Phase contrast microscope, Types of phase contrast, Parts of Phase contrast microscope, Application of Phase contrast microscope

1. Phase contrast MICROSCOPE
R. NITHYA M. Sc., M. Phil., (Ph. D)
ASSISTANT PROFESSOR IN BIOTECHNOLOGY,
SRI ADI CHUNCAHNAGIRI WOMEN’S COLLEGE, CUMBUM, THENI DT,
TAMIL NADU, INDIA.

2. PHASE CONTRAST MICROSCOPE
 The phase contrast microscope
is a light microscope.
 It is modification of compound
microscope.
 It contains all the component of a
compound microscope in addition to
an annular ring and a phase plate.
 It magnifies not only object but also
changes in brightness.

3. PRINCIPLE
The phase contrast microscope separates the illuminating background light
and the specimen scattering light.
Phase contrast microscope is used to visualize transparent, colourless,
unstained, living biological specimens.
These objects are called phase objects.
Light is bend (diffracted) and retarded based on the refractive index of the
object.
Highly refractive structures bend and retard light much.
This principle is used in phase contrast microscope.

5. Phase contrast microscope is the first
microscopic method which allow the
observation of living cell.
It was invented by Frits Zernike
and was awarded noble prize in 1953.

6. TYPES OF PHASE CONTRAST MICROSCOPE
There are two types of phase contrast microscope. They are
Positive phase contrast or Dark contrast
Negative phase contrast or light contrast
Positive phase contrast or Dark contrast
In positive phase contrast, the specimen is dark and the
background is bright. The specimen is surrounded by a bright halo.
Negative phase contrast or light contrast
In negative phase contrast, the specimen is bright and the
background is dark. The specimen is surrounded by a dark halo.

7. STRUCTURE
The phase contrast microscope has
the following parts.
Light source – Tungsten lamp
Annular diaphragm
Condenser
Objective lens with phase plate
Eye piece
Body tube
Coarse adjustment
Fine adjustment
Stage
Stage clips
Nose piece
Arm
Inclination joint
base

8. ANNULAR DIAPHRAGM
 The annular diaphragm is a plate
having circular thin area in the
centre surrounded by thick areas.
 Hence, light coming from the source
can pass through the diaphragm
in the form of a circle.

9. ANNULAR PHASE PLATE
 The annular phase plate is a
transparent disc with a groove.
 The size of the groove is identical
to the circular area of the sub-stage
diaphragm.
 The annular groove of phase plate
filters off 70 – 90% S –waves and leaves
D-waves as such.
 It is placed at the back focal plane
of the objective.

10. CONDENSER
 It gathers and focuses the reflected
light on the object.
 The condenser has a diaphragm.
 It allows the required intensity
(power, concentration, strength)
of light to pass through.

11. DIAPHRAGM
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. 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.

13. OBJECTIVE LENS
Metal cylinders attached below the nosepiece and contains especially ground
and polished lenses
It magnifies the object.
Onion cells

14. TYPES OF OBJECTIVE LENSES
LPO / Low Power Objective
Gives the lowest magnification, usually 10x
HPO / High Power Objective
Gives higher magnification usually 40x or 43x
OIO / Oil Immersion Objective
Gives the highest magnification, usually 97x or 100x, and is used cedar wood
oil or synthetic oil for better resolution.

15. EYE PIECE
It magnifies the image produced by the objective lens.

16. BODY TUBE
It is a tube with the objective lens at the lower end and the eye piece lens at
the upper end.

17. COARSE ADJUSTMENT
It moves the body tube up and
down rapidly to correct the distance
from the object to get focusing.
FINE ADJUSTMENT
It moves the body tube up
and down slowly to make exact
focusing.

18. SPECIMEN STAGE
It is a platform with a hole in the centre.
The light falls on the object through the hole.
The slide is placed on the stage.

19. STAGE CLIPS
Stage clips hold the slide firmly on the stage

20. NOSE PIECE
It is in the form of rotating disc having holes for fitting the objective lenses.

21. INCLINATION JOINT
It permits tilting of upper part
of the microscope to adjust to the
eye level.

22. BASE OR FOOT
The foot keeps the body in position.

23. USES OF PHASE CONTRAST MICROSCOPE.
• It is used to view the cellular parts of Protozoa, bacteria and others with out
staining.
.

24. • Employed to observe cells in the culture without fixation and
staining

25. Used to examine water and other fluids for the presence of minute transparent
microbes ( bacteria and flagellates)

26. Used to study phagocytosis, pinocytosis, cell divisions etc in
cells.

27. Used to study the behavior of chromosome during mitosis and
meiosis