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Phase Contrast Microscope: BSC Semester 2 Subject: Intro To Micro

The document discusses the principles and components of a phase contrast microscope, which uses special optical components to convert phase differences in light passing through transparent specimens into visible brightness differences, allowing observation of living unstained cells. Key components are an annular diaphragm and phase plate in the condenser and objective, which introduce a quarter wavelength phase shift to separated deviated and non-deviated light rays, producing an image with high contrast.

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29 views19 pages

Phase Contrast Microscope: BSC Semester 2 Subject: Intro To Micro

The document discusses the principles and components of a phase contrast microscope, which uses special optical components to convert phase differences in light passing through transparent specimens into visible brightness differences, allowing observation of living unstained cells. Key components are an annular diaphragm and phase plate in the condenser and objective, which introduce a quarter wavelength phase shift to separated deviated and non-deviated light rays, producing an image with high contrast.

Uploaded by

wadhwacommunisation0001
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Phase Contrast Microscope

BSc Semester 2
Subject: Intro to Micro
Introduction

• A microscope is an instrument which used to see object that are too small to be

seen by the naked eye.

• The object of magnification of cells and their components was achieved by the

lenses of various type or a combination of lenses which could magnify the minute

objects up to a particular limit, and therefore so many lenses were combined

together to form an instrument known as the microscope.

• PCM first described in 1934 by Dutch physicist Frits Zernike, whom awarded by

Nobel prize in physics in 1953.


Construction

• Compared to compound microscope two additional devices are equipped in PCM.


• 1) Annular Diaphragm: It is placed below the condenser. It permits a hollow
beam of light.
• 2) Phase contrast plate: It is placed at rear focal plane ( the location closer to
the ocular lens) of objective lens. It is a disc composed of glass having circular
coated area on it (MgF, Magnesium Fluoride is coated on this region) and is
responsible for creating phase difference of ¼ wavelength as compared to light rays
passing through rest of the plate.
Principle

• PCM 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.
• Light rays in phase produce brighter image.
• Light rays out of phase form darker image.
• Contrast is due to out of phase rays.
Knowledge of wave nature of light

• Wave of light consists of trough and crest.


• If two light waves are completely in phase, phenomenon of co-incidence occurs
between the waves. This forms a new light wave having greater amplitude.
• At the other extreme, if two waves are out of phase, this cancels the effect of each
wave and the resultant wave has zero amplitude. The phenomenon is called as
interference.
• Co-incidence of light waves leads to maximum brightness while interference of
light waves leads to darkness.
Working

• Unpigmented living cells are not clearly visible in the bright field microscope
because there is little difference in contrast between the cells and water.
• One solution to this problem is to kill and stain cells before observation to increase
contrast and create variations in color between cell structures.
• But if we want to visualize living cells it will not be possible with bright field
microscope.
• PCM is used for visualizing living unstained specimens.
• It converts slight differences in refractive index and cell density into easily
detected variations in light intensity and is an excellent way to observe living
cells.
Working

• When the light passes through an unstained cell, it encounters regions in the cell
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 light rays experience phase change or phase retardation
(deviation).
• Light rays pass through the area of less refractive index remain non-deviated (no
phase change).
Working

• Human eyes cannot detect these minute changes in the phase of light. The phase
contrast microscope has special devices such as annular diaphragm and phase plate,
which convert these minute phase changes into brightness (amplitude) changes, so
that a contrast difference can be created in the final image. This contrast difference
can be easily detected by human eyes.
• In phase contrast microscope, to get contrast, the diffracted waves have to be
separated from the direct waves. This separation is achieved by the sub-stage
annular diaphragm.
Working

• 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 deviation and they directly enter into the objective lens.
• The light rays passing through the denser region of the specimen get retarded and
they run with a delayed phase than the non-deviated rays.
• Both the deviated and non deviated light has to pass through the phase plate
kept on the back focal plane of the objective to form the final image. The
difference in phase (Wavelength) gives the contrast for clear visibility of the object.
Working

• Undeviated light rays strike a phase ring in the phase plate, while the deviated
rays miss the ring and pass through the rest of the plate.
• If the phase ring is constructed in such a way that the undeviated light passing
through it is advanced by ¼ wavelenght, the deviated and undeviated waves
will be about ½ wavelength out of phase and will cancel each other when they
come together to form an image. The phenomenon is called destructive
interference.
• The background formed by undeviated light is bright while the unstained
object appears dark and well defined.
Applications

• Phase contrast microscope enables the visualization of unstained living cells.

• It makes highly transparent objects more visible

• It is used to examine various intracellular components of living cells at relatively high


resolution.

• It helps in studying cellular events such as cell division.

• It is used to visualize all types of cellular movements such as chromosomal and


flagellar movements..
Advantages

• Living cells can be observed in their natural state without previous fixation or labeling.

• It makes a highly transparent object more visible.

• No special preparation of fixation or staining etc. is needed to study an object under a phase-

contrast microscope which saves a lot of time.

• Examining intracellular components of living cells at relatively high resolution.

• It made it possible for biologists to study living cells and how they proliferate through cell

division.

• Phase-contrast optical components can be added to virtually any brightfield microscope,

provided the specialized phase objectives conform to the tube length parameters, and the

condenser will accept an annular phase ring of the correct size.


Disadvantages

• Phase-contrast condensers and objective lenses add considerable cost to a


microscope, and so phase contrast is often not used in teaching labs except perhaps
in classes in the health professions.
• To use phase-contrast the light path must be aligned.
• Generally, more light is needed for phase contrast than for corresponding bright-
field viewing, since the technique is based on the diminishment of the brightness of
most objects.

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