DARK FIELD MICROSCOPY

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.

Dark-field microscopy is a technique that can be used for the observation of

living, unstained cells and microorganisms. In this microscopy, the specimen is
brightly illuminated while the background is dark. It is one type of light
microscope, others being bright-field, phase-contrast, differential interface
contrast, and fluorescence.
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 DARK FIELD MICROSCOPY

Dark-field microscopy uses a light microscope with an extra opaque disc (annular filter)
underneath the condenser lens, or a special condenser having a central blacked-out area, due to
which the light coming from the source cannot directly enter into the objective.

The path of the light is directed in such a way that it can pass through the outer edge of the
 condenser at a wide-angle and strike the sample at an oblique angle. Only the light scattered
by the sample reaches the objective lens for visualization. All other light that passes through
the specimen will miss the objective, thus the specimen is brightly illuminated on a dark
background.

Dark-field requires the use of a cone of light, no direct light enters the objective only reflected,
refracted and diffracted light and high magnification objectives require special condensers.

The condenser numerical aperture (NA) should be larger than that of the objective.

Dark-field requires a bright light source because only the refracted and diffracted light enters
the objective.

Glass slides used for dark-field should be very clean and between 1.1 and 1.2 mm thick.

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.

APPLICATIONS OF DARK FIELD MICROSCOPY

The dark ground microscopy has the following uses:

1. It is useful for the demonstration of very thin bacteria not visible under ordinary
illumination since the reflection of the light makes them appear larger.

2. This is a frequently used method for rapid demonstration of Treponema pallidum in

clinical specimens.

3. It is also useful for the demonstration of the motility of flagellated bacteria and
protozoa.

4. Darkfield is used to study marine organisms such as algae, plankton, diatoms, insects,
fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some
ceramics.

5. Darkfield is used to study mounted cells and tissues.

6. It is more useful in examining external details, such as outlines, edges, grain boundaries
and surface defects than internal structure.

7. A metallurgical darkfield microscope is used to observe hairline metal fractures.

8. Stereo darkfield microscope or gemological microscope used to study the diamonds

 and other precious stones.

9. A stereo dark-field microscope used to observe the shrimp or other invertebrates.

10. In the study of crystals and imaging of individual atoms, Dark-field studies in
transmission electron microscopy play a powerful role.

11. It helps to characterize the embedded nanomaterials in cells by combined with

hyperspectral imaging.

12. A biological darkfield microscope used to observe the blood cells.

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.

7.Dark-field microscopy is a very simple yet effective technique.

8.It is well suited for uses involving live and unstained biological samples, such as a smear
from a tissue culture or individual, water-borne, single-celled organisms.

9.Considering the simplicity of the setup, the quality of images obtained from this technique is
impressive.

10.Dark-field microscopy techniques are almost entirely free of artifacts, due to the nature of
the process.

11.A researcher can achieve a dark field by making modifications to his/her microscope.

12.esolution by dark-field microscopy is somewhat better than bright-field microscopy.

13.It improves image contrast without the use of stain, and thus do not kill cells.

14.Direct detection of non-culturable bacteria present in patient samples.

15.No sample preparation is required.

16.It requires no special setup, even a light microscope can be converted to dark field.

Limitations of Dark-Field Microscopy

1.Necessity to examine wet, moist specimens containing living organisms very quickly,
because visualization of the moving bacteria is essential to detection.

2.The sample must be very strongly illuminated, which can cause damage to the sample.

3.Besides the sample, dust particles also scatter the light and appear bright.

4.Sample material needs to be spread thinly, dense preparations can grossly affect the contrast
and accuracy of the dark field’s image.

5.The main limitation of dark-field microscopy is the low light levels seen in the final image.

6.The sample must be very strongly illuminated, which can cause damage to the sample.

7.Dark-field microscope creates prone to degradation, distortion, and inaccuracies images.

8.If the specimen’s density differs across the slide or is not thin enough, it can create artifacts
throughout the image.

9.The slides of bad quality can grossly affect the contrast and accuracy of a dark field image.

10.Before use make sure slide, stage, nose, and light source are free from dust.

11.An intense amount of light is required for a dark field microscope to work.

12.It cannot measure the specimen accurately.

13.Liquid bubbles can be formed during uses of oil or water on the condenser and/or slide,
which is almost impossible to avoid.