2. SUBMITTED BY:-
SUBMITTED
TO:
REMI YAMACITHA. J,
III RD BIOTECH ‘B’ SEC,
CB19S076899,
BON SECOURS
COLLEGE
FOR WOMEN.
DR. J.REVATHY,
ASS.PROFESSOR,
DEPT. OF BIOTECH,
BON SECOURS
COLLEGE
FOR WOMEN.
4. INTRODUCTION:
-
The light microscope is an instrument used by
researchers in many different fields to magnify
specimens to as much as a thousand times their
original size.
In its simplest form, it is composed of a clear lens
that magnifies the sample and a light source to
illuminate.
5. Phase contrast microscope:-
The easiest and most common way
to image biological samples is using
phase contrast, which is a special
contrast- enhancing imaging method
for transmitted –light microscopes
invented by Frits Zernike(1888-
1966) in 1932 and introduced into
microscopic practice by August
Kohler(1866-1948) and Loos in
1941.Soon it revolutionized
biological and medical research and
earned its inventor the Noble prize in
physics in 1953.
6. INSTRUMENTATIO
N:-
Unstained living cells absorb practically no light. Poor
light absorption results in extremely small differences
in the intensity distribution in the image. This makes
these cells barely, or not at all visible in a brightfield
microscope.
Phase-contrast microscopy is an optical microscopy
technique that converts phase shifts in the light
passing through a transparent specimen to brightness
changes in the image.
It was first described in 1934 by Dutch physicist Frists
7. PRINCIPL
E:-
When a light passes through an object that is
more optically dense than its
environment(background), the wavefronts are
retarded with respect to the unaffected bypassing
background light.
In a phase-contrast microscope, these phase
shifts are converted into changes in amplitude,
which can be observed as differences in image
8. CONSTRUCTIO
N:-
Phase-contrast microscope 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.
9. THE ANNULAR
DIAPHRAGM:-
It is situated below the condenser, 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 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.
The annular phase plate is placed at this back focal
plane.
10. THE PHASE
PLATE:-
It is either a negative phase plate having a thick
circular area or a positive phase plate having a
thin circular groove.
The phase plate is a transparent disc, thin or thick
area in the phase plate is called conjugate area.
With the help of annular diaphragm ad phase
plate, the phase contrast is obtained in this
microscope.
This is obtained by separating the direct rays
from the diffracted rays.
The direct rays passes through the annular
groove whereas the diffracted light rays pass
through the region outside the groove.
11. PROTOCO
L:-
In order to achieve a phase
contrast image your microscope
must be set-up with the proper
phase contrast components.
These components must be
centered to the corresponding
phase annulus.
12. In order to centre for phase contrast phase follow the
instructions below:-
If possible align your microscope for kohler
illumination.
Remove one eyepiece and insert the phase contrast
centering telescope.
Focus the phase contrast microscope so that the
phase plate and the phase annulus are sharp and in
focus.
Start with the lowest magnification phase objective.
If it is 10x Ph1 put this objective in place and the
corresponding Ph1 phase annulus.
Observe the phase plate and the phase annulus
through the phase telescope.
13. Images (a) &(c) are an example of a system that is
not centered and aligned for phase contrast. Images
shows perfectly centered system.
The segmented circle of lights falls directly into the
black ring.
While looking through you will see something like the
image below:-
14. In order to centre your system if it is not aligned you
will need to rotate the adjustment screws. Some
condensers have these built into the condenser,
typically two silver plungers sticking out of the
condenser, other systems require you to insert
centering tools.
Once you have centered the first objective you can
move onto your next objective.
Once complete remove the phase contrast
centering telescope and re-insert the eyepiece.
Once your microscope is fully centered for phase
contrast it should hold its position. There really
should be no need to re-centre unless the system is
tampered with or it is a written protocol. You should
check for proper centeration periodically by inserting
your phase centering telescope.
15. WORKING
:-
A. Partially coherent illumination produced by the
tungsten-halogen lamp is directed through a collector
lens ad focused on a specialized annulus (labelled
condenser annulus) positioned in the substage
condenser front focal plane.
B. Wavefronts passing through the annulus illuminate
the specimen and either pass through undeviated or
are diffracted and retarded in phase by structures and
phase gradients present in the specimen.
C. Undeviated and diffracted light collected by the
objective is segregated at the rear focal plane by a
phase plate and focused at the intermediate image
plane to form the final phase-contrast image
observed in the eyepiece.
16. APPLICATIO
N:-
To produce high-contrast images of transparent
specimens, such as
Living cells(usually in culture)
Microorganisms
Thin tissue slices,
Lithographic patterns,
17. Fibers,
Latex dispersions,
Glass fragment, and
Subcellular-particles (including nuclei and
other organelles).
Applications of phase-contrast microscopy in
biological
Research are numerous.
18. CONCLUSIO
N:-
The phase contrast
microscope opened up an
entire world of microscopy,
providing incredible definition
and clarity of particles never
seen before.
These transparent specimens
could not be explored
because they do not have the
capacity to absorb light.
