Page 104 - Fundamentals of Light Microscopy and Electronic Imaging

SPATIAL RESOLUTION 87

Air Oil
NA = 0.95 NA = 1.4
72° 67°

n = 1 n = 1.515

Figure 6-2
Effect of immersion oil on increasing the angular extent over which diffracted rays can be
accepted by an objective lens. Numerical aperture is directly dependent on the wavelength
and the sine of the half angle of the cone of illumination accepted by the front lens of the
objective. For dry lenses, NA is limited, because rays subtending angles of 41° or greater are
lost by total internal reflection and never enter the lens (dotted line). The practical limit for a
dry lens is 39°, which corresponds to an acceptance angle of 72°, and an NA of 0.95. By
adding high-refractive index immersion oil matching that of the glass coverslip (n 1.515), an
oil immersion objective can collect light diffracted up to 67°, which corresponds to NA 1.4.

(1.515), refraction of specimen rays at the coverslip-air interface is eliminated, the
effective half angle is increased, and resolution is improved. The reader can refer to
Pluta (1988) for more details on this important phenomenon.

SPATIAL RESOLUTION

For point objects that are self-luminous (fluorescence microscopy, dark-field micro-
scopy), or for nonluminous points that are examined by bright-field microscopy in
transmitted light where the condenser NA is ≥ the objective NA, the resolving power of
the microscope is defined as

d 0.61λ/NA,

where d is the minimum resolved distance in m, λ is the wavelength in m, and NA is
the numerical aperture of the objective lens.
In the case of bright-field microscopy, where the condenser NA objective NA
(the condenser aperture is closed down and/or an oil immersion condenser is used in the
absence of oil), the resolution is given as
122λ
.
d = .
condenser NA + objective NA