Darkfield Microscopy At High Magnifications - OPTIKA MICROSCOPES B-500DK Manual De Instrucciones

3.1 DARKFIELD MICROSCOPY AT HIGH MAGNIFICATIONS

For more precise work and blacker backgrounds, you may choose a condenser designed especially
for darkfield, i.e. to transmit only oblique rays. There are several varieties: "dry" darkfield condensers
with air between the top of the condenser and the underside of the slide–and immersion darkfield
condensers which require the use of a drop of immersion oil (some are designed to use water in-
stead) establishing contact between the top of the condenser and the underside of the specimen
slide. The immersion darkfield condenser has internal mirrored surfaces and passes rays of great
obliquity and free of chromatic aberration, producing the best results and blackest background.
Perhaps the most widely used darkfield condenser is the paraboloid, consisting of a solid piece of
glass ground very accurately into the shape of a paraboloid.
As discussed above, the dry darkfield condenser is useful for objectives with numerical apertures
below 0.75, while the paraboloid and cardioid immersion condensers (Figure 3) can be used with
objectives of very high numerical aperture (up to 1.4). Objectives with a numerical aperture above
1.2 will require some reduction of their working aperture since their maximum numerical aperture
may exceed the numerical aperture of the condenser, thus allowing direct light to enter the objecti-
ve.
For this reason, many high numerical aperture objectives designed for use with darkfield as well as
brightfield illumination are made with a built-in adjustable iris diaphragm that acts as an aperture
stop.
This reduction in numerical aperture also limits the resolving power of the objective as well as the
intensity of light in the image. Specialized objectives designed exclusively for darkfield work are
produced with a maximum numerical aperture close to the lower limit of the numerical aperture of
the darkfield condenser. They do not have internal iris diaphragms, however the lens mount diame-
ters are adjusted so at least one internal lens has the optimum diameter to perform as an aperture
stop.
The cardioid condenser is very sensitive to alignment and must be carefully positioned to take
advantage of the very sharp cone of illumination, making it the most difficult darkfield condenser to
use. In addition, the condenser produces a significant amount of glare, even from the most minute
dust particles, and the short focal length may result in poor illumination on objects that exceed a few
microns in size or thickness. When choosing microscope slides for quantitative high-magnification
darkfield microscopy, make certain to select slides made from a glass mixture that is free of fluore-
scent impurities.
Page 8