#microbiology #biology

1. Dr. Sarah Abdulsalam

2. The Fluorescence Microscope
 British scientist Sir George G. Stokes first described
fluorescence in 1852
 A fluorescence microscope is an optical microscope
that uses fluorescence and phosphorescence instead
of, or in addition to, reflection and absorption to
study properties of organic or inorganic substances.
 Fluorescence : is the emission of light by a substance
that has absorbed light or other electromagnetic
radiation.

4.  Fluorescence microscopy is a specially modified
compound microscope furnished with an ultraviolet
(UV) radiation source and a filter that protects the
viewer’s eye from injury by these dangerous rays.
 Some organisms fluoresce naturally because of the
presence within the cells of naturally fluorescent
substances such as chlorophyll.
 Those that do not naturally fluoresce may be
stained with a group of fluorescent dyes called
fluorochromes.

5.  The name of this type of microscopy originates
from certain dyes (acridine, fluorescein) and
minerals that possess the property of
fluorescence.
 This means that they emit visible light when
bombarded by shorter UV rays.
 This has been widely used in diagnostic
microbiology to detect both the antigen and
antibodies, may they be in pure form or mixed
form.

6. Principle
 The fluorescence microscope depends on two
intrinsic properties of the substance to be
observed
 – FLUORESCENCE
 – PHOSPHORESCENCE

7. Applications of fluorescence microscope
in clinical samples?
1) Fluorescent staining is commonly used to improve
tuberculosis diagnosis efficiency as well as for
malaria diagnosis.
2) Early detection of bacteria in blood cultures, and to
detect and identify nucleic acids by color.
3) Chromosomal anomalies ( FISH)
4) Fluorescent antibodies provide a wide variety of
immunologically specific, rapid diagnostic tests for
infectious diseases. can observe in live cells

8. ELECTRON MICROSCOPY
 In 1938 Von Borries and Ruska built the first
practical electron microscope.
 The electron microscope use electron beams
and magnetic fields to produce the image
instead of light waves and glass lenses used
in the light microscopes.
 Resolving power of electron microscope is far
greater than that of any other compound
microscope. This is due to shorter
wavelengths of electrons. The wavelength of
electrons are about 100,000 times smaller
than the wavelength of visible light.

11. Method For Electron
Microscope
 The specimen to be observed is prepared as
extremely thin dry film on small screens.
 These are then introduced into the instrument at
a point between the magnetic condenser and
the magnetic objective.
 The magnified image is viewed on a fluorescent
screen through an airtight window.
 The image can be recorded on a photographic
plate by a camera built into the instrument.

12. Types of electron microscopy
 Mainly 2 types:
1) Transmission Electron Microscope
(TEM) - allows one the study of the
inner structures.
2) Scanning Electron Microscope (SEM) -
used to visualize the surface of objects.

13. PRINCIPLE OF WORKING OF TEM
 Electrons possess a wave like character.
 Electrons emitted into vacuum from a
heated filament with increased
accelerating potential will have small
wavelength.
 Such higher-energy electrons can
penetrate distances of several microns
into a solid.
 If these transmitted electrons could be
focused - images with much better
resolution.
 Focusing relies on the fact that, electrons
also behave as negatively charged
particles and are therefore deflected by
electric or magnetic fields.

14. What is SEM?
 The scanning electron microscope (SEM) uses
a focused beam of high-energy electrons to
generate a variety of signals at the surface of
solid specimens. The signals that derive from
electron-sample interactions reveal information
about the sample.

15. PRINCIPLE OF SEM
 Accelerated electrons in an
SEM carry significant
amounts of kinetic energy,
and this energy is dissipated
as a variety of signals
produced by electron-sample
interactions when the incident
electrons are decelerated in
the solid sample.
 These signals include
secondary electrons that
produce SEM images.

16. SEM WORKING
 The electron gun produces an electron beam which is
accelerated by the anode.
 The beam travels through electromagnetic fields and lenses,
which focus the beam down toward the sample.
 A mechanism of deflection coils enables to guide the beam
so that it scans the surface of the sample in a rectangular
frame.
 When the beam touches the surface of the sample, it
produces:
 – Secondary electrons (SE)
 – Back scattered electrons (BSE)
 – X - Rays...
 The emitted SE is collected by SED and convert it into signal
that is sent to a screen which produces final image.

18. Differences between SEM and
TEM
TEM SEM
1. Electron beam passes through thin
sample.
1. Electron beam scans over surface of
sample.
2. Specially prepared thin samples are
supported on TEM grids
2. Sample can be any thickness and is
mounted on an aluminum stub.
3. Specimen stage halfway down
column.
3. Specimen stage in the chamber at the
bottom of the column.
4. Image shown on fluorescent screen. 4. Image shown on TV monitor.
5. Image is a two dimensional projection of
the sample.
5. Image is of the surface of the sample

19. ADVANTAGES & DISADVANTAGES OF TEM
 Advantages:
1) TEMs offer very powerful magnification and resolution.
2) TEMs have a wide-range of applications and can be utilized
in a variety of different scientific, educational and industrial
fields
3) TEMs provide information on element and compound
structure .
4) Images are high-quality and detailed.
 Disadvantages:
1) TEMs are large and very expensive.
2) Laborious sample preparation.
3) Operation and analysis requires special training.
4) Samples are limited to those that are electron transparent.
5) TEMs require special housing and maintenance.
6) Images are black and white .

20. BIOLOGICAL APPLICATIONS OF TEM
1) In medicine as a diagnostic tool – important in
renal biopsies.
2) Cellular tomography, Used for obtaining
detailed 3D structures of subcellular
macromolecular objects.
3) Cancer research - studies of tumor cell
ultrastructure .
4) Toxicology – to study the impacts of
environmental pollution on the different levels of
biological organization.

21. ADVANTAGES & DISADVANTAGES OF SEM
 Advantages
1) It gives detailed 3D and topographical imaging and the
versatile information garnered from different detectors.
2) This instrument works very fast.
3) Modern SEMs allow for the generation of data in digital form.
4) Most SEM samples require minimal preparation actions.
 Disadvantages
1) SEMs are expensive and large.
2) Special training is required to operate an SEM.
3) The preparation of samples can result in artifacts.
4) SEMs are limited to solid samples.
5) SEMs carry a small risk of radiation exposure associated
with the electrons that scatter from beneath the sample
surface.

22. BIOLOGICAL APPLICATIONS OF SEM
1) Virology - for investigations of virus structure
2) Cryo-electron microscopy – Images can be made of
the surface of frozen materials.
3) 3D tissue imaging -
 – Helps to know how cells are organized in a 3D
network
4) Forensics - SEM reveals the presence of materials on
evidences that is otherwise undetectable
5) SEM renders detailed 3-D images
A. – extremely small microorganisms
B. – anatomical pictures of insect, worm, spore, or other
organic structures