Introduction:
First microscope was invented by Antoni van Leeuwenhoek in 1673.
Over the years, microscopes have evolved from the simple, single-lens instrument of Leeuwenhoek, with a magnification of 300 X, to the present-day electron microscopes capable of magnifications greater than 250,000X.
Microscopes are designated as either light microscopes or electron microscopes.
Light microscopes use visible light or ultraviolet rays to illuminate specimens. They include brightfield, darkfield, phase-contrast, and fluorescent instruments.
The microscope which forms a bright image against a dark background is called a “Darkfield Microscope.”
A Darkfield Microscope is similar to the ordinary light microscope; however, the condenser system is modified so that the specimen is not illuminated directly.
The condenser directs the light obliquely so that the light is deflected or scattered from the specimen, which then appears bright against a dark background.
Living specimens may be observed more readily with darkfield than with brightfield microscopy.
Principle:
A dark field microscope is arranged so that the light source is blocked off, causing light to scatter as it hits the specimen.
This is ideal for making objects with refractive values similar to the background appear bright against a dark background.
The introduction of a condenser and/or stop below the stage ensures that these light rays will hit the specimen at different angles, rather than as a direct light source above/below the object.
The result is a “cone of light” where rays are reflected and/or refracted off the object, ultimately, allowing the individual to view a specimen in a dark field.
Diagram:
Uses:
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.
This is a frequently used method for rapid demonstration of Treponema pallidum in clinical specimens.
It is also useful for the demonstration of the motility of flagellated bacteria and protozoa.
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.
Darkfield is used to study mounted cells and tissues.
It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.
Advantages:
Dark-field microscopy is a very simple yet effective technique.
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.
Considering the simplicity of the setup, the quality of images obtained from this technique is impressive.
Dark-field microscopy techniques are almost entirely free of artifacts, due to the nature of the process.
A researcher can achieve a dark field by making modifications to his/her microscope.
Limitations:
The main limitation of dark-field microscopy is the low light levels seen in the final image.
The sample must be very strongly illuminated, which can cause damage to the sample.
Key Points to remember:
The dark-ground microscopy makes use of the dark-ground microscope, a special type of compound light microscope.
The dark-field condenser with a central circular stop, which illuminates the object with a cone of light, is the most essential part of the dark-ground microscope.
This microscope uses reflected light instead of transmitted light used in the ordinary light microscope.
It prevents light from falling directly on the objective lens.
Light rays falling on the object are reflected or scattered onto the objective lens with the result that the microorganisms appear brightly stained against a dark background.