the index differences into intensity variations that can be visually observed and
recorded.- Differential interference contrast(DIC)microscopyuses plane-polarized light and
light-shearing prisms, known as Nomarski prisms, to exaggerate minute differ-
ences in thickness gradients and in refractive index variations of a specimen. When
light passes through a specimen, DIC microscopy utilizes the phase differences
generated in the regions where there is a thickness gradient. This process adds
bright and dark contrast to images of transparent specimens. DIC allows a fuller
use of the numerical aperture of the system than in other observation methods.
Thereby the microscope achieves an excellent resolution and the user can focus on
a thin plane section of a thick specimen without getting interference from images
that lie above or below the plane being examined. One limitation is that because
DIC utilizes polarized light, plastic Petri dishes cannot be used. - Polarized light microscopy is an image contrast-enhancing technique that,
compared to other observation methods, dramatically improves the quality of an
image obtained from birefringent materials. In polarized light microscopy the
specimen is viewed between crossed polarizing elements inserted into the
optical path before the condenser lens and after the objective lens. Muscle tissue
and structures within the cell that have birefringent properties (e.g., plant cell
walls, starch granules, and protein structures formed during cell division) rotate
the plane of light polarization and thus appear bright on a dark background.
8.1.3 Numerical Aperture
Analogous to the discussion in Chap. 3 about the light capturing capability of an
opticalfiber, thenumerical aperture(NA) of an objective lens of a microscope
measures the ability of the lens to gather light and to resolvefine detail at afixed
distance from a specimen. Given thatαis the half-angle of the light cone captured
by an objective lens of diameter D and focal length f as shown in Fig.8.5, and
letting n be the index of refraction of the medium between a specimen and the lens
(referred to as theimmersion medium), then the NA is defined by
NA¼n sinaD=2f ð 8 : 1 ÞThe immersion medium commonly is either air (n = 1.00) or a transparent
medium such as water (n = 1.33), glycerin (n = 1.47), orimmersion oil(n = 1.51)
that is used to increase the NA. Values of NA range from 0.025 for very low
magnification objectives up to 1.6 for high-performance objectives utilizing spe-
cialized transparent immersion oils. For an illustration, consider a series of objec-
tives (shown in blue in Fig.8.5) that have the same lens diameter D but varying
values of the focal length. As the focal length decreases, the angleαincreases and
the light cone shown in yellow becomes wider. Thus, the lenses with a shorter focal
240 8 Microscopy