Bright Field Microscope: Definition,

Parts, Working Principle, Application.

A brightfield microscope is a type of microscope that uses visible light
and an objective lens to magnify and illuminate a sample. It is called a
“brightfield” microscope because the sample is illuminated from below
and appears bright against a dark background. Brightfield microscopes
are commonly used in biology and medicine to observe cells, tissues,
and other biological samples.

To use a brightfield microscope, the sample is placed on a transparent

glass stage and illuminated from below by a light source, such as a
bulb or LED lamp. The light passes through the sample and is focused
onto the objective lenses by a condenser lens. The objective lenses
magnify the image and the eyepieces, also known as ocular lenses,
further magnify the image for the observer.

Brightfield microscopes are widely used in research, education, and

other fields because they are relatively simple and inexpensive.
However, they have some limitations, such as the inability to
distinguish between different types of molecules or structures that are
the same color. Other types of microscopes, such as fluorescence
microscopes or electron microscopes, may be used to overcome these
limitations.

Principle of Brightfield Microscope

The principle of a brightfield microscope is based on the use of visible
light and an objective lens to magnify and illuminate a sample. When a
sample is placed on the stage of a brightfield microscope and
illuminated from below, the light passes through the sample and is
focused onto the objective lenses by a condenser lens. The objective
lenses magnify the image and the eyepieces, also known as ocular
lenses, further magnify the image for the observer.

The sample appears bright against a dark background, hence the

name “brightfield” microscope. This contrast is created by the
difference in light intensity between the illuminated sample and the
surrounding area. The contrast can be adjusted by using a diaphragm,
which is an adjustable aperture located below the condenser that
controls the amount of light that reaches the sample.
In order for a specimen to be the focal point and generate an image
under a Brightfield Microscope, it must travel through a uniform beam
of the illuminating light. The microscope will produce a contrasted
image by differential absorption and differential refraction.

The specimens are initially stained to introduce colour for

straightforward contracting characterisation. The coloured specimens
will have a refractive index that distinguishes them from their
surroundings through both absorption and refractive contrast.

The operation of the microscope is dependent on its ability to produce

a high-resolution image from an appropriately supplied light source
that is focussed on the image, thereby providing an image of high
quality.

The item is seen under oil immersion or with a coverslip after being
placed on a microscope slide.

Light Path of Brightfield Microscope

 A stained specimen or sample is placed over the specimen stage.
 A condenser lens containing an aperture diaphragm is located under
the stage, will focus the light ray on the sample or specimen.
 The light rays will pass through the specimen sample and then it will
be collected by an objective lens located over the stage.
 The objective lens will form a magnified image of the specimen
and then transmit it to the eyepiece, where viewers will observe a
dark image against the brightfield.
 During the transmission through the specimen, some of the light
rays are absorbed by the stains, pigmentation, or dense areas of
the specimen.

Types of Bright Field Microscope

Brightfield microscopy is a microscopy technique that uses a bright
background to create contrast between the sample and the
background, making it easier to see the sample. There are several
types of brightfield microscopes that are commonly used, including the
following:
1. Compound microscope: A compound microscope is a type of
brightfield microscope that uses multiple lenses to magnify the
image of the sample. It consists of two sets of lenses: an objective
lens that is located close to the sample and a eyepiece lens that is
located near the viewer. Compound microscopes are often used to
study small samples such as cells, tissues, and microorganisms.
2. Stereo microscope: A stereo microscope is a type of brightfield
microscope that uses two eyepieces to provide a three-dimensional
image of the sample. It is often used to study large or three-
dimensional specimens, such as plants or insects, or to perform
tasks that require fine manipulation, such as dissection or
microsurgery.
3. Inverted microscope: An inverted microscope is a type of
brightfield microscope that is used to study samples that are
mounted on a slide or other flat surface. The sample is placed on
the stage of the microscope and viewed from below, rather than
from above as in a regular microscope. Inverted microscopes are
often used to study cells or tissues that are grown in culture or to
study large samples that cannot be easily mounted on a slide.
4. Polarizing microscope: A polarizing microscope is a type of
brightfield microscope that uses polarizing filters to study the
optical properties of a sample. It is often used to study minerals,
crystals, and other materials that have birefringence, or the ability
to change the polarization of light as it passes through the sample.
Overall, brightfield microscopy is a widely used technique for studying
small or transparent specimens, and there are several types of
brightfield microscopes that are used for different applications. Each
type of brightfield microscope is designed to meet the specific needs of
the researcher and the sample being studied.

Bright Field Microscope

Bright field microscope labeled

A brightfield microscope is a type of microscope that uses visible light

and an objective lens to magnify and illuminate a sample. The main
parts of a brightfield microscope include:

1. Head: The top portion of brightfield microscope is known as

the head.
2. Arm: The holding portion of the microscope is known
as Arm. Which we used to carry the microscope from one place to
another. This portion provides support to the both optical and
mechanical components.
3. Body tube: A long tube connected the both ocular lens and
objective lens, called as body tube
4. Objective lenses: These lenses are located at the bottom of the
microscope and are used to magnify the sample. Brightfield
microscopes usually have multiple objective lenses with different
magnification levels.
5. Stage: The stage is a platform located below the objective lenses
where the sample is placed. It usually has a transparent glass
bottom to allow light to pass through the sample.
6. Condenser: The condenser is a lens located below the stage that
focuses light onto the sample. It helps to illuminate the sample and
increase the contrast of the image.
 7. Diaphragm: The diaphragm is a adjustable aperture located
below the condenser that controls the amount of light that
reaches the sample. It helps to fine-tune the contrast of the
image.
8. Eyepieces: The eyepieces, also known as ocular lenses, are
located at the top of the microscope and are used to magnify the
image formed by the objective lenses.
9. Stage clips: The stage clips are located over the specimen
stage. It holds the specimen slide.
10. Focus knobs: The focus knobs are used to adjust the
distance between the objective lenses and the stage to bring the
sample into focus. Bright-fields microscope also contains two
focusing knobs, called fine adjustment knob and the coarse
adjustment knob. These knobs help to adjust the focus of
objective lens.
11. Illuminator: The illuminator is a light source located below
the stage that provides the necessary light for the sample. It can
be a bulb or a LED lamp.
12. Nosepiece: The nosepiece is a rotating mechanism that
holds the objective lenses and allows the user to switch between
different magnification levels.
13. Base: The base is the bottom part of the microscope that
provides support and stability. It also houses the illuminator and
the electrical components of the microscope.
14. Stage knob: This microscope also contains a stage knob,
which controls the stage.

Bright field Microscope Magnification

The magnification of a brightfield microscope is determined by the
combination of the objective lenses and the eyepieces. The objective
lenses are located at the bottom of the microscope and are used to
magnify the sample. Brightfield microscopes usually have multiple
objective lenses with different magnification levels, such as 4x, 10x,
40x, and 100x. The eyepieces, also known as ocular lenses, are located
at the top of the microscope and are used to further magnify the image
formed by the objective lenses.

Total Magnification power = Magnification of the objective lens

x Magnification of the eyepiece
To calculate the total magnification of a brightfield microscope, the
magnification of the objective lens and the eyepiece must be
multiplied together. For example, if a microscope has a 10x objective
lens and a 10x eyepiece, the total magnification would be 10x * 10x =
100x.

It is important to note that the magnification of a microscope is not the

only factor that determines the resolution of the image. The quality of
the optics, the wavelength of the light used, and the sample itself also
affect the resolution of the image. A microscope with a high
magnification but poor optics or a low-quality sample may produce a
blurry or low-contrast image.

Application of Bright-Field Microscope

Brightfield microscopes are widely used in a variety of applications,
including:

1. Biology and medicine: Brightfield microscopes are commonly used in

biology and medicine to observe cells, tissues, and other biological
samples. They are used to study the structure and function of cells,
tissues, and organisms, as well as to identify and classify different
types of cells or tissues.
 Used in blood counting.
 Used to examine the bacterial cells.
 Used to examine the fungal cells.
 It also used in forensic laboratories.
 Used to study plant cells.

2. Used in agricultural laboratories.
2. Pharmaceutical industry: Brightfield microscopes are used in the
pharmaceutical industry to analyze the purity and quality of drugs.
They can be used to identify contaminants or impurities in drug
samples and to ensure that the drugs meet quality standards.

1. Food industry: Brightfield microscopes are used in the food

industry to detect contaminants or defects in food products. They
can be used to inspect food products for bacteria, fungi, or other
contaminants that may pose a health risk.
2. Materials science: Brightfield microscopes are used in materials
science to study the structure and properties of materials. They can
be used to examine the microstructure of metals, ceramics,
polymers, and other materials.
3. Geology: Brightfield microscopes are used in geology to study
rocks and minerals. They can be used to identify different types of
minerals and to examine the microstructure of rocks.
4. Forensic science: Brightfield microscopes are used in forensic
science to analyze evidence from crime scenes. They can be used to
identify fibers, hairs, and other small particles that may be used as
evidence in criminal investigations.
5. Education: Brightfield microscopes are widely used in education to
introduce students to the principles of microscopy and the structure
and function of cells and tissues. They are relatively simple and
inexpensive, making them a popular choice for educational
purposes.

Advantages of bright field microscope

1. Simplicity: Bright-field microscopes are relatively simple to use and
operate, making them a popular choice for many educational and
research settings.
2. Wide availability: Bright-field microscopes are widely available
and can be found in many research and educational labs, making
them a convenient choice for many scientists and students.
3. Good for observing detail: Bright-field microscopes can
produce high-resolution images and are good for observing fine
details in specimens.
4. Versatility: Bright-field microscopes can be used to view a wide
variety of specimens, including biological samples, minerals, and
other materials. It is capable of displaying both stained and
unstained images.
5. Low cost: Bright-field microscopes are generally less expensive
than other types of microscopes, such as confocal microscopes or
electron microscopes.
6. Easy to modify: The brightness and contrast of the image can
be easily adjusted using the light source and condenser, making
it easy to optimize the image for different samples. The
microscope can be altered and modified for improved viewing,
such as by inserting a camera to create a digital microscope or by
using fluorochromes on the specimen and observing in a dark
environment to create a darkfield microscope.
7. Color Change: The optics of the microscope have no effect on
the specimen’s hue.
1. Body tube: A long tube connected the both ocular lens and
objective lens, called as body tubBody tube: A long tube connected
the both ocular lens and objective lens, called as body tube.

Disadvantages of Bright-Field Microscope

1. Limited contrast: Bright-field microscopy relies on the absorption
of light by the sample, so it is not very effective for observing
samples that do not absorb light well or that have similar refractive
indices to the surrounding medium. This can make it difficult to
distinguish between different structures within the sample.
2. Poor resolution: Bright-field microscopy has a relatively low
resolution compared to other types of microscopy, such as phase
contrast microscopy or electron microscopy. This means that it may
not be possible to see very small structures or details within the
sample.
3. Limited sample preparation: Bright-field microscopy requires
samples to be thinly sectioned and mounted on a microscope slide,
which can be time-consuming and may not be suitable for all types
of samples.
4. Limited visualization of fluorescent or transparent
samples: Bright-field microscopy is not well-suited for observing
fluorescent or transparent samples, as it relies on the absorption
of light by the sample. For these types of samples, other
techniques, such as fluorescence microscopy or differential
interference contrast microscopy, may be more appropriate.
5. Limited depth of field: Bright-field microscopy has a limited
depth of field, which means that objects at different depths within
the sample may be out of focus. This can make it difficult to get a
clear image of samples with multiple layers or structures at
different depths.
6. Aperture diaphragm: Therefore, the iris diaphragm is
recommended over the aperture diaphragm, which may generate
excessive contrast that distorts the image.
7. Living Sample: It cannot be used to observe living organisms
such as bacterial cells. Under the brightfield microscope, only
fixed specimens may be observed.
8. Magnification Power: The brightfield microscope’s maximum
magnification is 100x, but it can be adjusted to 1000x, which is
the optimal magnification for viewing bacterial cells.
9. Other Disadvantages:
 1. Due to its poor contrast, the majority of specimens must be
dyed to be visible
10. The use of oil immersion may cause visual distortion.
11. Using a coverslip may harm the specimen.
12. Staining may introduce undesirable details or contaminate
the specimen
13. Staining the specimen prior to viewing it under a brightfield
microscope is laborious.
14. For magnification, the microscope requires a powerful light
source, which can produce excessive heat that can harm or kill
the specimen.
Enhancements of Bright-Field Microscope
 The iris diaphragm reduces or increases the quantity of the light
source.
 Employing an oil-immersion objective lens and covering the
specimen with a glass cover containing a particular immersion oil.
Immersion oil has the same refractive index as glass and improves
the observed specimen’s resolution.
 Use of staining techniques for microbiological samples, such as
simple stains (methylene blue, safranin, crystal violet) and
differential stains (negative stains, flagellar stains, endospore
stains).
 Use a coloured (often blue) or polarising filter on the light source
to highlight elements that are not visible in white light. Filters are
very beneficial for mineral samples.

Darkfield Microscope: Definition,

Principle, Application.

The concept of darkfield microscopy dates back to the late 19th

century, when German scientist Adolf Fick first described the technique
in his book “Die Mikroskopie des Lebenden Auges” (The Microscopy of
the Living Eye). Fick used a simple microscope with a special
condenser lens to illuminate samples from the side, creating a dark
background behind the sample.
Over the next few decades, darkfield microscopy was further
developed and refined, and the technique became more widely used in
the study of biological samples. In the 1930s, American scientist Calvin
S. Snyder developed a special darkfield condenser for use in
microscopy, which made it easier for researchers to use the technique
in their studies.

Today, darkfield microscopy is widely used in a variety of fields,

including biology, medicine, and materials science. It is a valuable tool
for studying samples that are transparent, have low contrast, or are
difficult to observe with other types of microscopy.

What is a Darkfield Microscope?

 A darkfield microscope is a type of microscope that uses darkfield
illumination to observe samples. In darkfield microscopy, the
sample is illuminated from the side or back, rather than from above,
as in a traditional brightfield microscope. This creates a dark
background behind the sample, making it easier to see details and
structures that might be difficult to see with a brightfield
microscope.
 Darkfield microscopy is particularly useful for observing samples
that are transparent or have low contrast with the background, such
as living cells or small organisms. It can also be used to study
samples with a high refractive index, such as crystals or minerals.
 To use a darkfield microscope, a special condenser lens is used to
focus the light on the sample from the side or back. This creates a
halo of light around the sample, which is then focused onto the
eyepieces or a camera by the objective lens. The resulting image
shows the sample as a bright object against a dark background,
making it easier to see details and structures that might be
otherwise difficult to see.
 Darkfield microscopy is commonly used in biological and medical
research, as well as in the study of materials science and other
fields. It is a useful tool for studying samples that are difficult to
observe with other types of microscopy, such as brightfield or
fluorescence microscopy.
 Darkfield microscope allows a viewer to observe the specimen
bright whereas the surrounding appears as dark.
 Using a dark field microscope we can see the living and unstained
cells
  The darkfield microscope can reveal considerable internal
structure in microorganism.
 For a dark field microscope, we can use three types of condenser
such as abbe condenser, cardioids condense, paraboloids
condenser.
 The condenser lens of a dark field microscope creates a hollow
cone of light which is opposite of a bright field microscope.
 An opaque disc is located underneath the condenser lens, which
only allows the scattered lights from the object, and prevents the
straight lights which are transmitted through the specimen. So
that we can see a dark background and the bright object.
 Most of the compound and stereo microscope can be converted
into dark field microscope.
Principle of Darkfield Microscope
Dark-field microscopy employs a light microscope with an extra
opaque disc beneath the condenser lens or a modified condenser with
a central blacked-out section, which prevents light from the source
from entering the objective directly.

The light is intended to pass through the outer edge of the condenser
at a wide angle and strike the sample at an angle. For visualisation,
only the light scattered by the sample reaches the objective lens. All
extraneous light that goes through the specimen and misses the target
illuminates the specimen brightly against a black background.

Types of Specimens for Dark Field

The finest specimens for dark field are those with scattered refractive
objects separated by empty space. If there are too many items or if a
thick solid specimen emits light into the microscope, there will be no
dark field. Very thin histological sections can be utilised if they are
unstained or very partially stained, such as with silver stains. Dark field
microscopy is applicable to fluids from animals and plants, cell
cultures, microorganisms, edibles, fibres, crystals, colloids, and
submicroscopic particles. Also acceptable are autoradiography and
gold labelling preparations.
What is Critical Angle In Dark Field Microscopy?
In a dark field, critical angle is vital. The dark field condenser generates
an extremely oblique light angle. If this angle exceeds the critical angle
at any interface, the light will be completely internally reflected. The
specimen’s immersion medium is crucial for this reason. The critical
angle for glass to air is 41 degrees, whereas it is 61 degrees for glass
to water. Low-power dark field condensers work well for water-based
specimens. Due to the aforementioned reason, a high-powered dark
field condenser may not be useful with a water-immersed specimen.
Even with low power dark field condensers, it is always preferable to
immerse the condenser in oil as long as the critical angle is not
exceeded.

Parts of Darkfield Microscope

1. Dark Field Condensers

Dark field is the only non-Kohler lighting broad field method we shall
investigate. In dark field microscopy, the objective lens receives no
direct light from the condenser. The objective admits only light that is
reflected, refracted, or diffracted by the specimen. The dark field
condenser emits a bright ring. The angle of the light relative to the
surface of the slide is highly oblique.

This oblique light is concentrated on the specimen. It then diverges so

dramatically that no direct light can penetrate the objective. This sort
of illumination is a cone of light that is hollow. The old microscopists
employed only one source of oblique illumination to resolve N.
Spencrii. Strongly tilting the mirror of the microscope to one side
generated this effect.

The dark field condenser illuminates the specimen obliquely from all
360 degrees. To prevent direct light from entering the objective lens,
the numerical aperture of the condenser must be greater than that of
the objective lens. This is not an issue for dry objectives with low
magnification when a 0.95 NA condenser is utilised. However, this is a
concern for NA goals with a high priority. Here, the condenser must
have a very high NA, such as 1.45, and must be utilised with a
maximum NA objective of 1.25.
a. Low Magnification Dark Field Condensers

A low magnification dark field condenser is only an ordinary brilliant

field condenser with an opaque disc of the appropriate diameter put in
its front focal plane. The opaque disk’s diameter must be precisely
sufficient to prevent any direct light from entering the objective.
Obviously, this means that objectives with a higher NA require larger
drives.

Numerous microscope manufacturers provide “universal” condensers

with one or more dark field discs that are compatible with objectives of
varying numerical aperture (NA). By increasing or lowering the
condenser, one can increase or decrease the apparent size of an
opaque disc. Thus, a single disc may be useful for a limited number of
objective NAs. If the condenser has a numerical aperture (NA) more
than 0.95, better results can be obtained at low magnification if the
condenser is lubricated to the slide, even if the objective is used dry.
As follows, you can create your own dark field opaque disc for low
power objectives:

1. Set up Kohler illumination

2. Observe the back focal plane of the objective.
3. Adjust the aperture iris till it just fits outside of the objective
aperture.
4. Measure the diameter of the aperture iris.
5. Create a disc of this diameter that is opaque.
6. Place the disc as close to the condenser iris as feasible beneath
the condenser.
7. If the field is grey, the disc is too tiny, and if the specimen cannot
be lighted, the disc is too large
b. High Numerical Aperture Dark Field Condensers

In 1855, Francis H. Wenham and George Shadbolt developed the first

condenser designed exclusively for dark field. Using a parabolic glass
reflector, this condenser produced a hollow cone of light. A reflecting
condenser does not cause chromatic aberrations, unlike a refracting
condenser, and its parabolic shape lowers spherical aberrations. The
end result is a more tightly concentrated point of light. Over time,
further dark field condenser designs arose. Figure 9.3 and Table 9.1
illustrate some of the most prevalent.
The requirement to keep the objective NA below the condenser NA led
to the manufacturing of ever-higher NA condensers and the
development of the “funnel stop” for objective lenses. A funnel-shaped
cone is put into the objective lens to restrict its aperture.

Modern objective lenses do not utilise funnel stops. Instead, certain

high NA oil goals incorporate an iris diaphragm. This iris decreases the
NA of the objective lens. Always lubricate high magnification dark field
condensers with the specimen slide. Due to the fact that the angle of
incidence of light exiting the top of the condenser is far greater than
the critical angle for glass to air, no light will emerge from the
condenser unless immersion oil is applied to its surface.

2. Eyepiece

The eyepiece is the small lens through which researchers and

scientists can see images of utilised biological specimens.

3. A Light Source

Important for the magnification of biological specimens is a light

source. If there is a light source, the dark field microscope’s condenser
scatters the light rays. Light scattering is entirely dependent on the
light source. The only light source in electron microscopy techniques is
high-accelerating electrons. The electrons contribute to the process of
light ray scattering.

4. Objective Lens

The objective lens of a dark field microscope is located in the dark

hollows of the apex cone.
Path of light in Darkfield Microscope

1. Light enters the microscope to illuminate the specimen.

2. A special disc, called patch stop blocks some light from the light
source, leaving an outer ring of illumination.
3. A wide phase annulus can also be reasonably substituted at low
magnification.
4. Now the condenser lens focuses the light towards the specimen.
5. The light enters the specimen.
6. Most of the light rays are directly transmitted, while some of
them are scattered from the sample
7. Only Scattered light started to enter the objective lens and
creates an image of the specimen.
8. While the directly transmitted light simply misses the lens and is
not collected due to a direct-illumination block and they omitted.
Application of Darkfield Microscope
1. The darkfield microscope is used to identify bacteria like thin and
distinctively shaped.
2. We Can observe the living and unstained cells by using a dark field
microscope.
1. Considerable internal structure in microorganisms can be revealed
by the dark field microscope.
2. A biological darkfield microscope used to observe the blood cells.
3. Used to identify algae.
4. A metallurgical darkfield microscope is used to observe hairline
metal fractures.
5. Stereo darkfield microscope or gemological microscope used to
study the diamonds and other precious stones.
6. A stereo dark-field microscope used to observe the shrimp or other
invertebrates.
7. In the study of crystals and imaging of individual atoms, Dark-field
studies in transmission electron microscopy play a powerful role.
8. It helps to characterize the embedded nanomaterials in cells by
combined with hyperspectral imaging.
Advantages of Darkfield Microscope
1. No need to stain the specimen.
2. It is useful for those specimens that are transparent and absorb
little or no light.
3. Marine organisms such as algae, plankton, diatoms, insects, fibers,
hairs, yeast, and protozoa can observe under a dark-field
microscope.
4. Dark-field microscope can use to study for minerals and crystals,
thin polymers, and some ceramics.
5. It is useful to study the external structure of the specimen in great
detail.
6. It can be used for the research of live bacteria and mounted cells
and tissues.
Disadvantages of Dark-field Microscope
1. Dark-field microscope creates prone to degradation, distortion, and
inaccuracies images.
2. If the specimen’s density differs across the slide or is not thin
enough, it can create artifacts throughout the image.
3. The slides of bad quality can grossly affect the contrast and
accuracy of a dark field image.
4. Before use make sure slide, stage, nose, and light source are free
from dust.
5. An intense amount of light is required for a dark field microscope to
work.
6. It cannot measure the specimen accurately.
7. Liquid bubbles can be formed during uses of oil or water on the
condenser and/or slide, which is almost impossible to avoid.
Challenges of Using Darkfield Microscopy
When preparing specimens for darkfield microscopy, care must be
given since characteristics above and below the plane of focus can
scatter light and degrade the image. When imaging, the cleanliness of
the slide is a critical factor, but in darkfield, it is even more crucial
because every piece of debris will be highlighted and can obscure the
image.

Other obstacles you may encounter when configuring a microscope for

darkfield include:
 Insufficient illumination to render the specimen visible, or the
specimen is visible but extremely dim. Darkfield microscopy requires
a great deal of light since so much of it is obscured by the cone.
Don’t be hesitant to increase the power, but keep in mind that some
samples may not tolerate lengthy exposure to this quantity of light.
 There is a dark area in the centre of the field of view, but peripheral
objects are well-lit and appear normal. This is typically caused by
incorrect condenser alignment or focus. This issue will be resolved
by aligning and focussing the condenser. This solution applies to the
majority of situations where the sample is in focus but the
illumination appears uneven.
 The backdrop has colours, or the background is unevenly lighted with a
grey cast. This occurs frequently when the sample is excessively
thick or mounted in contaminated media. Remounting the sample
ought to be useful.
Why Is Darkfield Microscopy a Good Imaging
Technique?
Darkfield microscopy is a good imaging technique because it allows
researchers to observe details and structures in a sample that might
be difficult to see with other types of microscopy, such as brightfield or
fluorescence microscopy.

Some specific advantages of darkfield microscopy include:

1. High contrast: By illuminating the sample from the side or back,

darkfield microscopy creates a bright image of the sample against a
dark background, making it easier to see details and structures that
might be difficult to see with other techniques.
2. Improved resolution: The high-contrast image produced by darkfield
microscopy can help to improve the resolution of the image,
allowing researchers to see smaller details and structures in the
sample.
3. Good for transparent samples: Darkfield microscopy is particularly
useful for studying transparent samples, such as living cells or small
organisms, which can be difficult to see with other techniques.
4. Can be used with a variety of samples: Darkfield microscopy can be
used with a wide range of samples, including biological, mineral,
and materials science samples.
Overall, darkfield microscopy is a valuable tool for researchers looking
to study samples that are transparent, have low contrast, or are
difficult to observe with other techniques. It can provide high-contrast,
high-resolution images that can help researchers to better understand
the structure and properties of their samples.

Phase Contrast Microscopy: definition,

parts, uses, working principle.
What is phase contrast microscopy?
A phase contrast microscopy converts slight differences in refractive
index and cell density into easily detected variation in light intensity to
observe living cells.

his microscope is used for visualization of cell culture and live cells.
Living cells can be observed without any staining.

Unstained specimens have absorbed no light, as a result it creates

extremely small differences in the intensity distribution in the image.
Therefore in a bright field microscope, the specimen is not clearly
visualized.

Because a small phase shifts occurred, when light passes through

specimens, which we can’t see with our eyes.

In a phase contrast microscopy, these phase shifts are transformed

into changes in amplitude, which can be observed as differences in
image contrast.

In 1932, Dutch physicist Frits Zernike of the University of Groningen

discovered the phase concept. In 1935, he described its usage in
microscopy. In 1953, he was awarded the Nobel Prize in physics for
this achievement. Using a specific disc in the condenser, Zernike
separated straight light from the specimen from diffracted light from
the specimen.

Utilizing a unique plate on the back focal plane of the objective lens, he
increased the phase difference between direct and diffracted light.
Increased interference between direct and diffracted light in the
intermediate picture plane produced visible amplitude contrast for the
microscopist.

Principle of Phase Contrast Microscope

The condenser of a phase-contrast microscope has an annular stop an
opaque disk with a thin transparent ring that produces a hollow cone of
light.

As this cone passes through a cell some light rays are bent due to
variation in density and refractive index within the specimen and are
retarded by 1/4 wavelength. The deviated light is focused to form an
image of the object.

The undeviated light rays strike a phase ring in the phase plate a
special optical disks located in the objective, while the deviated rays
miss the ring and passed through the rest of the plate. The undeviated
light which strikes the phase ring gets advance by 1/4 wavelength
when passing through this ring.

The deviated and undeviated waves become 1/2 wavelength to each

other and will cancel each other to come together to form an image.
Therefore deviated and undeviated lights from different image.
The background formed by undeviated light is bright while the
unstained object appears dark and well-defined.

Light Path of Phase Contrast Microscope

1. The light rays enter the annular diaphragm from its source.
2. Then it passes through the condenser lens, which focused the rays
on the specimen.
3. The light transmitted through the specimen and then enter the
objective lens where an image of the specimen is created.
4. As the light transmitted through the specimen it creates a
deviated and undeviated light rays.
5. The deviated light rays miss the phase ring over the objective
lens.
6. Whereas the undeviated light rays strike a phase ring. As a result,
deviated and undeviated rays formed different images.
 7. The undeviated light rays formed the background of specimen’s
image.
Types of Phase Contrast Microscope
There are two main types of phase contrast microscopes:

1. Brightfield phase contrast microscopes: These microscopes use a

standard brightfield illumination system, in which the sample is
illuminated from above and the image is viewed through the
eyepieces or a digital camera. The phase contrast effect is achieved
by using a special phase contrast condenser and objective lens,
which are placed between the light source and the sample.
2. Differential interference contrast (DIC) microscopes: These
microscopes use a different approach to achieve the phase
contrast effect. Instead of using a phase contrast condenser and
objective lens, they use a special polarizing light source and a
pair of compensating prisms to create the phase contrast effect.
DIC microscopes are particularly useful for studying highly
transparent samples, such as cells and tissues, because they
provide a higher level of contrast enhancement than brightfield
phase contrast microscopes.
Overall, there are two main types of phase contrast microscopes:
brightfield phase contrast microscopes and differential interference
contrast (DIC) microscopes. Both types of microscopes use different
approaches to achieve the phase contrast effect and are useful for
studying small, transparent, or otherwise difficult-to-see specimens.

Operating Procedure of Phase Contrast

Microscopy
The operating procedure for phase contrast microscopy typically
involves the following steps:

1. Preparing the sample: The sample is typically prepared by

placing it on a microscope slide and covering it with a thin layer of
immersion oil or a coverslip. The sample should be as thin as
possible in order to maximize the contrast enhancement provided
by the phase contrast light.
2. Setting up the microscope: The microscope is typically equipped
with a phase contrast condenser and objective lens, as well as a
special phase contrast light source. The condenser and objective
 lens are adjusted to optimize the phase contrast effect, and the light
source is turned on.
3. Focusing the microscope: The microscope is focused using the
fine focus knob, with the light source turned off. This allows the
operator to find the plane of focus for the sample.
4. Illuminating the sample: The light source is turned on and the
sample is illuminated with phase contrast light. The sample is
then viewed through the eyepieces or a digital camera, and the
focus is fine-tuned using the fine focus knob.
5. Analyzing the sample: The operator can then analyze the
sample and make observations about its structure and features.
It may be helpful to take photographs or video of the sample for
later analysis.
How does phase contrast microscopy help
scientists to visualize difficult specimens?
 To make phase shifts apparent in phase-contrast microscopy, it is
necessary to separate the illuminating (background) light from the
specimen-scattered (foreground) light and to modify them
differently.
 The condenser focuses the ring-shaped illuminating light (green)
that flows through the condenser annulus onto the specimen. A
portion of the illumination is scattered by the specimen (yellow).
The remaining, undamaged light serves as background
illumination (red). When studying an unstained biological
specimen, the scattered light is normally feeble and phase-
shifted by 90° relative to the background light (owing to the
typical specimen thickness and the refractive index difference
between biological tissue and the surrounding medium). This
causes the foreground (blue vector) and background (red vector)
to have similar intensities, resulting in a lack of picture contrast.
 Picture contrast is raised with a phase-contrast microscope in two
ways: by generating constructive interference between dispersed
and background light rays in specimen-containing parts of the
field of view, and by limiting the quantity of background light that
reaches the image plane.
 First, the background light is phase-shifted by 90° using a phase-
shift ring, eliminating the phase difference between the
background and scattered light beams.
 This phase shift causes background and scattered light rays
originating from regions of the field of view that contain the sample
(i.e. the foreground) to constructively interfere, making these
regions brighter than regions that do not contain the sample when
the light is focused on the image plane (where a camera or
eyepiece is placed).
 Finally, a grey filter ring is used to decrease the background by 70-
90 percent; this technique optimises the dispersed light generated
by the illumination light while decreasing the quantity of
illumination light that reaches the image plane.
 The phase-shift and grey filter rings will only be illuminated by the
background light, while the dispersed light that illuminates the full
surface of the filter will be dimmed and phase-shifted to a lower
degree.
 That which has been described exemplifies negative phase
contrast. When inverted, the phase shift is +90 degrees,
therefore the background light is now positive. This means the
ambient light will be 180 degrees out of phase with the scattered
light.
 In the second illustration, we can see how subtracting the
scattered light from the background light yields an image with a
darker foreground and a lighter backdrop.
Parts of Phase Contrast Microscope
In contrast to dark field, which may be performed using homemade
equipment, phase contrast requires precise optical elements in both
the condenser and objective. These components must correspond with
one another. No single condenser element is compatible with every
objective element. The phase contrast condenser will be equipped with
a variety of phase annuli. These will coincide with the phase plates
found in phase objectives.

Phase Annulus in the Condenser

 The Phase Annulus is a black disc with a transparent ring or slit that
resides in or underneath the condenser.
 Its aim is to provide a focused cone of light on the specimen. In
contrast to black field lighting, phase annulus light enters the
objective.
 Different purposes demand annuli of varying sizes that correspond
to the objectives (in practise one disc may support more than one
objective e.g. Motic Phase 2 disc supports both the 20X and 40X
objectives).
 Zernike experimented with other slit patterns, but the circular
disc is the most frequent one in use today because it was the
simplest to align with the phase plate so that halo artefacts are
distributed in an angular direction.
 The phase annulus also functions to provide partially coherent
light. Coherence occurs when the majority of light of a particular
colour is in phase, or when the majority of light waves are in
phase with one another

Function

 The Phase Annulus is a transparent ring within an opaque disc. Its

aim is to produce a circle of light in the condenser’s front focal
plane.
 Therefore, the condenser generates a hollow cone of illumination
that is concentrated on the specimen plane. As the numerical
aperture (NA) of the objective grows, the phase annulus must also
expand in diameter.
 Laser light is totally coherent, but it turns out that it is excessively
coherent, causing optical noise by exposing faults and dust in the
optical system.
 The other function of the phase annulus is to align the phase disc
at the back of the objective focal plane with the phase plate so
that direct light can be aligned and directed.
 A phase annulus is not strictly necessary if the phase plate in the
objective is designed to affect only the 0 order diffraction spot in
the back focal plane of the objective.
 A ring of illumination and a complementary circular phase-
altering ring in the objective back focal plane have been proven
to produce the optimum results.

2. Phase Plate in the Objective

 The Phase Plate is a transparent plate with a circular ring. Typically,

the ring is a groove carved into the plate and filled with a phase-
advancing or phase-delaying material (usually a dielectric).
 Due to the presence of a substance that modifies the light’s
amplitude, the ring may look darker or brighter than the remainder
of the plate (a neutral density material usually a thin film of
evaporated metal).
 The position of the ring is over the condenser’s picture of the
annulus. The ring and annulus are conjugate.
 The remainder of the plate complements the annulus and phase
ring. Consequently, the phase plate has conjugate and
complementary regions. In lieu of a separate plate, the phase ring
may be incorporated within the objective’s lens.
Positive phase contrast is the most prevalent type of microscopy.
With positive phase contrast, denser organelles in the cell, such as the
nucleus and mitochondria, look black against a light background.
Positive phase contrast is frequently employed to examine cultured
cells and aquatic microbes.

Negative phase contrast causes the thicker sections and organelles

of a cell to look bright against a dark background. The direct light is
slowed by one-fourth of a wave, resulting in constructive interference
that illuminates features against a dark background. Negative phase is
ideal for observing certain protists, such as vorticella. Negative phase
contrast renders the nuclei of cheek cells brilliant white.

3. Phase Telescope

 The eyepiece of the phase telescope focuses an image of the back

focal plane of the objective onto the retina of a person.
 Observing the back focal plane of the objective is required for
aligning the phase annulus to the phase ring. On some microscopes,
the phase telescope is replaced by an integral Bertrand lens.
4. Green Filter

 A phase contrast system is based on the use of green light

(Fraunhofer E line at 525 nm). The green line is used by
manufacturers to decide how much material to put in the phase ring
for phase advancement or retardation.
 This illumination is due to the green filter. However, phase contrast
works adequately under white light.
5. Centering Tools

 The phase annulus in the condenser and the phase ring in the
objective must be identical. Tools are provided for moving the
phase annulus relative to the phase ring while observing the two
with the phase telescope.
Other parts of a Phase contrast microscope;

 Eyepiece
 head
 arm
 base
 nosepiece
 objective lense
 condenser lense
 specimen stage.
 stage clips
 Aperture

Working Mechanism of the Phase Apparatus

Each component of the phase apparatus has a distinct effect. All of
these processes combine to produce phase contrast. Depending on the
design of the equipment, many forms of phase contrast can be
produced.

Phase Annulus in the Condenser

 The annulus produces a hollow cone of illumination which converges

on the specimen. Contrary to the dark field hollow cone, this ring of
direct light reaches the objective lens’ aperture.
 The direct light generates a ring of illumination at the objective’s
back focal plane. This ring of light is precisely the same size as
the phase ring on the phase plate.
 In the specimen plane, the ring of light is at a focal point and, as
a result, is a circular illumination patch that is solid.
 A portion of this light is diffracted by the specimen, and the
diffracted light spreads across the entire rear focal plane of the
objective and hence the entire phase plate (including over the
phase ring).
Phase Plate in the Objective

 The phase ring has a thickness that differs from the rest of the
phase plate such that it alters the phase of the direct light by 1/4
wave relative to the diffracted light (usually advancing the phase of
the direct light).
 Additionally, the amplitude of the direct light is altered by making
the phase ring darker than the remainder of the phase plate. There
is usually a significant lot more direct light than diffracted light,
making it darker.
 This direct light could overpower any amplitude difference caused
by interference. The typical specimen retards the phase of
diffracted light by 1/4 wave.
 The additional 1/4 wave advance provided to direct light by the
phase ring leads direct and diffracted light to be approximately 1/2
wave out of phase. This configuration is optimal for maximising
interference in the intermediate image plane.
Types of Phase Contrast

Depending on the structure of the phase plate in the objective, as

shown in Figure, several types of phase contrast are available.

1. In “positive” phase contrast, the phase of the direct light is

advanced by 1/4 wave (- type). This results in destructive
interference and dark details on a bright background. The most
prevalent form of phase contrast.
2. In “negative” phase contrast, direct light is slowed by a quarter-
wave in phase (+ type). This results in luminous details on a dark
background due to constructive interference.
3. In positive or negative phase contrast, the phase plate might
be one of two varieties. Either the straight light (type A) or the
diffracted light (type B) can be absorbed (B type). A – is the most
prevalent type. Both A and B type plates are assessed by the
transmission percentage of the ring area, with 20 percent being the
norm.
What is the negative and positive phase
contrast?
 In the positive phase contrast, the object appears as dark gray on a
brighter grey background.
 In the negative phase contrast, the object appears as brighter on a
dark background.
Applications of Phase Contrast Microscope
Phase contrast microscopy is widely used in a variety of scientific and
medical applications, including:

1. Cell biology: Phase contrast microscopy is often used to study living

cells, tissues, and organisms in order to understand their structure
and function. This technique can be used to visualize cell shapes,
 organelles, and other subcellular structures, as well as to study the
movement and behavior of cells over time.
2. Microbiology: Phase contrast microscopy is used to study
microorganisms, such as bacteria and viruses, in order to
understand their characteristics and behavior. This technique can
be used to identify and characterize different types of
microorganisms, as well as to study their growth and reproduction.
3. Genetics: Phase contrast microscopy can be used to study DNA and
other genetic material within cells and tissues, providing valuable
insights into the mechanisms of gene expression and regulation.
4. Materials science: Phase contrast microscopy is used to study the
structure and properties of materials at the microscopic level, such
as metals, ceramics, and polymers. This technique can be used to
understand how these materials behave under different conditions
and to identify and characterize small features within the sample.
Overall, phase contrast microscopy is a valuable tool for scientists and
medical professionals because it allows them to visualize and study
small, transparent, or otherwise difficult-to-see specimens in greater
detail, providing valuable insights into the structure and function of
these materials.

Advantages
There are several advantages to using phase contrast microscopy in
scientific and medical applications, including:

1. Non-invasive: Phase contrast microscopy allows scientists to study

living cells and tissues without the need to stain or dye them, which
can be toxic or harmful to the sample. This makes it a useful tool for
studying living cells and tissues, as well as for identifying and
characterizing small structures or features within a sample.
2. High contrast: Phase contrast microscopy enhances the contrast
between different parts of the sample, making it easier to see small
or transparent structures within the sample. This makes it
particularly useful for studying small or difficult-to-see specimens,
such as microorganisms or subcellular structures.
3. Wide field of view: Phase contrast microscopy allows scientists to
study a wide area of the sample at once, providing a broad view of
the overall structure and organization of the sample. It produces a
resolution and high contrast images of the specimen.
4. Versatility: Phase contrast microscopy can be used with a wide
range of samples, including cells, tissues, microorganisms, and
 materials, making it a valuable tool for a variety of scientific and
medical applications. It is useful for studying on thin specimen.
5. Capture Photo: The modern form of this microscope can capture
photos or can record videos.
6. Intercellular Components: Intercellular components of living cells can
be observed with high resolution. This breakthrough allowed
biologists to examine living cells and the process of cell division.
7. Easy to setup: To add phase-contrast optical components, all you
need are brightfield microscopes, specialised phase objectives that
fit within the tube length restrictions, and a condenser that will
accommodate an annular phase ring of the appropriate size.
8. Phase Contrast Microscopy can create a visible image of a highly
transparent objects.
Overall, phase contrast microscopy is a powerful tool that allows
scientists and medical professionals to study small, transparent, or
otherwise difficult-to-see specimens in greater detail, providing
valuable insights into the structure and function of these materials.

Disadvantages
There are a few limitations to using phase contrast microscopy in
scientific and medical applications:

1. Limited to transparent or semi-transparent specimens: Phase contrast

microscopy is most effective at enhancing the contrast of
transparent or semi-transparent specimens, such as cells and
tissues. It is less effective at enhancing the contrast of opaque or
highly reflective specimens, such as metals or solid materials. Thick
organisms or specimens can not be observed by this microscope, a
distorted image can appear.
2. Requires specialized equipment: Phase contrast microscopy requires
the use of specialized light sources and optics, which can be
expensive and complex to set up. This can limit the accessibility of
this technique to some laboratories or institutions.
3. Limited resolution: Phase contrast microscopy has a relatively low
resolution compared to other techniques, such as electron
microscopy. This can limit the ability to visualize small structures or
features within the sample in great detail. The Annuli or rings of
phase contrast microscope limit the aperture to some extent, which
decreases the resolution of image.
4. Potential for artifacts: Phase contrast microscopy can sometimes
produce artifacts, such as halos or rings around structures within
 the sample. These artifacts can distort the appearance of the
sample and can make it difficult to accurately interpret the results.
5. Poor photomicrography: With the uses of white or green lights
Images appear as grey or green, which results a poor
photomicrography.
6. Light Path: Light paths need to be parallel for phase-contrast to
work.
7. Cost: Due to the high expense of phase-contrast condensers and
objective lenses, they are rarely employed in educational
laboratories outside of the medical field.
8. Light Intensity: Since phase contrast relies on the reduction of most
objects’ brightness, it requires more light than the analogous bright-
field seeing method.
Overall, while phase contrast microscopy is a valuable tool for studying
small, transparent, or otherwise difficult-to-see specimens, it has a few
limitations that should be considered when choosing the appropriate
technique for a particular application.

What is Phase Contrast? And When to use it?

Phase contrast is a specialized technique used in microscopy to
enhance the contrast of small, transparent, or otherwise difficult-to-see
specimens by highlighting subtle differences in the refractive index of
different materials within the sample. This is achieved by illuminating
the sample with a special type of light, called “phase contrast light,”
which is able to highlight these differences and make them more
visible.

Phase contrast microscopy is typically used when studying small,

transparent, or otherwise difficult-to-see specimens, such as cells,
tissues, microorganisms, and small structures or features within
materials. It is particularly useful for studying living cells and tissues
because it allows scientists to visualize these specimens without the
need to stain or dye them, which can be toxic or harmful to the
sample.

Overall, phase contrast microscopy is an important tool for scientists

and medical professionals because it allows them to visualize and
study small, transparent, or otherwise difficult-to-see specimens in
greater detail, providing valuable insights into the structure and
function of these materials.
How Is Phase-Contrast Implemented?
When implementing phase-contrast microscopy, there are two primary
challenges: how to phase shift the scattered light or the direct light,
but not both, and how to illuminate the sample with well-ordered
phase light. Before the invention of the laser, there were no sources of
light with a uniform phase (1960s).

It was known that squeezing light through a small pinhole produced an

expanding light wave with a well-organized phase, but at the cost of a
significant loss of intensity. Using a lens, this circular wave was easily
transformed into a flat wave. Utilizing a circular ring (annulus) of
illumination, phase-contrast compromises between light intensity and
uniform phase. This annulus behaved similarly to a ring of pinholes,
with each orientation around the ring having the same phase despite
the phase varying sporadically.

To phase shift either the scattered or direct light, a phase-shifting optic

(such as a glass disc) is positioned in the light path where it will have a
disproportionate effect on the direct light. A well-organized light plane
begins on the left in below Figure. The phase and direction of the
diffracted (solid lines to the right of the sample) and direct (dashed)
light vary as the light strikes the sample. The objective lens
concentrates scattered light into orderly waves, whereas direct light is
concentrated at the optical centre, where phase-shifting material is
located. This restores the phase relationship between dispersed light
and direct light, allowing the detector to generate contrast through
interference.