High Magnification Darkfield Microscopy - Optika Italy B-510 Serie Manual De Instrucciones

12.3 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 speci-
men 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 accu-
rately 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 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
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 ob-
jects 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 distor-
tion, 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 condens-
ers 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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