Detailed study about phase contrast and flurosence microscope

1. PHASE CONTRAST
MICROSCOPY &
FLUORESCENT
MICROSCOPY
Vivek kumar
MSC MICROBIOLOGY
1ST YEAR
Bangalore university

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).

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

8. Light source
Annular ring
Condenser
Specimen plate
(interference)
Objective lens
Phase ring
Imge plane

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

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.

14. FLUORESCENCE MICROSCOPY
.

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

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

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

24. Thank you