compound microscope florescent microscope DGI Phase contrast Electron microscope and its types TEM SEM

1. COMPOUND MICROSCOPE
OR
LIGHT MICROSCOPE
PRESENTED BY
MEGHA SHRIDHAR

2. INTRODUCTION
The term “compound” in compound microscopes
refers to the microscope having more than one
lens.
Devised with a system of combination of lenses, a
compound microscope consists of two optical
parts, namely the objective lens and the ocular
lens.

3. PRINCIPLE
A beam of visible light from the base is
focused by a condenser lens onto the
specimen. The objective lens picks up the
light transmitted by the specimen and
creates a magnified image of the specimen
called the primary image inside the body
tube.

4. HOW IT WORKS or PROCEDURE
• The specimen or object, to be examined is usually mounted
on a transparent glass slide and positioned on the specimen
stage between the condenser lens and objective lens.
• A beam of visible light from the base is focused by a
condenser lens onto the specimen.
• The objective lens picks up the light transmitted by the
specimen and creates a magnified image of the specimen
called the primary image inside the body tube. This image is
again magnified by the ocular lens or eyepiece.
• When higher magnification is required, the nose piece is
rotated after low power focusing to bring the objective of a
higher power (generally 45X) in line with the illuminated
part of the slide.
• Occasionally very high magnification it required (e.g. for
observing bacterial cell). In that case, an oil immersion
objective lens (usually 100X) is employed.

6. Magnification of compound
microscope
In order to ascertain the total magnification when
viewing an image with a compound light microscope,
take the power of the objective lens which is at 4x, 10x
or 40x and multiply it by the power of the eyepiece
which is typically 10x.
Therefore, a 10x eyepiece used with a 40X objective
lens will produce a magnification of 400X. The naked
eye can now view the specimen at magnification 400
times greater and so microscopic details are revealed.

9. Parts of a Compound Microscope
Eyepiece And Body Tube.
• The eyepiece is the lens through which the viewer looks to
see the specimen.
• It usually contains a 10X or 15X power lens.
• The body tube connects the eyepiece to the objective
lenses.
Objectives and Stage Clips
• Objective Lenses are one of the most important parts of a
Compound Microscope.
• They are the closest to the specimen.
• A standard Microscope has three to four Objective Lenses
which range from 4X to 100X.
• Stage Clips are metal clips that held the slide in a place.

10. Arm and Base
• The Arm connects the Body Tube to the base of
the Microscope.
• The Base supports the Microscope and its where
Illuminator.
Illuminator and Stage
• The illuminator is the light source for a
microscope.
• A compound light microscope mostly uses a low
voltage bulb as an illuminator.
• The stage is the flat platform where the slide is
placed.

11. Nosepiece and Aperture
• Nosepiece is a rotating turret that holds the objective lenses.
• The viewer spins the nosepiece to select different objective
lenses.
• The aperture is the middle of the stage that allows light from
the illuminator to reach the specimen.
Condenser, Iris diaphragm, and Diaphragm
• A condenser gathers and focuses light from the illuminator
onto the specimen being viewed.
• Iris diaphragm adjusts the amount of light that reaches the
specimen.
• The diaphragm is a five holed disk placed under the stage.
• Each hole is of a different diameter. By turning it, you can
vary the amount of light passing through the stage opening

12. Applications
• A compound microscope is of great use in pathology labs so
as to identify diseases.
• Various crime cases are detected and solved by drawing out
human cells and examining them under the microscope in
forensic laboratories.
• The presence or absence of minerals and the presence of
metals can be identified using compound microscopes.
• Students in schools and colleges are benefited by the use of
a microscope for conducting their academic experiments.
• It helps to see and understand the microbial world of
bacteria and viruses, which is otherwise invisible to the
naked eye.
• Plant cells are examined and the microorganisms thriving on
it can be ascertained with the help of a compound
microscope. Thereby, a compound microscope has proved to
be crucial to biologists

13. Advantages
• Simplicity and its convenience.
• A compound light microscope is relatively
small, therefore it’s easy to use and simple to
store, and it comes with its own light source.
• Because of their multiple lenses, compound
light microscopes are able to reveal a great
amount of detail in samples.

14. B) DGI MICROSCOPE
or
DARK GROUND ILLUMINATION
MICROSCOPE
or
DARK FIELD MICROSCOPE

16. INTRODUCTION
This is similar to the ordinary light microscope;
however, the condenser system is modified so
that the specimen is not illuminated directly.
The condenser directs the light obliquely so that
the light is deflected or scattered from the
specimen, which then appears bright against a
dark background. Living specimens may be
observed more readily with dark field than with
bright field microscopy.

18. PRINCIPLE
The dark-ground microscopy makes use of the dark-
ground microscope, a special type of compound light
microscope.
The dark-field condenser with a central circular stop,
which illuminates the object with a cone of light, is the
most essential part of the dark-ground microscope.
This microscope uses reflected light instead of transmitted
light used in the ordinary light microscope.
It prevents light from falling directly on the objective lens.
Light rays falling on the object are reflected or scattered
onto the objective lens with the result that the
microorganisms appear brightly stained against a dark
background.

19. Uses of Dark-field Microscope
The dark ground microscopy has the following uses:
• 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.
• Darkfield is used to study marine organisms such as algae, plankton,
diatoms, insects, fibers, hairs, yeast and protozoa as well as some
minerals and crystals, thin polymers and some ceramics.
• Darkfield 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.

20. Advantages of Darkfield
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.
• Considering the simplicity of the setup, the
quality of images obtained from this technique is
impressive.

21. Limitations of Darkfield
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.

23. Definition
• 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 while
phosphorescence is a specific type of
photoluminescence related to fluorescence.

24. Principle
• Light of the excitation wavelength is
focused on the specimen through the
objective lens. The fluorescence emitted by
the specimen is focused on the detector by
the objective. Since most of the excitation
light is transmitted through the specimen,
only reflected excitatory light reaches the
objective together with the emitted light.

27. Parts of Fluorescence Microscope
1) A light source: Lasers are mostly used for
complex fluorescence microscopy techniques,
while xenon lamps, and mercury lamps, and
LEDs with a dichroic excitation filter are
commonly used.
2) The excitation filter: The exciter is typically a
band pass filter that passes only the
wavelengths absorbed by the fluorophore

28. 3) The dichroic mirror: A dichroic filter or thin-
film filter is a very accurate color filter used to
selectively pass light of a small range of colors
while reflecting other colors.
4) The emission filter: The emitter is typically
a band pass filter that passes only the
wavelengths emitted by the fluorophore and
blocks all undesired light outside this band –
especially the excitation light.

29. Advantages of Fluorescence
Microscope
• Fluorescence microscopy is the most popular
method for studying the dynamic behavior
exhibited in live-cell imaging.
• This stems from its ability to isolate individual
proteins with a high degree of specificity amidst
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.

31. Limitations of Fluorescence
Microscope
• Fluorophores lose their ability to fluoresce as
they are illuminated in a process called
photo-bleaching.
• Photo-bleaching occurs as the fluorescent
molecules accumulate chemical damage from
the electrons excited during fluorescence.
• Cells are susceptible to photo toxicity,
particularly with short-wavelength light.

32. Phase contrast microscopy

33. Principle
When light passes through cells, small phase
shifts occur, which are invisible to the human
eye. In a phase-contrast microscope, these
phase shifts are converted into changes in
amplitude, which can be observed as
differences in image contrast.

35. Parts of Phase Contrast Microscope
Phase-contrast microscopy is basically a specially designed light
microscope with all the basic parts in addition to which an
annular phase plate and annular diaphragm are fitted.
The annular diaphragm:
• It is situated below the condenser.
• It is made up of a circular disc having a circular annular
groove.
• The light rays are allowed to pass through the annular
groove.
• Through the annular groove of the annular diaphragm, the
light rays fall on the specimen or object to be studied.
• At the back focal plane of the objective develops an image.

36. Applications of Phase contrast
Microscopy
To produce high-contrast images of transparent
specimens, such as
• living cells (usually in culture),
• microorganisms,
• thin tissue slices,
• lithographic patterns,
• fibers,
• latex dispersions,
• glass fragments, and
• subcellular particles (including nuclei and other
organelles).

37. Advantages
• 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 under a phase-contrast
microscope which saves a lot of time.
• Examining intracellular components of living cells at
relatively high resolution. eg: The dynamic motility of
mitochondria, mitotic chromosomes & vacuoles.
• It made it possible for biologists to study living cells
and how they proliferate through cell division.

38. Limitations
• 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.

40. Definition
An electron microscope is a microscope that uses
a beam of accelerated electrons as a source of
illumination.
It is a special type of microscope having a high
resolution of images, able to magnify objects in
nanometres

41. Working Principle of Electron
microscope
Electron microscopes use signals arising from the interaction of an
electron beam with the sample to obtain information about structure,
morphology, and composition.
Working
1. The electron gun generates electrons.
2. Two sets of condenser lenses focus the electron beam on the
specimen and then into a thin tight beam.
3. To move electrons down the column, an accelerating voltage (mostly
between 100 kV-1000 kV) is applied between tungsten filament and
anode.
4. The specimen to be examined is made extremely thin, at least 200
times thinner than those used in the optical microscope. Ultra-thin
sections of 20-100 nm are cut which is already placed on the
specimen holder.

42. 5. The electronic beam passes through the specimen
and electrons are scattered depending upon the
thickness or refractive index of different parts of the
specimen.
6. The denser regions in the specimen scatter more
electrons and therefore appear darker in the image
since fewer electrons strike that area of the screen. In
contrast, transparent regions are brighter.
7. The electron beam coming out of the specimen
passes to the objective lens, which has high power
and forms the intermediate magnified image.
8. The ocular lenses then produce the final further
magnified image

43. Types of Electron microscope
There are two types of electron microscopes,
with different operating styles:
1. The transmission electron microscope (TEM)
2. The scanning electron microscope (SEM)

44. The transmission electron
microscope (TEM)
The transmission electron microscope is used to view
thin specimens through which electrons can pass
generating a projection image.
TEM is used, among other things,
• to image the interior of cells (in thin sections),
• the structure of protein molecules
• the organization of molecules in viruses and
cytoskeletal filaments,
• the arrangement of protein molecules in cell
membranes

47. The scanning electron microscope
(SEM)
• Conventional scanning electron microscopy depends
on the emission of secondary electrons from the
surface of a specimen.
• it provides detailed images of the surfaces of cells and
whole organisms that are not possible by TEM.
• It can also be used for particle counting and size
determination, and for process control.
• It is termed a scanning electron microscope because
the image is formed by scanning a focused electron
beam onto the surface of the specimen.

50. Parts of Electron microscope
Electron gun
• The electron gun is a heated tungsten filament, which
generates electrons.
Electromagnetic lenses
• Condenser lens focuses the electron beam on the
specimen. A second condenser lens forms the electrons
into a thin tight beam.
• The electron beam coming out of the specimen passes
down the second of magnetic coils called the objective
lens, which has high power and forms the intermediate
magnified image.
• The third set of magnetic lenses called projector (ocular)
lenses produce the final further magnified image.
Each of these lenses acts as an image magnifier all the while
maintaining an incredible level of detail and resolution.

51. Specimen Holder
The specimen holder is an extremely thin film of
carbon held by a metal grid.
Image viewing and Recording System.
The final image is projected on a fluorescent
screen.
Below the fluorescent screen is a camera for
recording the image.

52. Applications
1. Electron microscopes are used to investigate the
ultrastructure of a wide range of biological and
inorganic specimens including microorganisms, cells,
large molecules, biopsy samples, metals, and
crystals.
2. Industrially, electron microscopes are often used for
quality control and failure analysis.
3. Modern electron microscopes produce electron
micrographs using specialized digital cameras and
frame grabbers to capture the images.
4. Science of microbiology owes its development to
the electron microscope. Study of microorganisms
like bacteria, virus and other pathogens have made
the treatment of diseases very effective.

53. Advantages
• Very high magnification
• Incredibly high resolution
• Material rarely distorted by preparation
• It is possible to investigate a greater depth of
field
• Diverse applications

54. Limitations
• The live specimen cannot be observed.
• As the penetration power of the electron beam is very
low, the object should be ultra-thin. For this, the
specimen is dried and cut into ultra-thin sections before
observation.
• As the EM works in a vacuum, the specimen should be
completely dry.
• Expensive to build and maintain
• Requiring researcher training
• Image artifacts resulting from specimen preparation.
• This type of microscope is a large, cumbersome extremely
sensitive to vibration and external magnetic fields.