2. INTRODUCTION TO PHASE CONTRAST
MICROSCOPY
Phase contrast microscopy, first described in 1934
by Dutch physicist Frits Zernike
Contrast-enhancing optical technique that can be
utilized to produce high-contrast images of
transparent specimens
such as living cells (usually in culture),
microorganisms, thin tissue slices, fibers, glass
fragments, and subcellular particles (including
nuclei and other organelles).
3.
4. .
The phase contrast microscopy is a special
adaptation of the light microscopy & helps to obtain
a clear picture of living or unstained cells.
The adaptors convert minute difference in phase
changes in transmitted light due to refractive
indices of all cell organelles in to perceptible
shades of grey
This allow organelles of the living cell to become
visible with fair contrast in them.
5. ANNULAR RING AND PHASERING
ANNULAR RING : is between condenserand light
source
PHASE RING : is between objective lens and
image plane
SPL PROPERTY : both the rings allow partial light
to pass through it and the rest is blocked
6. WORKING PRINCIPLE
Regions of different composition likely to have
different Refractive indices. Normally such
differences cannot be detected by our eyes.
However, PCM converts differences in μ into
differences in intensity (relative brightness and
darkness) Which are visible to eye. •
Interference of light: interaction of two light waves
which leads to the formation of resultant wave
constructive interference
destructive interference
7. .
Light passes through the condenser via annular
ring.
After reaching the specimen plate two types of beams
are formed
IF THERE IS NO SPECIMEN IN LENS
1 Surrounding wave (S)
2 Particle wave (P)
P=S
NO INTERFERENCE
9. .
IF LENS CONTAIN SAMPLE :
Light beams gets diffracted because of different
density at different regions of sample
1 Surrounding wave (S)
2 Diffaractive wave (D)
P=S+D
Either constructive interference or destructive
interference may occur
10.
11. .
The ring shaped illuminating light that passes the
condenser annulus is focused on the specimen by
the condenser • Some of the illuminating light is
scattered by the specimen .The remaining light is
unaffected by the specimen and forms the
background light. In a phase contrast microscope
The image contrast is improved by two different
methods
• Negative Phase contrast
• Positive Phase contrast
12. .
Negative Phase contrast produces destructive interference
• Thus , the image obtained is
• Inner region of the sample - bright
• Outer region of the sample - darker
• Surrounding lens - opaque
Positive phase contrast produces constructive interference
Thus, the image of the specimen obtained is
* Inner region of the sample –darker
* Outer region of the sample – bright
* Surrounding lens - opaque
13. ADVANTAGES
Small unstained specimens such as a living cell can
be seen.
It makes Highly Transparent objects more visible.
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.
15. DISCOVERY
British scientist Sir George G.Stokes first described
fluorescence in 1852
He observed that the mineral fluorspar emitted red
light when it was illuminated by ultraviolet excitation
Stokes noted that fluorescence emission always
occurred at a longer wavelength than of the
excitation light
This shift towards longer wavelength is known as
Stokes Shift
16. INTRODUCTION TO FLUORESCENCE MICROSCOPY
Fluorescence microscopy is an one of the light
microscope
It refers to any microscope that users fluorescence
to generate an image
It produces 3d image
The technique is used to study specimen , which
can be made to fluorescence
17.
18. HOW DOES FLUORESCENCE OCCURS
Florescence is a phenomenon that takes place
when a substances absorbs light at a given
wavelength and emits light at another wavelength.
Florescence occurs as an electron, which has been
excited to a higher, and more unstable energy state
, relaxes to its ground state and gives off a photon
of light
19.
20. WORKING PRINCIPLE
The sample to be analyzed is placed on a lens and
the sample is coated with a fluorescence materials
The light is illuminated through the lens with the
higher energy source. The illumination light is
absorbed by the fluorophores
The sample causes them to emit a longer lower
energy wavelength light
This fluorescence light can be separated from the
surroundings radiation with filters
21. DICHROIC FILTER
A dichroic filter or thin film filter , is a very accurate
color filter used to selective pass light of a small
range of colors while reflecting other colors
22. .
The light from the light source is passed through
the excitation
The specific wavelength of light is passed through
the sample via dichromatic filter
The objective lens focuses the light to the specimen
The light emitted from the specimen is filtered by
barrier filter
23. APPLICATIONS
Imaging structural components of small specimens
, such as cells
Conducting viability studies on cell populations (are
they alive or dead)
Imaging the genetic material within a cell (DNA and
RNA)
To differentiate different types of cells