Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 136

well-known phenomenon of stimulated emission. In SRS, two laser beams at
o p and o S (pump and Stokes beam, respectively) coincide on the sample

(Fig. 3a ). Only when the difference frequency, Do ¼ o P o S (Raman shift),

matches a particular molecular vibrational frequency

, amplification of the
Raman signal is achieved by virtue of a stimulated process. Consequently, the
intensity of the Stokes beam experiences a gain, and the intensity of the pump beam
experiences a loss, as shown in Fig. 3b . If the Stokes beam is not monochromatic, it
is also possible to collect directly a SRS spectrum (Fig. 3c, d ).
The intensity of the pump beam can be lost also due to higher order phenomena,
like the coherent anti-Stokes Raman scattering (CARS) schematized in Fig. 3a, b .
In this process, two photons of the pump beam are adsorbed, a photon is emitted
by stimulation of the Stokes beam, and as a result a photon is emitted coherently
with frequency

O

. Being a coherent process, CARS signal interferes
with other coherent 3-photon processes that can produce photons of frequency

o AS ¼ o P þ O

o AS ,
causing a more complicated peak shape than the simple lorentzian one expected
for homogeneously broadened Raman peaks. Nevertheless, CARS spectra have
been successfully used, e.g., to study the structure of the immature and mature form
of the red fluorescent protein DsRed; the Raman peaks, obtained after a fitting
procedure of the CARS spectra, have been compared to the known ones of S65T-
GFP [ 18 ]. The advantages of the CARS technique over simple Raman stem
from the nonlinearity of the process. This allows to increment the Raman signal
over the fluorescence (linear) background, e.g., even when exploiting the double
resonance for both the incoming and the scattered light for selective probing of
different DsRed species in resonance with their visible absorption [ 18 ].
The SRS can be exploited also in time-dependent, femtosecond-stimulated
Raman spectroscopy (FSRS). In this technique, femtosecond-pulsed lasers are
used, and the narrow pump beam and the broad Stokes probe are delayed by
a known amount of time from an actinic pump, which excites the system in
a higher-energy electronic state (Figs. 3e, f ). Therefore, low-frequency vibrational
motions can be sampled with time steps shorter than the vibrational period. In this
aspect, this technique is similar to other femtosecond-resolved techniques: as
examples, a pump-probe technique, with both pump and probe in resonance with
the absorption of EGFP, was used to test the coherent dynamics of single-electron
vibronic wave packets following ultrafast excitation [ 19 ], highlighting two modes
at 497 cm 1 (period 67 fs) and 593 cm 1 (period 59 fs); later, diffractive-optic
ultrafast transient grating spectroscopy was used to observe EGFP modes below
100 cm 1 , one of which (at 95 cm 1 ) was assigned to an intramolecular torsional
motion in the excited state. However, FSRS has the advantage of collecting
information on the structure of the molecule within its dynamics in the excited
state, through its Raman spectra, as will be shown in Sect. 4.4 .
A similar technique, albeit simplified and with poorer time resolution, has been
exploited by using an IR absorption probe delayed from the actinic probe; this
technique, called time resolved infrared (TRIR), has been used, e.g., to determine
the excited-state structure of the GFP chromophore [ 20 ]. A scheme of the set-up
used to study the proton pathways in the excited state proton transfer (ESPT) of