Biomedical Engineering Reference
In-Depth Information
The second harmonic imaging microscopy (SHIM) obtains the half wavelength signal
which is due to the nonlinear optical effect of the specimen for imaging.
The final part of this chapter shows the flowing image in the plants using the magnetic
resonance imaging (MRI) as well as static water and lipids.
4.1 Out of Focus Imaging
The earliest method for observing phase variations is imaging at various focal positions. This
earliest method is known as out of focus contrast. Let us consider an object in focus. It is
utterly transparent but has a boundary separating it from water with the same refractive indices
in and out of the boundary. If in true focus we shall see nothing in a transparent unstained
specimen. Just out of focus it will have contrast which reverses as we go through true focus to
the other side off the true focus. The contrast is due to partial coherence of the light waves
originating from parts of the object prior to and after the object being observed [1] . Using
Adobe Photoshop, it is possible to increase the contrast of such images considerably.
Such a method has been used by some Indian botanist scientists to observe enhanced
contrast in pollen grains mounted in water. In that case, there is a difference in
concentration of materials in the pollen grain and the outside which is pure water and this
enhances the contrast due to refraction at the boundaries as well.
4.2 Dark Field
An excellent method for visualizing boundaries is dark field. In this method, direct light
from the microscope condenser is excluded from entering the objective [2,3] . A high
aperture may be used for illumination up to 1.42 in commercial systems and the diffracted
light only admitted into the objective, which can then be with an aperture of up to 1.4.
Central dark field can also be used when the direct light is occluded in the objective itself
and the diffracted light gathered. The limit for this technique comes when the light enters
the specimen horizontally and then only diffracted light enters the objective. This method
much advocated by Siedentopf [4] can reveal any particle however small provided it has a
different refractive index than the medium it is in. Variations of this methodology can be
introduced by allowing some direct light or by using annular illumination of varying
aperture as through the Heine condenser made for many years in Leitz.
4.3 Phase Contrast Techniques
Zernike made a significant discovery in making it possible without staining to make a refractive
index change visible under the microscope under one condition [5,6] whichisveryimportant.
This condition is that the refractive index varies rapidly between the object being examined in
the specimen and its surrounds; ideally a sharp boundary is the most desired. This method was
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