Phase Imaging in Plant Cells and Tissues - Biomedical Optical Phase Microscopy and Nanoscopy - page 54

10 µ m
Figure 4.2
Inner epidermal nucleus from Allium cepa bulb scale.
that the image is only ideal for one wavelength and is suitable only for thin objects. The
annular illumination introduces a large depth of focus to the images which can be
problematic in thick specimens.
This methodology was discovered by Zernike in 1930s [5 9] . It is much used in optical
microscopy and also it has had applications in X-ray and electron microscopy. The method
basically relies on illuminating the specimen with an annular beam of light although earlier
methods used axial illumination and also a double annular illumination as in the interphako
Zeiss microscope now obsolete. The usual method is a dark contrast system where the
specimen has a darker image than the surrounds. The method works best for thin specimens
and those of small extent.
The main disadvantages of this approach are related to the artifacts that are caused by
decay of contrast from the edges and a halo caused by absorption of light in this
method [1] . The depth of focus is greater than in classical microscopy and results in
disturbing fringes causing problems when imaging thick specimens.
A new variation of through-focus imaging introduced by Nugent and colleagues and also by
PhaseView in France works well for small angles and works excellently for specimens
where the aperture of illumination is low [10] . The images are good for high apertures too
but the accuracy declines dramatically with increased aperture. It is manufactured by
Ultima in Australia and PhaseView in France.
The phase ring used in classical phase microscopy is usually designed for a single
wavelength but there have been versions which were achromatic and even apochromatic
corrected for three colors.