Different types of microscopes

1. MICROSCOPY
Dr. Pulipati Sowjanya
Professor & Head
Dept. of Pharmaceutical Biotechnology
Vignan Pharmacy College
Vadlamudi, Guntur (Dt)

2. MICROSCOPY
• Microscope was invented by Antony Van
Leeuwenhoek in 17th century.
• Required for the morphological studyof
microorganisms.
• USES - to magnify the image.
- to achieve maximum resolution.
- to provide sufficient contrast for observation

3. Resolution: the extent to which details in the
magnified object are maintained.
Resolving Power (RP) : the smallest distance by
which 2 points can be separated and still be observed as 2
distinct / different points.
RP (eye) – 200 µm
RP (visible light) – 300 nm
RP (electron microscope) – 0.1 nm
DEFINITIONS

4. Types of Microscopes
In light microscopy the magnification is obtained by optical
lenses using light waves. This includes
• Bright-Field microscopy
• Phase contrast microscopy
• Fluorescent microscopy
Electron microscope – Uses beam of electrons to produce the
image. The specimen samples can be examined by either
transmission or scanning electron microscopy
Microscopes are broadly categorized into light
(optical) & electron depending upon magnification

5. Bright-Field Microscopy
• Microorganisms do not absorb much light, but when they
are stained with dye, it increases their light absorbing
ability.
• This results in greater contrast & colour differentiation.
• This type of microscopes produce about 1,000 to 2,000
magnification.
• If magnifies greater than 2,000 the image becomes fuzzy.
The microscopic field is brightly lightened and the
microorganisms appear dark because they absorb
some of the light.

6. Bright-Field Microscope
A Bright-Field Microscope

7. Dark-Field Microscopy
• The light microscope is equipped with a special kind of
condenser that transmits a hallow cone of light from the
source of illumination.
• Most of the light directed through condenser does not enter
the objective, but some of the light rays will be scattered if the
transparent medium contains objects such as microbial cells.
• The objects are brightly illuminated
against dark background.

8. Dark-Field Microscopy
• This diffracted light will enter
the objective & reach the eye;
thus the object (microbial cell)
will appear bright in dark
microscopic field.
• Dark-Field microscopy is
valuable for the examination of
unstained microorganisms
suspended in fluid wet mount
& hanging drop preparations.
Dark-Field Microscope

9. Dark-Field Microscopy
Treponema pallidum; a spirochete
that causes syphilis
Spirillum volutans; a very large
bacterium with flagellar bundles
Clostridium botulinum; a bacterium
causes botulism

10. Dark-Field Microscopy
• Very useful in finding extremely small, unstained
and / or moving objects.
• Organelles like cilia, flagella, vacuoles and cell nuclei can
be clearly seen.
USES

11. Fluorescence Microscopy
• Such substances are called fluorescent & the phenomenon is
termed as fluorescence.
• The microorganisms are stained with a fluorescent dye and
then illuminated with blue light.
• The blue light is absorbed and green light emitted by the dye.
• Chemical substances absorb light but only some
substances will emit light of a longer wavelength
and a lesser energy content.

12. • Acridine orange : Orange
• Auramine-Rhodamine : Yellow
• Calcofluor white: White
• Fluorescein Isothiocyanate (FITC) :
Green
FLUOROCHROMES
Fluorescence Microscopy
Fluorescence Microscopy

13. Fluorescence Microscopy
• The fluorescent dyes are combined with antibodies which are
specific to microorganisms.
• Antibodies to which fluorescent dye is attached are known as
labelled antibodies.
• The labelled antibodies are then mixed with a suspension of
bacteria & examined by fluorescent microscopy.
• The bacterial cell combined with labelled antibody will be visible in
microscope,

14. Fluorescence Microscopy

15. Phase Contrast Microscopy
• It is a valuable microscope
for studying living unstained
cells. It is widely used in
applied & theoretical
biological studies.
Phase Contrast Microscopy

16. Phase Contrast Microscopy
• It uses a conventional light microscope fitted with a phase-
contrast objective system and makes it possible to distinguish
unstained structures within a cell.
• Principle: Light passing through one material and into another
material of slightly different refractive index or thickness will
undergo a change in phase.
• Advantage : since staining is not involved, live
organisms can be observed.

17. Phase Contrast Microscopy

18. Electron Microscopy
• Invented by Knoll & Ruska in 1936.
• Uses electrons in place of light.
• Electrons are focused by
electromagnetic field.
• Image is formed on a fluorescent screen or is taken
on a photographic material.
• Resolving power is 100,000 times more than
light microscope.

19. Electron Microscopy
• 1. Transmission EM ( TEM ) 2. Scanning EM ( SEM
)
• SEM allows the study of cell surfaces with greater contrast &
higher resolution than TEM.
Disadvantages :
• Only dead & dried objects can be examined, since the medium is vacuum.
• Cell morphology is distorted.
Types of Electron Microscopy

20. Electron Microscopy
Transmission Electron Microscopy
• It provides tremendous useful magnification because of
much higher resolution obtained by short wavelength of
the electron beam and magnifies the specimen.
• The electron microscope uses electron beams &
magnetic fields to produce the image, whereas the light
microscope uses light waves & glass lenses.
• The electron microscope produces a wavelength of 0.05A0. It is
possible to resolve objects as small as 10 A0.

21. Electron Microscopy
Transmission Electron Microscopy
• The resolving power of the electron
microscope and it produces magnification
upto 4,00,000 times.
• The specimen should be prepared as
extremely thin dry film and is introduced
into the instrument at a point between the
magnetic condenser and the magnetic
objective.
• The magnified image may be viewed on a
fluorescent screen.

22. Electron Microscopy
Scanning Electron Microscopy
• The specimen is subjected to a narrow electron beam
which rapidly moves over the surface of the specimen.
• This causes the release of a shower of secondary
electrons & other types of radiation from the specimen
surface.
• The intensity of these secondary electron depends on
the shape & the chemical composition of the irradiated
object.
• The secondary electrons are collected by a detector
which generates an electronic signal.
• Signals are scanned in a manner of a television system
to produce an image on a cathode tube.

23. Electron Microscopy
Scanning Electron Microscopy
• The scanning electron microscope lacks the resolving power obtainable
with the transmission electron microscope but has the advantage of
revealing a striking three dimensional picture.
Limitations:
• The specimen to be examined is kept in a chamber that is under very high
vacuum. So the cells cannot be examined in live state.
• The drying process alters some morphological characteristics.
• Low penetrating power of electron beam that requires very thin section of
specimen to observe the internal structures of the cell.

24. Comparison of different types of Microscopy
Type of
microscopy
Max.
magnification
Appearance of specimen Useful applications
Bright field 1,000-2,000
Specimens stained or unstained.
Bacteria stained and appear as
color of stain
Gross morphological features of
bacteria, yeasts, molds, algae &
protozoa
Dark field 1,000-2,000
Generally unstained, appears
bright or lightened in an dark back
ground
Microorganism that exhibit some
characteristic morphological
features. Ex.: Spirochetes
Fluorescence 1,000-2,000
Bright & colored based on color of
the fluorescent dye
Diagnostic techniques reveals
identity of organisms.
Phase contrast 1,000-2,000 Varying degrees of darkness
Examination of cellular structures
in living cells. Ex: yeasts, algae,
protozoa & some bacteria
Electron
2,00,000 –
4,00,000
Viewed on fluorescent screen
Examination of viruses & ultra
structures of microbial cells