- Dark field microscopyemploys oblique illumination to enhance contrast in
specimens that are not imaged well when using brightfield illumination. In this
observation method an opaque ring-shaped diaphragm with a small central
opening is used together with the condenser lens to direct a hollow inverted cone
of light onto the specimen at high azimuthal angles. Thus,first-order wave
fronts do not directly enter the objective lens, which creates an artificial dark
background in the microscope. The result is that the specimen features are seen
as bright objects on a black background. Darkfield microscopy is a popular tool
for biophotonics and medical investigations, such as viewing and imaging living
bacteria, cells, and tissues. - Phase contrast microscopy makes use of tiny refractive index differences
between cells and their surrounding aqueous solutions and within the cells
between the cytoplasm and the cell nucleus. This observation method is suitable
for viewing very thin colorless and transparent unstained specimens (e.g., cul-
ture cells on glass) and for live cells. Typically these specimens are approxi-
mately 5– 10 μm thick in the central region, but less than 1μm thick at the
periphery. Such specimens absorb extremely little light in the visible portion of
the spectrum and they cannot be viewed with the human eye when using bright
field and darkfield illumination methods. The phase contrast method translates
Table 8.1 Summary offive different microscopy observation methods
Microscopy
method
Features General applications
Brightfield
microscopy
- Common observation method
- Light illuminates the entirefield
of view - Cell staining adds color
Used to observe color and light
intensity information from a
stained specimen
Darkfield
microscopy
- Oblique illumination enhances
contrast - Transparent specimens seen as
bright objects on a black
background
Used for viewing and imaging
living bacteria, cells, and tissues
Phase contrast
microscopy
Uses tiny refractive index
differences between cells and their
surrounding aqueous solutions and
within cells
Suitable for viewing very thin
colorless and transparent unstained
specimens (e.g., culture cells on
glass) and for live cells
Differential
interference
contrast
microscopy
Uses plane-polarized light and
light-shearing prisms to exaggerate
minute differences in thickness
gradients and in refractive indices
of a specimen
Suitable for viewing very thin
colorless and transparent unstained
specimens and for live cells
Polarized light
microscopy
Uses crossed polarizing elements to
dramatically improve the quality of
an image obtained from
birefringent materials
Used for specimens such as plant
cell walls, starch granules, and
protein structures formed during
cell division
8.1 Concepts and Principles of Microscopy 239