Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 134

determined by the vibrational (and rotational) states of the sample. Also, the polari-
zation of the incoming and scattered light can be used to infer properties of the
sample (polarized or optically active Raman).
Typically, a sample is illuminated with a laser beam in the visible, near infrared,
or near ultraviolet range; since water and glass are transparent in these ranges, the
sample can be dissolved in water and cuvettes for fluorescence experiments can be
used. Light from the illuminated spot is collected with a lens system and sent
through a monochromator. Wavelengths close to the laser line (stray light), due to
elastic (Rayleigh) scattering, are filtered out, while the rest of the collected light is
dispersed onto a detector.
Spontaneous Raman scattering is typically very weak, and as a result the main
difficulty of Raman spectroscopy is separating the weak unelastically scattered light
from the intense Rayleigh scattered laser light. Historically, Raman spectrometers
used holographic gratings and multiple dispersion stages to achieve a high degree
of laser rejection. In the past, photomultipliers were the detectors of choice for
dispersive Raman setups, which resulted in long acquisition times. However,
modern instrumentation almost universally employs notch or edge filters for
laser rejection, FT (Fourier-transform based) or multichannel spectrographs, and
CCD detectors.
One of the main advantages of the Raman spectroscopy stems from the reso-
nance phenomenon: there is a huge increase of Raman intensity when the energy of
the incoming or of the scattered photons is close to a fundamental excitation of the
system (third panel in Fig. 1c ). Most importantly, the increase is specific for
the modes that interact with the in-resonance excitation, in particular for vibra-
tional modes localized on the part of the molecule responsible for the considered
electronic transition, which is, in fluorescent protein, the chromophore. Moreover,
the use of preresonance conditions (i.e., photons with energy slightly lower than
necessary for the excitation of a higher electronic energy level, so that it cannot
excite the fluorescence of the sample) makes it possible to selectively enhance
chromophore signal with negligible impact of the residual fluorescence, and the
vibrational spectrum of the chromophore can be investigated also while inside fully
folded proteins. For example, this technique helped to determine the protonation of
the chromophore before and after photoactivation in wild-type GFP ( wt GFP) [ 7 ].
Raman spectroscopy has also been used together with differential IR absorption to
collect evidence for isomerization and decarboxylation in the photoconversion of
the Red Fluorescent Protein DsRed [ 8 ].
In addition to yielding information on a chromophore's vibrational frequencies,
resonance Raman spectra can provide insight into the shape of the potential energy
surface of the excited state. The vibrational modes that are enhanced most strongly
are those for which the excited-state surface is shifted significantly with respect
to the ground state [ 9 ]. As an example, Schellenberg et al . observed that the
resonance Raman spectra of GFP and of the chromophore in solution were different
in transition intensities, suggesting that the protein modifies the excited-state
potential energy surface of the chromophore [ 9 ].