DARKFIELD MICROSCOPY

Brightfield microscopy uses light from the lamp source under the microscope stage to illuminate the
specimen. This light is gathered in the condenser, then shaped into a cone where the apex is
focused on the plane of the specimen. In order to view a specimen under a brightfield microscope,
the light rays that pass through it must be changed enough in order to interfere with each other (or
contrast) and therefore, build an image. At times, a specimen will have a refractive index very similar
to the surrounding medium between the microscope stage and the objective lens. When this
happens, the image can not be seen. In order to visualize these biological materials well, they must
have a contrast caused by the proper refractive indices, or be artificially stained. Since staining can
kill specimens, there are times when darkfield microscopy is used instead.
In darkfield microscopy the condenser is designed to form a hollow cone of light (see illustration
below), as apposed to brightfield microscopy that illuminates the sample with a full cone of light. In
darkfield microscopy, the objective lens sits in the dark hollow of this cone and light travels around
the objective lens, but does not enter the cone shaped area. The entire field of view appears dark
when there is no sample on the microscope stage. However, when a sample is placed on the stage it
appears bright against a dark background. It is similar to back-lighting an object that may be the
same color as the background it sits against - in order to make it stand out.

Illustration provided courtesy of Washington State University.

Darkfield Microscope Applications

Viewing blood cells (biological darkfield microscope, combined with phase contrast)

Viewing bacteria (biological darkfield microscope, often combined with phase contrast)

Viewing different types of algae (biological darkfield microscope)

Viewing hairline metal fractures (metallurgical darkfield microscope)

Viewing diamonds and other precious stones (gemological microscope or stereo darkfield
microscope)

Viewing shrimp or other invertebrates (stereo darkfield microscope)

Darkfield Microscope Options

Metallurigcal reflected light brightfield/darkfield microscope.

Metallurgical reflected and transmitted light brightfield/darkfield microscope.

Stereo microscope 420 with darkfield attachment.

Stereo Zoom SMZ-168 microscope with darkfield attachment.

Biological laboratory phase contrast microscope with darkfield for up to 40x.

Biological laboratory microscope BA210 with darkfield slider.

Biological student microscope 162 with darkfield attachment.

Already have a microscope, but your microscope manufacturer does not make a darkfield
stop? If there is a filter holder below your condenser, a darkfield stop we carry may work. Or
you can mount a coin or circle of another opaque material in the center of a clear disk and
put it in the filter holder.

Techniques

Darkfield
contrast technique where only light diffracted from the specimen is used to form the
image

OVERVIEW
TECHNOLOGY:
Darkfield microscopy creates contrast in transparent unstained specimens such as living
cells. It depends on controlling specimen illumination so that central light which
normally passes through and around the specimen is blocked. Rather than light
illuminating the sample with a full cone of light (as in brightfield microscopy) the
condenser forms a hollow cone with light travelling around the cone rather than through
it.
This form of illumination allows only oblique rays of light to strike the specimen on the
microscope stage and the image is formed by rays of light scattered by the sample and
captured in the objective lens. When there is no sample on the microscope stage the
view is completely dark.
Care should be taken in preparing specimens as features above and below the plane of
focus can also scatter light and compromise image quality (for example, dust,
fingerprints). In general, thin specimens are better because the possibility of diffraction
artifacts is reduced.
APPLICATIONS:
In darkfield microscopy, contrast is created by a bright specimen on a dark background.
It is ideal for revealing outlines, edges, boundaries, and refractive index gradients but
does not provide a great deal of information about internal structure. Ideal subjects
include living, unstained cells (where darkfield illumination provides information not
visible with other techniques), although fixed stains cells can also be imaged
successfully. Darkfield imaging is particularly useful in haematology for the examination
of fresh blood. Non-biological specimens include minerals, chemical crystals, colloidal
particles, inclusions and porosity in glass, ceramics, and polymer thin sections.
MICROSCOPE CONFIGURATION:
Almost any brightfield laboratory microscope can be easily converted for use with
darkfield illumination using special darkfield condensers (dry or oil type).