Biophotonics_Concepts_to_Applications

For example, consider a specimen consisting of many thin layers of material, as
shown in Fig.8.6. With a large depth offield (i.e., a thick plane of focus resulting
from a low NA), many layers of the specimen will be in focus at the same time.
Fewer layers can be focused simultaneously for a shorter depth offield (i.e., a
thinner plane of focus resulting from a high NA). Every other layer of the specimen
will be out of focus. In order to examine the other lower and higher layers of a thick
specimen, a microscope with a short DOF must be focused downward or upward
continuously. In microscopy the DOF typically is measured in units of micrometers.
As is shown in Fig.8.6, the DOF becomes larger as the NA decreases. Several
different formulas have been proposed to calculate the DOF of a microscope.
A commonly used equation is

DOF¼

nk

NA^2

ð 8 : 4 Þ

where n is the refractive index of the immersion material,λis the wavelength, and
NA is the numerical aperture of the objective.

Example 8.4 Consider two objective lenses that have NAs of 0.20 and 0.55,

respectively. What is the depth offield at a wavelength of 780 nm if the

immersion material is oil with an index n = 1.51?

Solution: From Eq. (8.4) the DOF for NA = 0.20 is found to be

DOF¼

nk

NA^2

¼

1 : 51 ð 0 : 780 lmÞ

ð 0 : 20 Þ^2

¼ 29 : 4 lm

Similarly, from Eq. (8.4) the DOF for NA = 0.55 is 3.89μm.

Low NA

depth of field

High NA

depth of field

Object

plane

Fig. 8.6 Depth offield
ranges for a layered specimen
for low and high NA values

8.1 Concepts and Principles of Microscopy 243