46 PART II A Deeper Look into a Light Microscope
Darkfield
Fine dendritic or minute point-shaped objects as well as minute and unstained
structures are contrasted using darkfield transmitted or reflected light illumination.
In darkfield microscopy, no light from the illuminator can pass into the
imaging system; only light diffracted by the structure is captured by the objective.
This is why the structures appear extremely rich in contrast – bright against
a background as dark as possible. This method is especially useful to detect tiny
and isolated structural details, such as bacteria. When working with reflected
light, darkfield illumination is used to identify grain boundaries in polished and
etched metal sections, for example, as well as to detect contaminants and flaws
in surfaces.
Phase Contrast
Transparent and thin objects appear with very low contrast in brightfield and are
consequently often barely visible. Here, phase contrast is the method of choice for
thin, unstained samples such as culture cells on glass. Remember that light has the
characteristics of a wave. When passing through a structure, the wave is slowed
down compared to the light passing through air. This generates a shift of the formerly
perfectly aligned waves – a phase shift. This can only happen when there
are density differences and thus works best for live or non-embedded samples.
Histological tissue sections will not show any contrast due to the homogeniza-
tion by the embedding medium. Phase contrast is an excellent method to in-
crease contrast when viewing or imaging living cells in culture, but typically results
in halos surrounding the outlines of edge features. These halos are optical
artifacts that often reduce the visibility of boundary details. The technique is not
useful for thick specimens such as plant or animal tissue sections.
Oblique Illumination
Oblique illumination is recommended to contrast objects that are too thick for
phase contrast. The term refers to using diaphragms to direct the illuminating
light beam onto the specimen at an acute angle. Due to the highly directed,
one-sided
illumination, one side of a structure boundary appears bright and the other
darker, which results in a shadow-casting relief image showing fine structural details.
In thick tissue, longer wavelength (red) light can be used – this increases the
penetration. However this light is not easily detected by our eyes; a camera is thus
required to capture the images.