What is Darkfield Condenser?
Darkfield is the only wide field method we will study that does not strictly use Köhler illumination. In darkfield microscopy, no direct light from the condenser enters the objective lens.
The only light that is reflected, refracted, or diffracted by the specimen enters the objective. The darkfield condenser produces a circle of light. The light is at an extremely oblique angle to the surface of the slide.
This oblique light comes to a focus on the specimen. It then diverges so strongly that no direct light enters the objective. This type of illumination is a hollow cone of light. That oblique illumination used by the old microscopists to resolve N. Spencrii came only from one direction.
Tilting the microscope’s mirror strongly to one side produced this effect. The darkfield condenser provides oblique illumination from 360 degrees around the specimen.
The numerical aperture of the condenser must be larger than the numerical aperture of the objective lens in order to prevent direct light from entering the objective lens. This is no problem for low magnification dry objectives if a 0.95 NA condenser is used.
This is a problem however for high NA objectives. Here the condenser must have a very high NA such as 1.45 and be used with an objective of no more than 1.25 NA.
Low Magnification Dark Field Condensers
A low magnification darkfield condenser can be nothing more than an ordinary bright field condenser with an opaque disk of the proper diameter placed in its front focal plane.
The diameter of the opaque disk must be just large enough to prevent any direct light from entering the objective. Of course, this means that larger disks are required for objectives of higher NA.
Many microscope manufacturers produce a “universal” condenser that has one or more dark-field disks that match the objectives of different NA. The apparent size of an opaque disk can be made larger or smaller by raising or lowering the condenser.
One disk may thus be useful for a small range of objective NAs. If the condenser has a NA greater than 0.95, better results at low magnification can be achieved if the condenser is oiled to the slide even though the objective is used dry.
You can make your own dark field opaque disk for low power objectives as follows:
- Set up Köhler illumination
- Observe the objective back focal plane
- Adjust the aperture iris to just barely fit outside the objective aperture
- Measure the aperture iris diameter
- Make an opaque disk of this diameter
- Place the disk below the condenser as near the condenser iris as possible
- Adjust the size of the disk: if the field is gray the disk is too small, if the specimen cannot be illuminated the disk is too large.
High Numerical Aperture Dark Field Condensers
The first condenser made specifically for darkfield was produced by Francis H. Wenham and George Shadbolt in 1855. This condenser used a parabolic glass reflector to create a hollow cone of light.
Unlike a refracting condenser, a reflecting condenser does not produce chromatic aberrations and a parabolic shape minimizes spherical aberrations.
The result is a more finely focused spot of light. Other darkfield condenser designs emerged over the years. Figure 9.3 and Table 9.1 describe some of the more common ones.
The need for keeping the objective NA lower than the condenser NA lead to the production of higher and higher NA condensers and to the introduction of the “funnel stop” for the objective lens.
The funnel stop is a funnel-shaped cone that is inserted into the objective lens to limit its aperture. Modern objective lenses do not use funnel stops. Instead, some high NA oil objectives have an iris diaphragm built into them.
This iris is used to reduce the NA of the objective lens. High magnification darkfield condensers must always be oiled to the specimen slide.
This is because the angle of incidence of light leaving the top of the condenser is much greater than the critical angle for glass to air; thus, no light emerges from the condenser until it has immersion oil applied to its surface.