Principles Of Darkfield Illumination - OPTIKA MICROSCOPES B-500DK Manual De Instrucciones

3.0 PRINCIPLES OF DARKFIELD ILLUMINATION

Darkfield microscopy is a specialized illumination technique that capitalizes on oblique illumination
to enhance contrast in specimens that are not imaged well under normal brightfield illumination con-
ditions.
All of us are quite familiar with the appearance and visibility of stars on a dark night, this despite their
enormous distances from the earth. Stars can be seen because of the stark contrast between their
faint light and the black sky.
This principle is applied in darkfield (also called darkground) microscopy, a simple and popular
method for making unstained objects clearly visible. Such objects are often have refractive indices
very close in value to that of their surroundings and are difficult to image in conventional brightfield
microscopy. For instance, many small aquatic organisms have a refractive index ranging from 1.2
to 1.4, resulting in a negligible optical difference from the surrounding aqueous medium. These are
ideal candidates for darkfield illumination.
Darkfield illumination requires blocking out of the central light which ordinarily passes through and
around (surrounding) the specimen, allowing only oblique rays from every azimuth to "strike" the
specimen mounted on the microscope slide. The top lens of a simple Abbe darkfield condenser is
spherically concave, allowing light rays emerging from the surface in all azimuths to form an inverted
hollow cone of light with an apex centered in the specimen plane. If no specimen is present and the
numerical aperture of the condenser is greater than that of the objective, the oblique rays cross and
all such rays will miss entering the objective because of their obliquity. The field of view will appear
dark.
The darkfield condenser/objective pair illustrated in Figure 3 is a high-numerical aperture arran-
gement that represents darkfield microscopy in its most sophisticated configuration, which will be
discussed in detail below. The objective contains an internal iris diaphragm that serves to reduce the
numerical aperture of the objective to a value below that of the inverted hollow light cone emitted by
the condenser. The cardioid condenser is a reflecting darkfield design that relies on internal mirrors
to project an aberration-free cone of light onto the specimen plane.
When a specimen is placed on the slide, especially an unstained, non-light absorbing specimen, the
oblique rays cross the specimen and are diffracted, reflected, and/or refracted by optical discontinui-
ties (such as the cell membrane, nucleus, and internal organelles) allowing these faint rays to enter
the objective. The specimen can then be seen bright on an otherwise black background. In terms of
Fourier optics, darkfield illumination removes the zeroth order (unscattered light) from the diffraction
pattern formed at the rear focal plane of the objective. This results in an image formed exclusively
from higher order diffraction intensities scattered by the specimen.
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