Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 15

contrast making it ideal for uses such as counting and tracking cells. The automated
outlining (third row) of thresholded regions (second row) of a certain size range is
produced. The lower contrast in brightfield does not allow easy contiguous identification of
the cell perimeter. With DIC, the shadow gradient presents a significant issue.
1.4.3 Fluorescence Overlay
As with other transmitted light microscopy techniques, phase contrast images can easily be
overlaid with fluorescence images providing a general morphology of the sample on which
the specific fluorescence labels can be interpreted. The transmitted and fluorescence images
are taken sequentially using the same camera and digitally overlaid. As with many
transmitted light and fluorescence overlays, the best results are often produced by scaling
the transmitted image so that it is relatively dark so as not to overwhelm the fluorescence
that is to be overlaid. Phase also provides a high contrast method to focus on and select live
samples avoiding the use of phototoxic intense fluorescence illumination when no
information is obtained with it.
Phase optics has a minor effect on the fluorescence image. Obviously, the ring annulus in
the condenser is irrelevant for epi-fluorescence configuration and hence presents no issue.
The phase plate is normally fixed in the objective and so fluorescence emission light passes
through it. The light that passes through the phase ring itself will be attenuated, but the
overall effect is quite minor but can be significant in very demanding applications where
the loss of a few percent of the signal is noticeable. The light will also be partially phase
shifted by the plate; since this is not coupled with spatially arranged illumination this is of
limited consequence.
Confocal microscopes are rarely equipped with phase optics. Point scanning confocals
could be affected a little more seriously than widefield as the beam pivots near the phase
plate and a coherent light source is used so the aberrations could be more apparent. Also,
the means of phase contrast formation described here is not compatible with a point-based
assembly so a phase contrast transmitted correlate to a confocal image cannot be built with
a nondescanned transmitted photo multiplier tube (PMT). DIC is typically used for
transmitted light contrast overlay on a confocal, and this works well in point scanning with
laser illumination that provides polarized light. DIC also offers the advantage of allowing
the removal of the analyzer and Wollaston prism offering maximum photon collection in
fluorescence imaging mode.
1.5 Limitations of Phase Optics
The phase contrast method presents enormous utility but does have some limitations. The
interpretation of the image is not trivial—it is really optical path length difference not