Principles Of Darkfield Illumination - Optika Italy B-510 Serie Manual De Instrucciones

12.2 Principles of darkfield illumination
Darkfield microscopy is a specialized illumination technique that
capitalizes on oblique illumination to enhance contrast in spec-
imens that are not imaged well under normal brightfield illumi-
nation conditions.
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 be-
tween their faint light and the black sky.
This principle is applied in darkfield (also called darkground) mi-
croscopy, 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 in-
stance, many small aquatic organisms have a refractive index
ranging from 1.2 to 1.4, resulting in a negligible optical differ-
ence 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 Fig. 23 is a
high-numerical aperture arrangement that represents darkfield
microscopy in its most sophisticated configuration, which will be discussed in detail below. The objective contains an inter-
nal 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 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 sec-
tions 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
F ig. 23 F ig. 23