This document provides information about different types of microscopy. It begins with an introduction to microscopy, then discusses the history and key figures in the development of the microscope. It describes different types of microscopes including light/bright field microscopy, dark field microscopy, phase contrast microscopy, fluorescence microscopy, and electron microscopy. For each type, it provides details on the optical principles, components, and applications. The document aims to inform the reader about the basic concepts and techniques of microscopy.

1. MRS. MERLIN DAYANA. E
BSC (N) NURSING TUTOR,
GANGA COLLEGE OF NURSING
COIMBATORE
MRS. MERLIN DAYANA. E
BSC (N) NURSING TUTOR,
GANGA COLLEGE OF NURSING
COIMBATORE

2. Introduction
Microscopy
History of Microscopy
Properties of
Microscopy
Types of Microscopy
Handlig and care of
Microscopy

3. INTRODUCTION

4. MICROSCOPY
Definition :
 A microscope ( Greek: micron=
small and scopos = to look)
 Microscope: Is an interment for
viewing objects that are too small to
be seen by the naked or unaided
eye.
 Microscopy: The science of
investigating small object using
such an instruments is called
microscopy.

5. HISTORY OF MICROSCOPY
HANS & ZACHARIS JANSSEN
In 1590 , both are Dutch eyeglass
makers inventors.
The first compound microscopes
are invented by Hans & Zacharias
janssen. That is tube with lenses at
each end.

6. HISTORY OF MICROSCOPY
•Anton van
leeuwenhook
•Is generaly credited
with bringing the
microscopeto attention
of biologists.
•In 1661 he discoverd
bacteria,free living &
parasitic microscopic
protists,sprem
cells.blood cells.
•He is the father of
microbiology.
Anton van Leeuwenhoek
 Is generally credited with bringing
the microscope to attention of
biologists.
 In 1661 he discovered bacteria,
free living & parasitic microscopic
protists,sprem cells. blood cells.
 He is the father of microbiology.

7. HISTORY OF MICROSCOPY
Robert hook
In 1635 - 1703, English chemist ,
mathematician , physicists &
inventor.
He improved the compound
microscope.

8. PROPERTIES OF MICROSCOPY
Good magnification
A good microscope should have at least three
properties
Good resolution
Good contrast

9. PROPERTIES OF MICROSCOPY
• Good resolution:-
Resolution power refers
to the ability to produce
separate images of closely
placed objectives.
That they can be
distinguished as two
separate entities.
The resolution power of:-
Unaided human eye is about 0.2mm
(200μm)
Light microscope is about 0.2μm
Electron microscope is about 0.5nm.

10. PROPERTIES OF MICROSCOPY
Good contrast :-
Contrast is improved by
staining the specimen.
When the stain binds to the
cells, the contrast is increased.

11. PROPERTIES OF MICROSCOPY
Good magnification :-
Ocular lens with a
magnification power of
10X
Objective lens :-
Scanning (4X)
Low power(10X)
High power(40X)
Oil immersion (100X)

12. Simple
microscopy
Bright field or
light microscopy
Phase
contrast
microscopy
Electron microscopyTYPES OF
MICROSCOPE

13. SIMPLE MICROSCOPE:
Simple microscope:
contains only one
lenses.
Ex. Magnifying
glass.

14. COMPOUND MICROSCOPY
Compound microscope:
A system of two lens
that works together
Compound microscope:
A system of two lens that
works together.

15. BRIGHT FIELD OR LIGHT
MICROSCOPE
 Light microscope forms a dark image against a brighter
background, hence the name Bright field
Mechanical part
Magnifying part
Illuminating part

16. BRIGHT FIELD OR LIGHT
MICROSCOPE

17. MECHANICAL PART
base :
It holds various part of microscope, such as the light
source , the fine and coarse adjustment.
c- shaped arm :
 It holds the microscope, and it connects the ocular
lens to the objective lens.
Mechanical stage:
The arm bears a stage with stage clips to hold the
slides and the stage control knobs to move the slide
during viewing.
It has an aperture at the center that permit light to
reach the object from the bottom.

18. MAGNIFYING PART
ocular lens: the arm contains an eye piece that bears an ocular lens of
10X magnification power.
Microscope with two eye piece are called as binocular microscopes.
ocular lens: The arm contains an eye piece that
bears an ocular lens of 10X magnification power.
Microscope with two eye piece are called as
binocular microscopes.
Objective lens: The arm also contains a revolving nose
piece that bear three to five objectives with lenses of
different magnifying power (4X,10X,40X,and 100X).

19. ILLUMINATING PART
condenser:It is
mounted beneath the
stage which focuses a
cone of light on the
slide.
iris diaphragm: It
control the light pass
through the
condenser.
light source: It may
be a mirror or an
electric bulb.
fine and coarse
adjustment knob:
They sharpen the
image.

20. PRINCIPLE
Principle:
The rays emitted from the light source pass
through the iris diaphragm and fall on the
specimen.
The rays passing through the specimen is
gathered by the objective and a magnified
image is formed.
This image is further magnified by the
ocular lens to produce the final magnified
virtual image.

21. DARK FIELD MICROSCOPE

22. DARK FIELD MICROSCOPE

23. DARK FIELD MICROSCOPE

24. PRINCIPLE DARK FIELD MICROSCOPE
The dark field condenser has
a central opaque area that
blocks light from entering the
object lens directly and has a
peripheral annular hollow area
which allows the light to pass
through and focus on the
specimen obliquely.
Only the which is reflected
by the specimen enters the
objective lens where as the
un reflected light dose not
enter the object.
As a result, the specimen is
brightly illuminated; but the
background appears dark.

25. Application:
•Dark field microscope is
used to identify the living,
unstained cells and thin
bacteria like spirochetes
which is cannot visualized
by light microscope.
Application of dark filed microscopy

26. PHASE CONTRAST MICROSCOPE
•As per name, in
Phase contrast
microscope the
contrast is enhanced.
•This microscope
visualizes the unstained
living cells by creating
difference in contrast
between the cells and
water.
•It converts slight
differences in refractive
index and cell density
into easily detectable
variations in light
intensity.
•Contrast can be enhanced
by staining of the
specimens, but as staining
kills microbes, the
properties of living cells
cannot be studied.

27. PHASE CONTRAST MICROSCOPE

28. PRINCIPLES
The condenser is similar to that of dark field microscope, consist of an opaque central
area with a thin transparent ring, which procuces a hollow cone of light.
•As this cone of light passes through a cells, some light rays are bent due to variations in
density and refractive index within the specimen and are retracted by about one fourth of a
wave length.
•The un deviated light rays strike a phase plate, while the deviated rays miss the ring and
pass through the rest of the plate.
•The phase ring is constructed in such a way that the undeviated light passing through it is
advanced by one – fourth of a wavelength out of the phase and will be cancel each other
when they come together to form an image.
•The background, formed by un deviated light, is bright, while the unstained object
appears dark and well defined.

29. APPLICATION
To study unstained living cells.
Detailed examination of internal structures in living microorganisms.
To study flagellar movements and motility of bacteria and protozoan’s
To study intestinal and other live protozoa such as amoebae .
To examine fungi grown in culture.

30. FLUORESCENCE MICROSCOPY
Refers to any microscope that uses Fluorescence
property to generate an image.

31. When Fluorescence dyes are exposed to ultraviolet rays,
they become excited and are said to Fluorescence .i.e. they
covert this invisible, short wavelength rays into light of
longer wavelength.
The source of light may be a mercury lamp which emits
rays that pass through an excitation filter.
The excitation filter is so designed that it allows only short
wavelength UV light (about 400nm, called as the exciting
wavelength of light)to pass through; blocking all other long
wavelength rays.
PRINCIPLES

32. PRINCIPLES
The exciting rays then get reflected by a
dichromatic mirror in such a way that they fall on the
specimen which is priory stained with fluorescent dye.
The fluorescent dye absorb the exciting
rays of short wavelength, gets activated and
it turns emits fluorescent rays of higher
wavelength.
The barrier filter positioned after the objective lenses
removes any remaining UV light, which could damage
the viewer eyes, or blue and violet light, which reduce the
image contrast.

33. APPLICATION:
EpiFluorescence microscope:
It is the simplest form of Fluorescence microscope, which has the
following applications.
Auto Fluorescence microscope:
Some microbes directly fluoresce when UV lamp e.g. cyclospora.
Microbe coated with fluorescent dyes:
 Certain microbes fluoresce when they are stained with
fluorochrome dyes. E.g.
•Acridine orange: dye is used in QBC examination for the detection
of malarial parasites.
•Aura mine phenol: is used for the detection of tubercle bacilli.
Immunofluorescence:
•It uses florescent dye tagged immunoglobulin to detect cell surface
antigen or antibodies bound to cell surface antigens.

34. ELECTRON MICROSCOPY
An electron microscope uses accelerated e
electrons as a source of illumination.
Because the wave length of electrons can be up
to 100,000 time shorter than that of visible light
photons.
The Electron microscope has a much better
resolving power than a light microscope.
Hence, it can reveal the details of flagella,
fimbriae and intracellular structures if a cell.

35. Electron microscopes are of two types:
•A Transmission Electron microscope
•B scanning Electron microscope
ELECTRON MICROSCOPY

36. TRANSMISSION ELECTRON MICROSCOPE
Electrons are
generated by electron
gun, which travel in
high speed.
The medium of travel in
EM should be a fully
vacuum path because in
air path, electrons can get
deflected by collision with
air molecules.

37. TRANSMISSION ELECTRON
MICROSCOPE

38. TRANSMISSION ELECTRON
MICROSCOPE

39. ELECTRON PATHWAY
Electron pathway:
•Electron pass through a magnatic condenser and
then bombardon thin sliced mounted on the copper
slide.
•The specimen scatters electrons passing through it,
and then the electron beam is focused by magnetic
lenses to form an enlarged, visible image of the
specimen on a fluorescent screen.

40. MEASURES TO INCREASE
CONTRAST OF EM – STAINING:
 In electron microscope the stain is used are
solution of heavy metal salts like lead citrate and
uranyl acetate.
Negative staining:
 The specimen is spread out in a thin film with
heavy metal like phosphotungstic acid and uranyl
acetate.
Shadowing:
 The technique is particularly useful in studying
virus morphology, bacterial flagella, and plasmids.

41. SCANNING ELECTRON MICROSCOPY
scanning Electron microscope has been
used to examine the surface of
microorganisms in great detail.
It has a resolution of7nm or less.
The SEM differs from TEM, in producing
an image from electrons emitted by an
objects surface rather than from
transmitted electrons.

42. SCANNING ELECTRON
MICROSCOPY

43. INTERFERENCE MICROSCOPE
Uses a differential - I Interference
contrast (DIC) microscope.
Allows for detailed view of live,
unstained specimens.
Includes two prisms that add
contrasting colors to the image.
The image is colorful and three-
dimensional.

44. INTERFERENCE MICROSCOPE

45. RULES OF USING A MICROSCOPY
Always carry with 2 hands.
Only use lens paper for cleaning.
Do not force knobs.
Always store covered
Be careful of the cords.