Phase contrast microscopy is a technique that was invented in 1934 by Dutch physicist Frits Zernike, for which he received the Nobel Prize in Physics in 1953. It allows for high-contrast imaging of transparent specimens like living cells without staining. In phase contrast microscopy, variations in the refractive index within a specimen cause some light rays to be retarded, producing an image with areas of different intensities. This converts subtle differences in a sample's density and refractive index into detectable variations in light intensity. Phase contrast microscopy is useful for observing living cells and intracellular structures in their natural state without needing to kill, fix, or stain the specimen. While it provides high resolution living images, it has limitations such as inability to view thick specimens clearly and

1. Phase Contrast Microscopy
1

2. HISTORY:
❖ Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike
❖ For it's discovery Frits Zernike was awarded the Nobel Prize in Physics in 1953.
❖ Prior to the invention of phase contrast techniques, transmitted brightfield
microscopy was one of the most commonly utilized optical microscopy, especially
for fixed, stained specimens or other types of samples having high natural absorption
of visible light.
2

3. WHAT IS PHASE CONTRAST MICROSCOPY:
❖ The phase contrast microscopy is a contrast-enhancing optical technique that can
be utilized to produce high-contrast images of transparent specimens, such as living
cells , microorganisms, and subcellular particles (including nuclei and other
organelles).
❖ It is a type of light microscopy in which the contrast is created by introducing a
specialised optical elements such as phase condenser and phase objective lenses.
3

4. Principle:
When light passes through a living cells, the phase of light wave is changed
according to the cells refractive index: light passing through a relatively thick or
dense part of the cell,such as nucleus is retarted;its phase consequently ,is shifted
relatively to light that has passed through an adjacent thinner region.
4

5. Working:
❖ A phase-contrast microscope 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.
❖ The condenser of a phase-contrast microscope has an
annular stop, an opaque disk with a thin transparent
ring, which produces a hollow cone of light.
❖ As this cone passes through a cell, some light rays are
bent due to variations in density and refractive index
within the specimen and are retarded by about 1/4
wavelength.
❖ The deviated light is focused to form an image of the
object. Undeviated light rays strike a phase ring in the
phase plate, a special optical disk located in the
objective, while the deviated rays miss the ring and
pass through the rest of the plate.
5

6. ❖ If the phase ring is constructed in such a way that the undeviated light passing through it is
advanced by 1/4 wavelength, the deviated and undeviated waves will be about 1/2 wavelength
out of phase and will cancel each other when they come together to form an image.
❖ The background, formed by undeviated light, is bright, while the unstained object appears
dark and well-defined.
6

7. Why Phase Contrast Microscopy is important?
❖ In cases where high magnifications are needed, and the specimen is colorless or the color of the fine details of
the specimen does not show up well, the phase-contrast microscope is the ideal choice compared to bright field
microscopy.
❖ For instance, cilia and flagella can be viewed with bright field microscopy, but the sharp contrast can only be
seen via a phase-contrast microscope. Likewise, clear details of the amoebae can be seen via a phase-contrast
microscope.
7

8. ❖ Observing a living organism in its natural state and/or environment can provide far more information
than specimens that need to be killed, fixed or stain to view under a microscope. This is possible
using PCM.
❖ The cells are human glial brain tissue grown in monolayer culture bathed with a nutrient medium
containing amino acids, vitamins, mineral salts, and fetal calf serum.In brightfield illumination, the
cells appear semi-transparent with only highly refractive regions, such as the membrane, nucleus, and
unattached cells (rounded or spherical), being visible. When observed using phase contrast optical
accessories, the same field of view reveals significantly more structural detail . 8

9. ❖ Examining intracellular components of living cells at relatively high resolution. eg:
The dynamic motility of mitochondria, mitotic chromosomes & vacuoles.
❖ 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.
Mitotic metaphase chromosomes
of Aegilops biuncialis under
Phase Contrast Microscope.
9

10. Phase contrast microscopy boon or bane:
❖ The capacity to observe living cells and, as such, the ability to examine cells in a natural
state
❖ Observing a living organism in its natural state and/or environment can provide far more
information than specimens that need to be killed, fixed or stain to view under a
microscope
❖ High-contrast, high-resolution images
❖ Ideal for studying and interpreting thin specimens
❖ Ability to combine with other means of observation, such as fluorescence
❖ Modern phase contrast microscopes, with CCD or CMOS computer devices, can capture
photo and/or video images. 10

11. Disadvantages or limitations of phase contrast microscopy are:
❖ Annuli or rings limit the aperture to some extent, which decreases resolution.
❖ This method of observation is not ideal for thick organism or particles.Thick specimens
can appear distorted.
❖ Images may appear grey or green, if white or green lights are used, respectively,
resulting in poor photomicrography
❖ Shade-off and halo effect, referred to a phase artifacts
❖ Shade-off occurs with larger particles, results in a steady reduction of contrast moving
from the center of the object toward its edges
❖ Halo effect, where images are often surrounded by bright areas, which obscure details
along the perimeter of the specimen 11

12. Scientists efforts to make phase contrast microscopy:
❖ Phase contrast microscope was invented by Frits Zernike in 1932
❖ Zernike’s studies in optics that Ultimately led to his nobel prize in 1953.
❖ He first received evidence of the phase contrast phenomenon in a study of
diffraction gratings when he was able to selectively detect transparent materials
with different refractive indices.
12

13. ❖ In 1938 Zernike built a microscope based on phase contrast illumination, but it
initially received little attention.
❖ Later on it was german war machine that confiscated his invention and made a
series of microscopes, which demonstrated the true utility of zernike's technique.
❖ In his invention of phase contrast microscopy he used two scientist theory:
❖ He was familiar with Lord Rayleigh’s simple process of making optically sound
glass plate etchings using acid and utilized the technique to make phase strips, glass
plates with a single groove, one millimeter wide and etched to a depth of half a
wavelength.
❖ When he placed a phase strip in the spectrum of the faulty grating and inspected it
with a telescope, the stripes on the grating surface were clearly visible.
13

14. ❖ He was also familiar with Ernst Abbe's theoretical description of the microscope
image associates the transparent object under a microscope with a grating.
❖ Abbe studied gratings consisting of alternating opaque and transparent strips
(amplitude gratings), but he was more concerned with alternating thick and thin
strips (phase gratings).
❖ At last he came to a conclusion that for a phase object, when the phase strip is placed
in the focal plane of the microscope objective, the direct image of the light source is
brought into phase with the diffracted images of a phase object.
❖ The result is that the image viewed appears similar to that produced by an amplitude
object. The image in the eyepiece of the microscope appears in black and white
contrast, as if it were an absorbing object.
❖ Phase contrast microscopy was introduced into microscopic practice by August
Kohler and Loos in 1941. 14

15. Factor Phase Contrast Microscope Standard Normal Microscope
Refractive
Indexes
The specimens do not possess the
ability to absorb light. However, they
have distinct refractive indexes which
leads to scattering of light beams
The specimens reflect the light to form the
images. The scattering of light due to its
refractive index is not found.
Type of
Specimen
For culture microscopes of phase
contrast nature, thick specimens
produce clear and crisp images.
Moreover, staining is not required for
magnification of amplitude specimens.
In normal compound microscope, thin and
transparent specimens produce clear
images.
Staining of some pigment specimens is
necessary to produce magnification.
Color of
biological
samples
The actual color of sample cannot be
detected as the image visualised is
dark.
The actual color of sample can be
detected.
Difference between Phase Contrast Microscope and Standard Microscope:
15

16. Factor Phase Contrast Microscope Standard Normal Microscope
Visibility of
samples structures
It can visualise the images very
efficiently under its eyepiece. The best
trinocular microscope of phase-
contrast nature are recommended in
labs.
It does not provide a detailed view of
samples and the structure is unclear.
It can also be used in labs with the
addition of light components.
Magnification
Feature
The magnification is produced by
phase shifts. The light phase has to be
shifted in a good direction to produce
clear and crisp images.
The magnification is produced by
adjusting the dial on the microscope.
Affordability The phase contrast microscopes are
expensive due to some expensive
components.
Its cost is generally lower than the
cost of a phase contrast microscope.
Difference between Phase Contrast Microscope and Standard
Microscope (Contd.):
16

17. 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.
❖ Application of phase contrast microscopy equipment range from the study of
biological specimens, medical applications, study of live blood cells and other
biological and science applications.
17

18. Advantages:
❖ The living cells can be examined in their natural state without previously being killed,
fixed, and stained.
❖ The dynamics of ongoing biological processes can be observed and recorded in high
contrast with sharp clarity of minute specimen detail.
❖ The cells could be examined in their native states.
❖ The microscopy gives out high-contrast, high-resolution images of all biological
samples or specimens.
❖ It can capture photo and/or video images through its inverted microscope camera.
18

19. Limitations:
❖ The phase-contrast microscopy method of observation is not ideal for thick organisms
or particles of samples.
❖ The thick specimens can appear distorted under the inverted phase microscope.
❖ All obtained images may appear grey or green, resulting in poor photomicrography.
❖ The method gives out the Shade-off and halo effect, referred to as phase artifacts or
samples.
❖ In phase contrast microscopy, shade-off occurs with larger particles, resulting in a
steady reduction of contrast.
❖ The Halo effect is observed, where images are often surrounded by bright areas under
the trinocular compound microscope.
19

20. Reference:
❖ F. Zernike: Das Phasenkontrastverfahren bei der mikroskopische Beobachtung, Zeitschrift
für technische Physik 16:454-457 (1935)
❖ Smacgigworld, Differences between the Standard Normal Microscope and Phase Contrast
Microscope, available at : https://www.smacgigworld.com/blog/difference-standard-
normal-microscope-and-phase-contrast-microscope.php
❖ Efforts of scientist in making phase contrast microscopy
http://www.science.org/doi/10.1126/science.121.3141.345
20

21. THANK YOU
21