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Phase Contrast Microscopy –

Optical Components, Working
Principle and Applications
(Short Notes with PPT)

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Phase Contrast Microscopy

(Optical Components, Working
Principle and Applications of Phase
Contrast Microscope)
Working Principle of an Ordinary
Microscope:

In an ordinary microscope, the object is

viewed due to differences in colour
intensities of the specimen. To create the
colour intensities, the specimen is first
stained with suitable dyes which will impart
specific colour. In an ordinary microscope,
the contrast is obtained when the light rays
pass through a stained specimen because
different stains absorb different amounts of
light. These differential absorption
properties of stained specimen modify the
intensity or amplitude of the light waves
transmitted by different regions of the cells
and this ultimately creates contrast in the
image. Thus, staining is essential to create
contrast in an ordinary microscope.
Moreover, the unstained specimen cannot be
observed through an ordinary microscope.

Download the PPT (Phase Contrast

Microscopy)

Why Phase Contrast Microscope?

The Phase Contrast Microscope is used to

visualize unstained living cells. Most of
the stains or staining procedures will kill the
cells. Phase contrast microscopy enables the
visualization of living cells and life events.

History of Phase Contrast Microscope

The Phase Contrast Microscope was

developed by Zernike in early 1930s. The
invention of this microscope enables us to
visualize live cells and cellular processes.
Due to the remarkable contribution of phase
contrast microscopy in biological sciences,
the inventor was awarded Nobel Prize in
Physics in 1953.

Working Principle of Phase Contrast

Microscopy
The phase contrast microscopy is based on
the principle that small phase changes in
the light rays, induced by differences
in the thickness and refractive index
of the different parts of an object, can
be transformed into differences in
brightness or light intensity.

In simple terms, phase contrast microscopy

is the translation of invisible phase shifts
into visible differences of intensities.
The phase changes are not detectable to
human eye whereas the brightness or light
intensity can be easily detected by the human
eyes.

Optical Components of Phase Contrast

Microscope

The phase contrast microscope is similar to

an ordinary compound microscope in its
optical composition. Similar to a normal
microscope, it possesses a light source,
condenser system, objective lens system and
ocular lens system.

A phase contrast microscope differs from the

normal microscope in having TWO
additional components:

(1). Sub-stage Annular

Diaphragm

(2). Phase Plate

(1). Sub-stage Annular Diaphragm

It is located below the sub-stage condenser of

the microscope. The sub-stage annular
diaphragm helps to create a narrow,
hollow cone or ring of light to illuminate
the object.

(2). Phase Plate

The phase plate is also called as diffraction

plate or phase retardation plate. It is located
at the back focal plane of the objective lens.
The phase retarding components are coated
on this plate. The phase plate is a
transparent glass disc with one or few
channels. The channel is coated with a
material that can absorb light but cannot
retard it. The other portion (other than
channel) of the phase plate is coated with
light retarding materials such as Magnesium
fluoride. Phase plate helps to reduce the
phase of the incident light.

How Contrast is Created in Phase

Contrast Microscopy?

The unstained cells cannot create contrast

under the normal microscope. However,
when the lights pass through an unstained
cell, it encounters regions in the cells with
different refractive indexes and thickness.
When light rays pass through an area of high
refractive index, it deviates from its normal
path and such a light ray experiences phase
change or phase retardation. Light rays pass
through the area of less refractive index
remain undeviated (no phase change).

The difference in the phases between the

retarded and un-retarded light rays is about
¼ of original wave length (i.e., λ/4). Human
eyes are NOT able to detect this minute
changes in the phase of light and thus, such
small phase changes do not create any
contrast in the image. The phase contrast
microscope has special devices (annular
diaphragm and phase plate), which convert
these minute phase changes into amplitude
changes or brightness changes so that a
contrast difference can be created in the final
image. This contrast difference can be easily
detected by our eyes.

Let’s see how phase contrast microscope

achieves contrast in the image without
staining of the object. In phase contrast
microscope, in order to get contrast, the
diffracted waves have to be separated from
the direct waves. This separation is achieved
by the sub-stage annular diaphragm.

The annular diaphragm illuminates the

specimen with a hollow cone of light. Some
rays (direct rays) pass through the thinner
region of the specimen and do not undergo
any retardation and they directly enter into
the objective lens. The light rays passing
through the denser region of the specimen
get regarded and they run with a delayed
phase than the undeviated rays. The
retardation of the phase of light is about ¼
of the λ of the incident light. Both the
retarded and unretarded light has to pass
through the phase plate kept on the back
focal plane of the objective to form the final
image.

The phase plate is designed and positioned

in such a way that the retarded light rays will
pass through the area of phase plate where
light retarding materials are coated. When
the ¼ (or λ/4) retarded light is passed
through this region of phase plate, it is
further retarded by ¼ (or λ/4). With this, the
final change or retardation will be- ¼λ + ¼λ
= ½λ (or λ/4 + λ/4 = λ/2). The un-retarded
rays will pass through the channels of the
phase plate and their phase is not altered by
the phase plate.

When the unretarded and ½λ (or λ/2)

retarded light are recombined (at the focal
point), a negative or destructive
interference is created because the crest
and trough of the unretarded and retarded
light rays will cancel each other. With the
destructive interference, the image of the
specimen appears darker against a bright
background. On the other hand, if the un-
deviated light rays are passed through the
phase regarding material, the two rays will
be in the same phase and the result will be a
positive or constructive interference.
In constructive interference, the image of the
specimen becomes brighter against a dark
background. Thus in phase contrast
microscopy, the combination of destructive
and constructive interferences creates high
contrast in the final image.

Applications of Phase Contrast

Microscopy
The magnification and resolution phase
contrast microscope is similar to that of an
ordinary microscope. Still, PCM has many
applications in biological sciences. The
applications of phase contrast microscopy
can be summarized as:

Ø PCM enables the visualization of living

cells.

Ø It enables visualization of unstained

cells.

Ø PCM can be used to view various cell

organelles (mitochondria, nucleus, and
vacuoles).

Ø Helps to study the cellular events such

as cell division, phagocytosis, cyclosis etc.

Ø Used to visualize all types of cellular

movements such as chromosomal and
flagellar movements.

Ø Enable the study of cytoskeleton

dynamics during cell division,
phagocytosis etc.

Ø Enable the study of membrane

permeability of cells and different
organelles.

Ø Phase contrast microscopy is

extensively used to observe living cells in
tissue culture to monitor their growth

Advantages of Phase Contrast

Microscope
Ø Provide the clear image of unstained
cells.

Ø Avoid damages of the cells due to

chemical preparation and staining.

Ø Provide high contrast images

highlighting the fine details of the cells.

Ø The optical construction is relatively

simple.

Ø A compound microscope can be

elevated to phase contrast microscope
with minor additions.

Ø Enable prolonged observation of living

cells without losing the viability of cells.

Ø Live cell imaging and live process

monitoring are possible.

Ø Affordable cost.

Disadvantages / Limitations of Phase

Contrast Microscope
Ø Phase contrast microscope produces a
bright halo around the images. The
formation of the halo is due to the partial
or incomplete separation of direct and
deviated rays.

Ø PCM is only useful for viewing

individual cells or thin layer of cells.

<<< Back to Biophysics Lecture

Notes

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Download PPT of Phase Contrast

Microscopy

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