Page 107 - Fundamentals of Light Microscopy and Electronic Imaging

90 DIFFRACTION AND SPATIAL RESOLUTION

Figure 6-5
Effect of numerical aperture on spatial resolution. The diatom Pleurosigma photographed
with a 25 , 0.8 NA oil immersion lens using DIC optics. (a) Condenser aperture open,
showing the near hexagonal pattern of pores. (b) The same object with the condenser
diaphragm closed. The 1st-order diffracted rays from the pores are not captured by the
objective with a narrow cone of illumination. Spatial resolution is reduced, and the pores are
not resolved. Bar 10 m.

the condenser diaphragm limits the number of higher-order diffracted rays that can be
included in the objective and reduces resolution. In an extreme case, the condenser aper-
ture may be nearly closed in a mistaken effort to reduce light intensity. Then the half
angle of the light cone entering the lens is greatly restricted, and resolution in the image
is reduced significantly. The proper way to reduce light intensity is to turn down the
voltage supply of the lamp or insert neutral density filters to attenuate the illumination.

DEPTH OF FIELD AND DEPTH OF FOCUS

Just as diffraction and the wave nature of light determine that the image of a point object
is a diffraction disk of finite diameter, so do the same laws determine that the disk has a
measurable thickness along the z-axis. Depth of field Z in the object plane refers to the
thickness of the optical section along the z-axis within which objects in the specimen are
in focus; depth of focus is the thickness of the image plane itself. Our present comments
are directed to the depth of field. For diffraction-limited optics, the wave-optical value
of Z is given as

2
Z nλ/NA ,
where n is the refractive index of the medium between the lens and the object, λ is the
wavelength of light in air, and NA is the numerical aperture of the objective lens. Thus,