Chapter 3 Detailed Explanation
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3.10 Tips for Each Microscopy Method
3.10.1 Tips for Phase Contrast Microscopy
Phase contrast microscopy is suited for observing colorless and transparent specimens, unstained or lightly
stained specimens, specimens that have lost their color, and ultra-thin sections for electron microscopes.
Shaded or strongly stained specimens are not suited to phase contrast microscopy.
The appearance of the phase contrast image is influenced by factors such as the phase difference or shape of
the specimen and the characteristics of the objective. Be careful of the following when preparing specimens
and selecting Ph objectives.
Select specimens that do not de-center the Ph annular diaphragm.
When light-scattering specimens or specimens that have a lens or prismatic effect are observed, the Ph
annular ring becomes de-centered. Special care is needed with specimens such as thick live specimens,
coarse specimens, and specimens using microplates, which affect the lens or prism and de-center the Ph
annular ring, worsening the image.
Ph objectives and specimens
Ph objectives can be divided into “achromat”, “plan achromat”, “plan fluor”, and “plan apochromat” objectives
depending on their chromatic aberration and degree of field curvature correction. These lenses can also be
divided into several types depending on the characteristics of their internal phase ring. Satisfactory results
cannot be achieved if the specimen's degree of phase difference or the characteristics of the phase ring are
unsuitable. For details on the characteristics of the use of phase contrast objectives, see the following table.
•
When using a Ph objective for dark contrast, make sure the specimen's phase difference does not
exceed the objective's degree of tolerance (latitude) for phase difference. If the specimen's degree of
phase difference exceeds the degree of tolerance for phase difference, the image shines more brightly
than the background, making observation impossible.
•
When preparing a phase-difference specimen, you can adjust the phase difference by changing the
refractive index, for example by changing the specimen thickness, mounting agent, or culture fluid.
•
Even specimens that appear in low contrast when observed using a DLL objective might yield good
results when observed using a DM objective.
Characteristics of the use of phase contrast objectives
Phase contrast
objective
Appearance of
image
Contrast
Latitude
Example of application
Dark
contrast
DLL
DL
Generally, objects
with a large phase
difference appear
dark.
Accordingly, the
image appears
blackened in a
comparatively bright
field of view, resulting
in a similar image to
bright-field
microscopy.
Dark
contrast is
suited to
detailed
observation,
mainly via
micro-contr
ast.
Halftones
(wide scope
of use)
Low- and
mid-range
phase difference
and absorptive
objects (stained
objects)
Fungal spores, general live
cells, slightly thick
specimens, bacteria,
stained specimens, insect
eggs, fat globules, crystals,
and the like
DM
High-contrast
tones
(comparativel
y narrow
scope of use)
Low-range
transparent
objects
Bacterial or protozoan
flagella, fibrin fibers, fine
granules, sections with
appropriately selected
mounting agents, ultra-thin
sections, and the like
Bright
contrast
BM
Generally, objects
with a large phase
difference appear
bright.
Accordingly, the
image appears
brightened in a
comparatively dark
field of view, resulting
in a similar image to
dark-field
microscopy.
Bright contrast is suited to
observing the forms of
detailed fibers or granules,
detection, or calculation,
mainly via macro-contrast.
Mostly full-range
Bacterial or protozoan
flagella, fibrin fibers, fine
granules, blood-count
calculation, and the like
