High Magnification Darkfield Microscopy - Optika B-380 Serie Manual De Instrucciones

11.3 High magnification darkfield microscopy
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 instead) 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 (Fig. 23) can be used with objectives of very high nu-
merical 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 objective.
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 diameters 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 fluorescent impurities.
Careful attention should be paid to the details of oiling a high numerical aperture condenser to the bottom of the
specimen slide. It is very difficult to avoid introduction of tiny air bubbles into the area between the condenser top
lens and the bottom of the microscope slide, and this technique should be practiced to perfection. Air bubbles will
cause image flare and distortion, leading to a loss of contrast and overall image degradation.
Problems are also encountered when using microscope slides that are either too thick or too thin. Many darkfield
condensers contain the range of usable slide thickness inscribed directly on the condenser mount. If the slide is
too thick, it is often difficult to focus the condenser without resorting to a higher viscosity immersion oil. On the
other hand, slides that are too thin have a tendency to break the oil bond between the condenser and the slide.
It is a good idea to purchase precision microscope slides of the correct thickness to avoid any of the problems
mentioned above.
High numerical aperture condensers, whether intended for use dry or with oil, must be accurately centered in the
optical path of the microscope to realize optimum performance.
To achieve this, many darkfield condensers are built with a small circle engraved onto the upper surface to aid in
centering the condenser. Centering is performed with a low power (10x-20x) objective by imaging the engraved
circle and using the condenser centering screws to ensure the circle (and condenser) are correctly centered in
the optical path.
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