Phase Contrast Microscopy
• Invented by Frits Zernike (1934) .
• Optical microscopy technique utilized to produce high contrast images of transparent
specimens, such as living cells, microorganisms, thin tissue slices, fibres, latex
dispersions, glass fragments and subcellular particles.
• Unlike ordinary bright-field microscopy, cells need not be killed, fixed, or stained,
which makes it ideal for studying living organisms and their internal structures.
Principle
• When light passes through a transparent specimen,some light rays are slowed down
due to differences in refractive index and thickness within the specimen, causing
phase shifts.
• These phase changes are invisible to the eye.
• The phase contrast microscope uses a condenser annulus and a phase plate to
convert these phase shifts into amplitude (intensity) differences.
• It splits light into direct (undeviated) and diffracted (deviated) rays
o Direct (undeviated) light – passes straight through the specimen.
o Diffracted (deviated) light – bent by structures inside the specimen.
• When these two types of light are brought together, they interfere with each other:
o Waves are in phase, the image appears bright.
o Out of phase, the image appears dark.
• By amplifying these phase differences, the microscope produces a contrast image,
making transparent details visible.
Instrumentation of Phase Contrast Microscope
1. Light Source
i. Provides bright, steady illumination for the specimen.
ii. Common light Sources include halogen lamps or LEDs, which should
offer consistent and adjustable intensity.
iii. Light passes through the annular diaphragm in the condenser.
2. Condenser
i. Focuses and directs light onto the specimen.
ii. In phase contrast microscopes, the condenser includes a phase
annulus, which generates a hollow cone of light that illuminates the
specimen.
3. Phase Annulus (Annular Diaphragm):
i. A specialized aperture located in the condenser that creates a ring-
shaped illumination pattern.
ii. Enhances contrast by using specific light patterns to reveal details in
transparent specimens.
iii. It is made up of a circular disc having a ring shaped annular groove
(annular ring).
iv. The light rays are allowed to pass through the annular groove.
v. Through this annular groove of the light rays fall on the specimen or
object to be studied.
vi. Produces a hollow cone of light that illuminates the specimen. It
ensures only a ring of light rays passes through, which helps separate
direct and diffracted light for contrast formation.
4. Specimen Stage
i. Holds the slide containing the transparent specimen.
ii. Positions the specimen correctly in the light path.
5. Objective Lens
i. Special phase contrast objective with a phase plate.
ii. Magnifies the image of the specimen.
iii. The objective lens captures both direct and diffracted light,
converting phase differences into visible contrast.
iv. Types: Positive phase contrast (bright background, dark specimen) or
Negative phase contrast (dark background, bright specimen).
6. Phase Plate
i. The phase plate is a transparent disc located at the back focal plane
of the objective lens.
ii. It can be either:
a. Positive phase plate - has a thin circular groove
b. Negative phase plate - has a thick circular area
iii. The thick or thin region in the phase plate is called the conjugate area,
also referred to as the phase ring.
iv. The phase ring introduces a phase shift to the direct light, creating a
detectable phase difference between direct and diffracted rays.
v. Using the annular diaphragm and phase plate, direct and diffracted
light rays are separated:
a. Direct rays pass through the phase ring (aligned with the
annular groove)
b. Diffracted rays pass through the region outside the
groove
vi. Due to the different refractive indices of cell components, the
specimen shows varying degrees of contrast, allowing clear
visualization of transparent structures.
7. Ocular Lens
i. Magnifies the image formed by the objective.
ii. Allows the observer to see the final high-contrast image.
Working
• Light from the source passes through the annular diaphragm, forming a hollow cone
that illuminates the specimen. As light passes through, some rays remain undeviated
(direct light) while others are diffracted or refracted by the specimen. The objective
lens collects both types of light, and the phase plate shifts the phase of direct light by
½ wavelength (A/4). When these rays combine at the image plane, interference
occurs-producing bright and dark regions. This results in a high-contrast image of
transparent, unstained specimens, which is then viewed through the ocular lens.
Applications of Phase Contrast Microscope
• Observation of Living Cells
o Used to study living, unstained, and transparent specimens such as protozoa,
bacteria, and tissue culture cells.
o Reveals cell morphology and movement without killing the specimen.
• Study of Cell Organelles
o Allows visualization of nuclei, mitochondria, vacuoles, and other internal
structures in living cells.
o Helps in studying cell division, cytoplasmic streaming, and organelle behavior.
• Microbial Studies
o Used to examine bacteria, yeasts, and fungi in their natural state.
o Useful for observing motility, spore formation, and binary fission.
• Tissue Culture and Medical Research
o Helps monitor cell growth, differentiation, and viability in culture.
o Commonly used in cancer research, drug testing, and toxicology studies.
• Useful in examining thin tissue sections and chromosomal structures without
staining.
• Applied to study fibres, polymers, glass fragments, and latex particles, where staining
is not possible.
Advantages
• No staining needed; live cells can be observed.
• Gives high contrast for transparent specimens.
• Reveals internal cell structures clearly.
• Useful for studying cell movement and division.
Disadvantages
• Produces halos around images.
• Not suitable for thick specimens.
• Expensive and needs careful alignment.
• Gives qualitative, not quantitative, results.