Fluorescence Lifetime of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 68

1.3 Meaning of Absorption Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
1.4 Relevant Radiationless Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1.5 Other Mechanisms Which Compete with Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
1.6 Importance of Long Fluorescence Lifetimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
2 Measurement of Fluorescence Decays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.1 Fluorescence Lifetime Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.2 Fluorescence Lifetime Imaging Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.3 Lifetime Differences Between Microscopic and Cuvette Experiments . . . . . . . . . . . . . . 82
2.4 Search for Proteins with Improved Photophysical Properties . . . . . . . . . . . . . . . . . . . . . . . . 84
3 Fluorescence Lifetime of FPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.1 Blue Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.2 Cyan Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.3 Green and Yellow Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.4 Orange and Red Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

1 Principles of Fluorescence Decays

1.1 Introduction into the Photophysics of a Fluorophore:

The Jablonski Scheme

In this chapter, we will provide a treatment of fluorescence, which is based on basic
physical laws. Some aspects are also covered in other chapters; we will rely on the
work, which is presented elsewhere in this compendium.
The treatise can start with a consideration of a simplified Jablonski scheme
(Fig. 1 ). Such diagrams are often used to visualize the possible transition of an
isolated conjugated system. With respect to fluorescent proteins (FPs), this situation
corresponds to a chromophore taken out of the protein barrel and embedded in an
inert solvent or, even more stringent, into the vacuum. Both kinds of conditions were
experimentally established and gave insight into the photophysics of FPs [ 1 - 3 ].
We can divide the different photophysical pathways in those where radiation
is involved and those which are purely radiationless. The first group comprises
transitions such as absorption, fluorescence and phosphorescence, the second group
mainly consists of intersystem crossing (ISC), internal conversion (IC) and, if we
account for the interaction with other molecules, quenching. The latter processes
are not restricted to the first excited singlet and triplet state, S 1 and T 1 , respectively,
but occur also for higher excited states. Indeed, IC is the main decay mechanism,
which prevents to observe strong emission from higher excited states.
The Jablonski scheme in Fig. 1 shows more photophysical pathways, which
connect the different states. However, little is known about higher excited states
S n and T n , i.e., about their energies and their lifetimes. Absorption spectra of
synthetic chromophores show the excitation into higher singlet states at wave-
lengths l <

S 1 transition [ 4 ]. However,
these absorption bands are hidden in the spectra of proteins since the large number

300 nm. They are weaker than the S 0 !