Microscope Image Processing

as defined earlier in Eq. 12.2. According to the Beer–Lambert law, the relationship

between the exciting, emitted, and absorbed light can be expressed as [9]

I

A

¼log I =I

EX

ðÞ,orI ¼ I

EX

e

I

A

(12:21)

Thus Eq. 12.20 can be rewritten as [9]

I

EM

¼ fI

EX

1  e

I

A



(12:22)

From Eq. 12.22, it can be seen that when the absorption, I

A

, is zero, I

EM

is zero.

When I

A

approaches infinity, I

EM

¼ fI

EX

. Thus, when I

A

is small, the term

1  e

I

A

ðÞapproaches I

A

and I

EM

¼ I

A

fI

EX

. That is, the amount of light

absorbed by the fluorophore is related to the concentration of the fluorophore,

with I

A

/ kc, where k is a constant and c is the fluorophore concentration. Thus,

at low concentrations, the fluorescence intensity is directly proportional to the

concentration of the fluorophore, with