PHASE CONTRAST AND

INTERFERENCE MICROSCOPE
GROUP 3: ROLL NO: 21-30
PHASE CONTRAST MICROSCOPE
HISTORY
It was first described in the 1930s,
by Frits Zernike, a Dutch physicist
at the University of Groningen.
He created an optical design that
could transform differences in
phase to differences in amplitude.
 PHASE CONTRAST MICROSCOPE
 Many unstained biological specimens have little contrast with their surrounding
medium and are virtually transparent when observed under bright-field
illumination. Phase contrast microscopy improves the contrast and makes evident
the internal structures of cells which differ in thickness or refractive index.

WORKING PRINCIPLE
 The objects having different refractive indices are identified as they produce
different contrasts. The object with scattered light is identified from the
illuminating background light. If the light passes through a transparent object,
then due to the change of refractive index of the object, the pathway of light will
be deviated slightly, and the light wave is retarded.
•In case of denser particle (higher refractive index) in the object, the
deviation of light will be more, and the light wave is more retarded.
This is known as phase difference.

•Cellular objects having a higher refractive index than the

surrounding medium are dark in appearance, whereas objects having a
lower refractive index than the surrounding medium appear bright.
SYSTEM
A phase contrast microscope differs from bright field microscope
in having,
i. An annular diaphragm (phase condenser)

ii. Phase - plate (diffraction plate or phase retardation plate)

The phase plate is a transparent plate with an etched ring of
reduced thickness.
The purpose of the phase plate is to create a significant phase
difference between the deviated and un-deviated light so that there
is more contrast.
Applications
 This type of microscopy is useful in the examination of living, unstained material
as well as fixed specimens.
 Useful for study of structures and structural changes in larger microorganisms
and tissue cells but not for small or slender objects like spirochaetes.
 Useful in observing the growth of cells and cell cultures.

 Unstained protozoa or fungi are best seen in this microscope.

 Motility of the organism.

INTERFERENCE MICROSCOPE
 Interference microscopy involves the measurement of differences in the path
between two beams of light that have been split.

 In the interference microscope the retarded rays are entirely separated from the
direct or reference rays, allowing improved image contrast, color graduation,
refractive index, dry mass of cells (optical weighing), and section thickness.
 Principle
 Interference microscope is also similar to
Phase contrast microscope in producing high
contrast images but there is no similarity in
how the images are made. It uses polarized
light and two crystalline beam-splitting devices
called Wollaston prisms or Modified Wollaston
(Nomarski) prism, that give the image formed,
a shadow-cast effect delivering a three-
dimensional image.
The additional parts in an interference microscope
includes:
 A polarizer in front of the condenser to produce plane
polarized light,
 A condenser prism mounted close to the front
aperture of the condenser to act as a beam splitter.
 An objective prism mounted close to the back
aperture of the objective lens to recombine the two
beams in the objective back aperture.
 An analyzer to “analyze” rays of elliptically polarized
light coming from the objective and to transmit plane
polarized light that is able to generate an image in the
image plane.
Uses
 It is also used to view unstained living cells.
 It is used to measure the refractive index of cells

 It can be used for concentration of dry mass in cells (optical weighing) .

References
 Fundamentals of Light microscopy and Electronic Imaging by
Douglas B. Murphy
 Bancroft’s Theory and practice of Histological Techniques.
 Field guide to microscopy- Tomasz S. Tkaczyk
 Manual of Clinical Microbiology by James H. Jorgensen