Microscope and Microscopy
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Fluorescence Microscopy
A fluorescence microscope is basically a conventional
light microscope with added features and components
that extend its capabilities. Fluorescence microscopy
takes advantage of fluorescence, the ability of
substances to absorb short wavelength of light
(ultraviolet) and give off light at a longer
wavelength(visible).
Fluorescent microscope also produces a magnified
image of the sample, but the image is based on the
second light source -- the light emanating from the
fluorescent species -- rather than from the light
originally used to illuminate, and excite, the sample.
Principle of Fluorescence
1. Energy is absorbed
by the atom which
becomes excited.
2. The electron jumps
to a higher energy
level.
3. Soon, the electron
drops back to the
ground state, emitting
a photon (or a packet
of light) - the atom is
fluorescing.
Specimens usually stained with
fluorochromes.
Microorganisms stained with
fluorochrome are examined
under a fluorescence
microscope with an ultraviolet
or near-ultraviolet light source.
Fluorescence Microscopy (contd…)
Fluorochromes have special attractions for different
microorganisms.
Auramine O, which glows yellow when exposed to UV
light, is strongly absorbed by Mycobacterium tuberculosis,
the bacterium that causes tuberculosis.
Bacillus anthracis, the causative agent of anthrax, appears
apple green when stained with another fluorochrome,
fluoresceine isothiocyanate (FITC).
Rhodamine auramine stain FITC stain
Fluorescence Microscopy: Principal uses
Principal use of fluorescence microscopy is a
diagnostic technique called the Fluorescent-
antibody(FA) technique (immunofluorescence) to
rapidly detect and identify microbes in tissues or
clinical specimen.
Antibodies are natural defense molecules that are
produced by humans and many animals in
reaction to a foreign substance, or antigen.
The Fluorescent Antibody Technique---
Immunofluorescence (contd…)
Fluorescent antibodies for a particular antigen are obtained as follows:
An animal is injected with specific antigen, such as a bacterium,
the animal begins to produce specific antibodies against that
antigen.
After a sufficient time, the antibodies are removed from the serum
of the animal.
Next, a fluorochrome is chemically combined with the antibodies.
These fluorescent antibodies are then added to a microscope slide
containing an unknown bacterium. If this unknown bacterium is
the same bacterium that was injected into the animal, the
fluorescent antibodies bind to antigens on the surface of the
bacterium, causing the fluorescence.
Direct Fluorescent Antibody Technique
Electron Microscopy
◼ Uses electrons
instead of light.
◼ The shorter
wavelength of
electrons gives
greater
resolution.
EM—contd………
◼ An electron microscope is a type of microscope that uses electrons
to illuminate a specimen and create an enlarged image. Electron
microscopes have much greater resolving power than light
microscopes and can obtain much higher magnifications. Some
electron microscopes can magnify specimens up to 2 million
times, while the best light microscopes are limited to
magnifications of 4000 times.
Instead of using glass lenses, an electron microscope uses
electromagnetic lenses to focus onto a specimen a beam of
electrons traveling through an evacuated tube.
◼ Electron microscopes create images with
electromagnetic radiation .
◼ The greater resolution and magnification of the
electron microscope is due to the wavelength
of an electron being much smaller than that of
a light photon.
There are two types of electron microscopes:
The transmission electron microscope
The scanning electron microscope.
Transmission Electron Microscopy
(TEM): Distinguishing Features
A finely focused beam of electron from an electron
gun passes through a specially prepared, ultrathin
sections of specimens.
The electron beam that has been transmitted through a
specimen and carries information about the inner
structure of the specimen in the electron beam that reaches
the imaging system of the microscope
The specimen is usually placed on a copper mesh grid.
TEM(CONTD….)
◼ The variation in this information (the "image") is
then magnified by a series of electromagnetic lenses
until it is recorded by hitting a fluorescent screen,
photographic plate, or light sensitive sensor such as a
CCD (charge-coupled device) camera. The image
detected by the CCD may be displayed in real time
on a monitor or computer.
Transmission Electron Microscopy (TEM):
Distinguishing Features (contd…)
Electrons scatter when they pass through thin sections of a
specimen
Transmitted electrons (those that do not scatter) are used
to produce image.
TEM can resolve object as close together as 2.5nm, and
objects are generally magnified 10,00-100,000X
Salts of various heavy metals, such as lead, osmium,
tungsten, and uranium, are commonly used as stain.
Transmission Electron Microscopy (TEM)
Disadvantage of TEM
◼ Electrons have limited penetration power, only a very thin section of a
specimen (about 100nm) can be studied effectively.
◼ Specimens must be fixed, dehydrated, and viewed under a high
vacuum to prevent electron scattering. These treatments not only kill
the specimen but also cause some shrinkage and distortion, sometimes
to the extent that there may appear to be additional structures in a
prepared cell (artifacts).
Principal use:
◼ To examine viruses or the internal ultra structure in thin sections of
cells.
This colored TEM shows
a thin slice of Paramecium
with detail of internal
structure
Scanning Electron Microscopy (SEM)
An electron gun produces a beam of electrons that
scans the surface of a whole specimen.
Secondary electrons emitted from the specimen
produce the image.
Scanning Electron Microscopy (SEM)
◼ A scanning electron microscope provides striking three-
dimensional views of specimens
◼ An electron gun produces a finely focused beam of electron
called the primary electron beam. These electrons passed
through the electromagnetic lenses are directed over the
surface of the specimen
◼ The primary electron beam knocks electron out of the
surface of the specimen, and the secondary electron
produced are transmitted to an electron collector,
amplified, and used to produce an image of on a viewing
screen or photographic plate. The image is called scanning
electron micrograph.
◼ 1000-10,000;
resolution ,20 nm
◼ Principal Use:
– To study the surface
features of cells and
viruses