Spectral Versatility of Fluorescent Proteins Observed on the Single Molecule Level - Fluorescent Proteins: From Understanding to Design - page 211

3.5 Spectral Diffusion: Sampling the Parameter Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
3.6 Identification and Characterization of Distinct Spectral Forms . . . . . . . . . . . . . . . . . . . . . 228
4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

1 Introduction

The discovery, further development, and application of visible fluorescent proteins
(VFPs) as tools for visualizing biological processes have had tremendous impact on
cellular biology. In combination with optical microscopy techniques, VFPs allow
for the visualization of the subcellular localization of proteins and the dynamics of
their transport, trafficking, and interactions with other molecules and cellular com-
ponents. Indeed, the breakthrough advances enabled by VFPs have been recognized
by the award of the Nobel Prize in Chemistry in 2008. Furthermore, the exciting
recent advances in optical super-resolution techniques that allow for near-molecular
resolution optical imaging of biological systems [ 1 - 7 ] strongly rest on the unique
properties of switchable fluorescent dyes and switchable fluorescent proteins.
To date, a wide palette of fluorescent proteins has been discovered or engineered
for genetically encoded in vivo labeling. Besides proteins derived from the first
discovered green fluorescent protein (GFP) in the Aequoria jellyfish, the palette of
VFPs has been enormously extended by the discovery of new intrinsically fluores-
cent VFPs from other marine organisms [ 8 - 14 ] and by their optimization by
protein engineering [ 15 - 20 ]. The combination of these genetically encodable
markers with advanced microscopic and spectroscopic techniques has enabled the
quantitative measurement of protein-protein interactions at high spatial and tem-
poral resolutions. Variants of VFPs exhibiting different colors and photophysical
properties such as photoactivation [ 21 - 28 ], photobleaching [ 29 , 30 ], and phototox-
icity [ 31 , 32 ] have provided new windows into the cell.
However, many studies have established that VFPs exhibit intrinsically complex
photophysical behavior. To effectively and correctly exploit the enormous power of
VFPs to visualize biological processes, it is of paramount importance to compre-
hensively understand the intrinsic photophysical properties of these remarkable
fluorophores in detail.
In addition to the ensemble characterization of emitters, rapid advances in
ultrasensitive optical detection and spectroscopy have made it possible to visualize
and characterize emitters at the single molecule level [ 33 - 36 ]. In contrast to
ensemble measurements that yield information about the averaged properties of
the sample, single molecule studies yield information about individual molecular
entities and hidden subensembles that are not observable due to the ensemble
averaging effect. These molecular properties vary from molecule to molecule and
with time for individual molecules. As a result, single molecule studies on a
statistically relevant number of molecules yield distributions of parameters that in
the limit of very large numbers of molecules approach ensemble data, but which
contain a great deal of more detailed information about subensembles and individual