Biology Reference
In-Depth Information
One-Photon and Two-Photon Excitation
of Fluorescent Proteins
R. Nifos` and V. Tozzini
Abstract Fluorescent proteins (FPs) offer a wide palette of colors for imaging
applications. One purpose of this chapter is to review the variety of FP spectral
properties, with a focus on their structural basis. Fluorescence in FPs originates
from the autocatalytically formed chromophore. Several studies exist on synthetic
chromophore analogs in gas phase and in solution. Together with the X-ray
structures of many FPs, these studies help to understand how excitation and
emission energies are tuned by chromophore structure, protonation state, and
interactions with the surrounding environment, either solvent molecules or amino
acids residues. The increasing use of FPs in two-photon microscopy also prompted
detailed investigations of their two-photon excitation properties. The comparison
with one-photon excitation reveals nontrivial features, which are relevant both for
their implications in understanding multiphoton properties of fluorophores and for
application purposes.
Keywords Fluorescent proteins
Chromophore structures
Computational
studies
Isolated chromophores
Multiphoton spectroscopy
Structure-property
relationship
Spectral tuning
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2
av
GFP: Structure and Optical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.1 Structure and Chromophore Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2 Absorption, Excitation, and Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3 Chromophores of FPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4
Isolated Chromophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.1 Model Chromophores in Gas Phase and in Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.2 Chromophores of FPs: Computational Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
5
FPs Spectra: Spectral Tuning by the Protein Environment
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
R. Nifos` (
*
) and V. Tozzini
NEST CNR-NANO, Piazza San Silvestro 12, 56126 Pisa, Italy
e-mail: r.nifosi@sns.it
