From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 150

2. FLUORESCENCE GENERALITIES
2.1. Introduction
The relaxation of a fluorophore from an excited state to its ground state after
the absorption of electromagnetic radiation may result in the emission of
photons, which is called “luminescence”. If this transition occurs for an elec-
tron in the excited singlet state (with a spin opposite to that of a paired elec-
tron in the ground state), this emission is called “fluorescence”. The
fluorescent molecule is often promoted to the S 1 excited state. In most cases,
a rapid relaxation subsequently occurs from the lowest vibrational level of
the first excited state S 1 ; this is the internal conversion process that usually
occurs within approximately 10 -12 s and results in some energy loss from the
system, which is responsible for the energy difference (Stokes' shift) between
the absorption and emission spectra (see the Jablonski diagram in Fig. 5.4 ).
The energy of the emitted photon is dependent upon the ground state
toward which the transition occurs.

2.2. The absorption process
The energy of the excited photon must be equal to, or greater than, the en-
ergy difference ( E 0 and E 1 ) between the ground state (S 0 ) and the excited
state (S 1 ). The frequency of this photon is n¼ ( E 1 - E 0 )/ h , where h is the

Plank constant. When a photon is absorbed, its energy is transferred to
the valence electron and this electron is promoted to a higher electronic or-
bit, thus putting the molecule in the excited state. This absorption is very
fast, since it occurs within 10 -15 s.
Experimentally, the efficiency of light absorption at a wavelength l is
characterized by the absorbance A (l) related to the transmittance T (l)by

¼

I 0
I

A ðÞ¼ log log T ðÞ ½ 5 : 1

where I 0 is the intensity of a monochromatic incident light of wavelength l
passing through an isotropic sample containing absorbing molecules at a
concentration c (mol -1 ), I is the light intensity leaving the absorbing me-
dium, and l (cm) is the absorption path length (sample thickness) of the sam-
ple ( Fig. 5.5 ).
The absorbance follows the Beer-Lambert law

A ðÞ¼ e ðÞ lc ½ 5 : 2