Phase Contrast Microscopy - Biomedical Optical Phase Microscopy and Nanoscopy - page 11

The objective needs to be one specifically designed for phase contrast, but this typically
now adds little cost to the optic. If an objective without a phase plate is used even with the
phase condenser element, an image like brightfield will be obtained and represent the
equivalent of Figure 1.3 . Objectives capable of phase contrast generally have green writing
and indicate which condenser phase ring position offers compatibility. The physical
diameter of the ring in the phase plate decreases with increasing magnification and
Numerical Aperture (NA) of the lens and generally covers about 10% of the area of the
objective.

1.3.2 General Alignment

The components are easy to align. No rotary alignment between components or the samples
is necessary in setting up the scope and only a few adjustments are necessary to get a phase
image of optimal quality making the technique quick and simple to use.

Nearly all modern microscopes have a transmitted light path employing an
adjustable condenser giving K¨hler illumination and this is the starting point for phase
contrast. It is important to establish K¨hler illumination under brightfield for the objective to
be used to ensure the condenser is centered and positioned so as the image and illumination
planes are physically optimally arranged and homogeneous illumination be provided.

The correct phase ring within the condenser needs to be selected for the chosen phase
objective. Commonly, when a phase ring position on a condenser is selected, the condenser
aperture is moved out of the path or fully opened. If this is not the case and the condenser
aperture is not opened to the diameter of the phase ring, all illumination will be blocked.

A color filter (most commonly green) may be included in the path to ensure the
illumination reaching the sample is monochromatic or of a small spectral range. This helps
to produce maximum contrast by ensuring optimal phase shift by the phase plate; if white
light is used a range of phase shifts around

/4 will be produced as described in Eq. (1.4) .
Defined color illumination also reduces the degradation of the contrast and resolution in the
image from chromatic aberrations and allows inexpensive achromat lenses to be used.

λ

1.3.3 Phase Ring Alignment

The most critical alignment in phase contrast is the coincidence of the illumination ring and
phase plate in conjugate planes at the condenser aperture and back focal plane of the
objective, respectively. Figure 1.8 shows the impact of misalignment of the phase ring and
the consequent degradation of image contrast and quality. The image formed with
misaligned phase optics is less clear because the misaligned illumination is not adequately
attenuated by the phase plate, so it is bright and the imperfect contrast formation
overwhelmed.