Spectral Versatility of Fluorescent Proteins Observed on the Single Molecule Level - Fluorescent Proteins: From Understanding to Design - page 210

Spectral Versatility of Fluorescent Proteins
Observed on the Single Molecule Level

Christian Blum and Vinod Subramaniam

Abstract The photophysics of visible fluorescent proteins (VFPs) remains a topic
of intense research, driven by the widespread use of these proteins as reporters and
sensors in living cells. The photophysical complexity of these markers originates
from the multistep chemical reaction that forms the chromophore, and from the
embedding of the chromophore within the protein nanoenvironment. To accurately
interpret the biological and biochemical processes illuminated by the VFPs, it is
essential to understand the details of their photophysics. Certain aspects of VFP
photophysics can only be observed and understood at the single molecule level,
which removes the averaging effect inherent to ensemble studies. Here, we review
how spectrally resolved single molecule emission detection at room temperature
has helped to understand the complex photophysics of VFPs. We focus on the
detection of spectrally distinct subensembles, the spontaneous or light-induced
transition between these subensembles, and on subtle spectral variations induced
by changes in the local nanoenvironment of VFP chromophores.

Keywords Fluorescence spectra Photophysics Photochromism Single mole-

cule spectroscopy Spectral diffusion Spectral forms

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2 Basis for Photophysical Complexity of VFPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
3 Ultrasensitive Spectroscopy at the Single Molecule Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
3.1 Technical Principles of Single Molecule Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
3.2 Single VFP Intensity Trajectories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
3.3 Spectrally Resolved Single Molecule Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
3.4 Single VFP Emission Spectra for Target Molecule Identification . . . . . . . . . . . . . . . . . . 224

C. Blum ( * ) and V. Subramaniam
Nanobiophysics, MESA þ Institute for Nanotechnology, Faculty of Science and Technology,
University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands
e-mail: [e-mail]