Page 108 - Fundamentals of Light Microscopy and Electronic Imaging

OPTIMIZING THE MICROSCOPE IMAGE 91

the larger the aperture angle (the higher the NA), the shallower will be the depth of field.
The concept of depth of field is vivid in the minds of all of those who use cameras.
Short-focal-length (fast) lenses with small focal ratios ( f/4) have a shallow depth of
field, whereas the depth of field of long-focal-length (slow) lenses ( f/16) is relatively
deep. At one extreme is the pinhole camera, which has an infinitely small NA and an
infinite depth of field—all objects, both near and far, are simultaneously in focus in such
a camera.
The depth of field along the z-axis is determined by several contributing factors,
including principles of geometrical and physical optics, lens aberrations, the degree of
physiological accommodation by the eye, and overall magnification. These variables
and quantitative solutions for each are described in detail by Berek (1927), and are
reviewed by Inoué (in Pawley, 1995) and Pluta (1988). Calculated values of the wave
optical depth of field for a variety of objective lenses are given in Table 4-1.
The depth of field for a particular objective can be measured quickly and unam-
biguously using the microscope. A planar periodic specimen such as a diffraction grat-
ing is mounted obliquely on a specimen slide by propping up one end of the grating
using the edge of a coverslip of known thickness. When the grating is examined in the
microscope, it will be seen that only a narrow zone of grating will be in focus at any par-
ticular setting of the specimen focus dial. The depth of field z is then calculated from the
width w of the focused zone (obtained photographically) and the angle of tilt of the
grating through the relationship

Z nw tan ,

where n is the refractive index of the medium surrounding the grating.

OPTIMIZING THE MICROSCOPE IMAGE: A COMPROMISE
BETWEEN SPATIAL RESOLUTION AND CONTRAST

Putting these principles into practice, let us examine two specimens using transmitted
light illumination and bright-field microscope optics: a totally opaque object such as a
copper mesh electron microscope grid and a stained histological specimen. These
objects are called amplitude objects, because light obscuring regions in the object
appear as low-intensity, high-contrast regions when compared to the bright background
in the object image. In comparison, transparent colorless objects such as living cells are
nearly invisible, because the amplitude differences in the image are generally too small
to reach the critical level of contrast required for visual detection. We discuss methods
for visualizing this important class of transparent objects in Chapter 7.
With the microscope adjusted for Koehler illumination using white light, begin by
opening the condenser front aperture to match the diameter of the back aperture of the
objective lens to obtain maximum aperture angle and therefore maximal spatial resolu-
tion. This operation is performed using an eyepiece telescope or Bertrand lens while
inspecting the back focal plane of the objective lens. Since this plane is conjugate with
the condenser aperture, the edges of the condenser diaphragm appear when the
diaphragm is sufficiently stopped down. With the telescope lens focused on its edges,
open the diaphragm until its margins include the full aperture of the objective to obtain
the maximum possible aperture angle. Expecting maximum resolution in normal view-
ing mode, we are surprised to see that the image of the opaque grid bars is gray and not