Principles Of Darkfield Illumination - Optika Italy B-380 Serie Manuel D'utilisation

11.2 Principles of darkfield illumination
Darkfield microscopy is a specialized illumination tech-
nique that capitalizes on oblique illumination to enhan-
ce contrast in specimens that are not imaged well un-
der normal brightfield illumination conditions.
All of us are quite familiar with the appearance and vi-
sibility of stars on a dark night, this despite their enor-
mous distances from the earth. Stars can be seen be-
cause 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 me-
thod 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 in-
stance, many small aquatic organisms have a refrac-
tive index ranging from 1.2 to 1.4, resulting in a negli-
gible optical difference from the surrounding aqueous
medium. These are ideal candidates for darkfield illu-
mination.
Darkfield illumination requires blocking out of the cen-
tral 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 cen-
tered 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 Fig. 22 is a high-numerical aperture arrangement that repre-
sents 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 reflec-
ting 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 discontinuities (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.
Ideal candidates for darkfield illumination include minute living aquatic organisms, diatoms, small insects, bone,
fibers, hair, unstained bacteria, yeast, and protozoa.
Non-biological specimens include mineral and chemical crystals, colloidal particles, dust-count specimens, and
thin sections of polymers and ceramics containing small inclusions, porosity differences, or refractive index
gradients.
Care should be taken when preparing specimens for darkfield microscopy because features that lie above and
below the plane of focus can also scatter light and contribute to image degradation.
Specimen thickness and microscope slide thickness are also very important and, in general, a thin specimen is
desirable to eliminate the possibility of diffraction artifacts that can interfere with image formation.
High
Numerical
Aperture
Objective
Oblique light
cone
Concave
Mirror
Light from
Source
Cardioid condenser for darkfield
Page 17
Light to Eyepieces
Iris Diaphragm
Slide
Cardioid
Condenser
Convex
Mirror
Central
Stop
Fig. 23