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 DEPARTMENT OF HUMAN ANATOMY
FACULTY OF BASIC MEDICAL SCIENCE
AHMADU BELLO UNIVERSITY, ZARIA.

COURSE TITTLE:-PRINCIPLE OF MICROSCOPY, ELECTRON MICROSCOPY

AND ULTRASTRUCTURE( ANAT 314)

GROUP:- PRENSENTATION GROUP(4)

TOPIC:-PHASE CONTRAST MICROSCOPE, WORKING PRINCIPLE AND ITS

APPLICATION.

COURSE LECTURER:-PROFESSOR. S.S ADEBISI, & A.A OMONIYI.

PROF. S.S ADEBISI, DR. S.A MUSA

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 OUTLINES

 Introduction to phase contrast microscope

 What is a phase contrast microscope.
 Component of phase contrast microscope.
 Working Principle.
 Application and it Advantages
 Limitations.
 Conclusion.

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 INTRODUCTION TO PHASE CONTRAST MICROSCOPE
Abstract
 Phase Contrast Microscopy was introduced by Frits Zernike, a
Dutch physicist, in 1934. The microscopic technique is contrast-
enhancing, allowing users to view high-contrast images of living
cells, tissue slices, and transparent specimens.
 He first describe phase contrast microscopy in 1934
 He describe the use of the phase principle
in microscopy in 1935
 He built the first phase contrast microscopy in 1938
 This project earned him a Nobel prize in 1953

25/Aug/2025 fig 1. Frits Zernike (1888 – 1966) 4

  When living cells are unstained, they do not absorb much light. Due to
poor absorption of light, there's almost little difference in the distribution
of intensity in an image.
 As a result, the cells are either bearly visible or invisible under a
brightfield microscope that shows specimens by using transmitted light.
 That is why there's a need for phase contrast microscope. This is very
instrumental in the evolution of microscopy.
 Because the life of the organism is safe

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 WHAT IS A PHASE CONTRAST MICROSCOPY?

 As mentioned earlier, brightfield microscopes use transmitted light to

view cells. These microscopes don't work well when viewing transparent
specimens because there's little difference in the distribution of intensity
in an image. Phase contrast microscope is designed to overcome this
limitation by converting phase shift.

 Phase contrast microscope is an optical intrument designed to convert

the phase difference of the light passing through a specimen into an
intensity difference. This is known as converting phase shift.

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 25/Aug/2025

25/Aug/2025 fig 2. phase contrast microscope 7

 COMPONENT OF LIGHT PART OF PHASE CONTRAST
MICROSCOPE.
1. Illuminator
2. Annular diphragm (Annular ring)
3. Condenser lens
4. Stage
1/4λ.
5. Objective lens
6. Phase plate
7. Eye piece (ocular lens)

25/Aug/2025 fig 3. light component of pcm 8

 FUNCTIONS OF EACH COMPONENT
 Illuminator:-Light souce to illuminates the specimen
 Annular Diaphragm(Annular ring):-It is placed underneath the condenser,
it focuses hollow cone of light onto the specimen
 Condenser:-Focuses light onto the specimen, improving clarity.

25/Aug/2025 fig 4. Annular Diaphragm 9

 Stage:-Platform to hold the specimen slide.
 Objective lenses:-Provides varying magnification e.g ranging from 4x,10x, 20x, 40x
60x and 100x specifically designed for phsaes contrast microscope.
 Phase Plate:-Is an optical element in the component Phase contrast microscope
within the objective lens.
 It alters the phase and amplitude of light waves passing through the specimen,
creating an intensity difference between the direct and diffracted light rays by
advancing or retarding the original wave length by one quarter difference (λ/4).
 Eye piece:-Magnifies the image formed by the objective lens

25/Aug/2025 fig 5. Different magnifications of objective lens with phase plate 10

 WORKING PRINCIPLE OF PHASE CONTRAST MICROSCOPE

 Phase contrast microscopy is a useful technique for observing

transparent and unstained specimens that are in their natural
state.
 Steps of the working principle of Phase contrast microscope can
be summarized as follows
1. Illumination of the specimen
2. Reaction of light with the specimen
3. The work of the phase plate
4. Inteference and image formation

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  Illumination of the specimen:-When light pass through a
transparent or unstained specimen, they undergoes a phase shift,
meaning thier wave length slightly changes, this phase shift is
usually very small and not visible to the naked eyes.

fig [Link] slight differences in the light waves(diffracted b) passing through the object and the
undiffracted (c) light wave by the object is called phase sphift 12
25/Aug/2025
 Reaction of light with the specimen:-The Phase contrast microscope
is based on the principle of how small phase changes of the wave in the
light rays, caused by the differences in the refractive index of different
part of the specimen can be transformed into differences in brightness
or light intensity. This principle is known as converting phase shift or
principle of wave interference.

25/Aug/2025
fig 7. Refractive index of diffrerent cellular organells 13
  The work of the phase plate:-Phase plate is the most important
component among all, it is an optical element that manipulate the
phase of light coming from the specimen and this manipulation is
used to enhance contrast in imaging.
 It helps to seperate the direct undiffracted light wave from the
diffracted light wave(light rays bent by the specimen).
 It does this by increasing or decreasing the wave length by a
quarter of the original wave lenght.
 Interference and image formation:- In general knowledge
 Interference:- Refers to when two or more waves superpose
(combine) to form a resultant wave of a greater or lower amplitude
 In phase:- Means when trough and crest of a wave aligned
 Out phase:-Is when crest and trough of a wave misaligned
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CONSTRUCTIVE INTERFERENCE
Makes specimen appear brighter
It amplify the light waves

DESTRUCTIVE INTERFERENCE
Makes spciemen appear darker
Destroys the light wave

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  Image formation:-When deviated light having a phase difference
of (λ/4) pass through the phase plate they expirience a further
phase difference of (λ/4) so the final difference will be (λ/2).
 Contructive interference of wave length increases the amplitude
which will inturn increase the brightness of the specimen whereas
destructive interference will cause the darkness of the
background.
 For this reason, an observer make use of intensity
difference(brightness) to differenciate, the different components of
the specimen.

fig 8. 16
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 DIFFERENT SPECIMENS VIEWED UNDER PHASE CONTRAST MICROSCOPE

fig: 9 chloroplast fig 10. Paramecium cells

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 USES OF PHASE CONTRAST MICROSCOPE AND ITS
APPLICATION
 Phase contrast microscopes are primarily used to view unstained and transparent
spercimens, like living cells, microorganisms, and thin tissue slices, making internal
structures visible without need to stain the sample that are difficult to see with
standard brightfield micrscope.
APPLICATION OF PHASE CONTRAST MICROSCOPY
 Cell Biology:- Used to study the morphology, dynamics and behaviour of living
cells.
 Histology:- Utilized in histopathology enabling pathologist to study tissue
abnormalities and diagnose diseases.
 In-Culture Cell Analysis:- Cells in culture can be observed under a phase contrast
microscope to see their morphology and behavior without the need for staining.
 Nanotechnology:-manipulation of cell organelles, It aids in studying their
morphology, size, aggregation, and interactions with surrounding media.
 Live cell imaging - with real time-lapse imaging it enables the observation of
dynamic cellular processes in real-time.(e.g cell cycle) 18
 ADVANTAGES OF PHASE CONTRAST MICROSCOPY

 Enhanced contrast:- Phase contrast microscope significantly improves contrast

for transparent or low-contrast specimens.
 Non-destructive Techniques:-Phase contrast microscope does not requires
staining or other sample preparation techniques that may alter or damage the
specimen.
 Real-time observation:- The non-destructive nature of phase contrast
microscopy enables real-time observation of biological processes.
 High resolution:- Phase contrast microscopy can achieve high resolution
imaging, allowing for visualization of sub cellular structures with great detail.
 Versatility:-Can be applied to various types of specimens, including cultured
cells, tissue sections, microorganism and other transparent samples.
 Non-invasive:-This means the specimen remains unaltered after imaging.

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 LIMITATIONS
 This method is not ideal for thick organisms or specimens
 Images may appear grey or green( if white or green lights is used) resulting in
poor photomicrography.
CONCLUSION
 A phase contrast microscope allows for the observation of transparent and
unstained specimens by converting phase differences in amplitude, which then
create contrast(Brighness).
 The tecnique is particularly useful for studying living cells and microorganisms
that would be difficult to see with a standard brightfield microscpe.

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 REFERENCE
 F. Zernike: Das Phasenkontrastverfahren bei der mikroskopischen Beobachtung, Zeitschrift für
technische Physik 16:454-457 (1935)

 A. Köhler, W. Loos: Das Phasenkontrastverfahren und seine Anwendungen in der Mikroskopie, Die
Naturwissenschaften 29:49-61 (1941)

 W. Loos: Das Phasenkontrastverfahren nach Zernike als biologisches Forschungsmittel, Klinische

Wochenschrift (Journal of Molecular Medicine) 20(34):849-853 (1941)

 A. Köhler: Ein neues Beleuchtungsverfahren für mikrophotographische Zwecke, Zeitschrift für

wissenschaftliche Mikroskopie und mikroskopische Technik 10:433-440 (1893)

 phase contrast microscopy, molecular Expresions, optical Microscopy Primer,2003

 [Link] yolanda smith

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 THANKS

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