly

Morphology of spiral bacteria have to be examined in living state
Cytological changes, spore formation and germination have to
be examined in living state
Cell inclusion bodies e.g. vacuoles of lipid material can
be observed.
2. Dry, fix and stain films or smear

Preparation of the film or smear by sample

Fixation

Staining of solutions

Morphological characteristics observed frequenly

More clear visibility by coloration
Easy to distinguish different species by using various
staining solution
 Microbiological stains
A large number of colored organic compounds (dye) are used
for staining microorganisms.

Classification
On the basis of molecular structure
a) Triphenylmethane dyes
b) Oxazine dyes
c) Thiazine dyes
On the basis of chemical behavior
a) Acid -- the charge on the dye ion is negative
b) Base -- the charge on the dye ion is positive
c) Neutral – complex salt with dye acid with a dye base
e.g. Eosinate of methylene blue
 Staining process

Staining process is the ion-exchange reaction

between the stain and active sites at the surface
of or within the cell.

(Bacterial cell - )(Na + ) + (MB + )(Cl - )

Methylene blue

(Bacterial cell - )(MB + ) + (Na + )(Cl - )

 Staining process

1. Simple staining--- The coloration of bacteria by

applying a single staining
solution

2. Differential staining--- Visibility of differences

between bacterial cells or parts of
bacterial cell by more than one
staining reagent
 Gram staining

Gram staining, also called Gram's method,

is a method of staining used to differentiate
bacterial species into two large groups (gram-
positive and gram-negative).

The name comes from the Danish

bacteriologist Hans Christian Gram, who
developed the technique in 1884.
 Gram staining reagents

Crystal violet

Iodine solution

Alcohol (Decolorizing agent)

Safranin (or some other suitable counterstain)

Gram-positive ----- retain the crystal violet and hence
deep violet color

Gram-negative ---- lose crystal violet color and

counterstained by safranin and hence red in color
 Mechanism of Gram staining

Higher percentage of lipid in Gram-negative bacteria

cell wall

Ethanol treatment extract the lipids from cell wall

Increased porosity or permeability of the cell wall

CV-I can be extracted by ethanol and hence gram

negative organisms is decolorized

Finally take one the color of the safranin

counterstain
 Mechanism of Gram staining

Lower percentage of lipid in Gram-positive bacteria

cell wall

Ethanol treatment causes dehydration and hence

smaller pore size reduces permeability

CV-I can not be extracted by ethanol and hence remain

purple violet color
 Another Mechanism of Gram staining

Thick peptidoglycan layer in Gram-positive bacteria

cell wall

Ethanol treatment causes dehydration of peptidoglycan

resulting diminution pore size

CV-I are not able to come out this tightened

peptidoglycan layer

Remain purple violet color

 Another Mechanism of Gram staining

Smaller amount of peptidoglycan in Gram-

negative bacteria cell wall

Less extensively cross linked and larger pore size

Ethanol can be able to extract CV-I complex

Gram negative organisms is decolorized

Finally take safranin counterstain

Other staining techniques