Biology Reference
In-Depth Information
There are a number of advanced types of Raman spectroscopy aimed
at overcoming the difficulty given by the usually low cross-section for Raman
scattering. Among these, we mention surface-enhanced Raman scattering (SERS),
tip-enhanced Raman (TERS), stimulated Raman (analogous to stimulated emis-
sion), and hyper Raman.
SERS spectroscopy is a surface-sensitive technique that results in the enhance-
ment of Raman scattering by molecules in proximity of (adsorbed on or linked to)
metal surfaces [ 10 , 11 ]. This enhancement is particularly strong when the metallic
surfaces possess nanometric-size roughness or features, and it can reach values up
to 10 14 when appropriate nanoparticles are used and when Raman resonance is
exploited as well (surface enhanced resonant Raman scattering - SERRS) [ 12 ].
Two effects are operative in SERS: the electromagnetic (EM) enhancement mech-
anism is related to resonances between the surface plasmons of the metal
nanostructures and the impinging and scattered electromagnetic radiation during
the Raman process, giving rise to enhanced local electric fields; the EM enhance-
ment is more effective with certain configurations of the metallic surface, which
could happen in the so called “hot spots.” The second enhancement mechanism is
attributed to the so-called chemical or electronic effect, and is linked with the
interaction between the electronic states of the molecule and of the metals, which
produce more states through which the Raman scattering could occur and that give
rise to more opportunities for resonances.
SERS of EGFP, E 2 GFP and of their common chromophore allowed to address
their low-energy vibrational modes [ 13 ], which usually have a too low signal-to-noise
ratio to be detected with normal Raman scattering (see Sects. 4.2 and 4.3 ). More-
over, the high enhancement of the SE(R)RS sensitivity offers the possibility of
single-molecule (SM) detection [ 14 ]. Indeed, SM-SERRS has been used to study
EGFP, highlighting changes in the spectra with time scales ranging from the ms to
several seconds [ 15 ]. The origin of the fluctuations at the fastest time scales was not
clear; the spectral jumps in a time scale of seconds were interpreted as produced by
dynamic conversions between the protonated and the deprotonated forms of the
chromophore in individual EGFPs [ 15 ]. However, the strong local fields responsi-
ble for the enhancement in SERS can hinder the interpretation of the collected
time-resolved spectra, especially in mutants with many possible structural changes
such as reversible switchable fluorescent proteins (RSFPs).
2.3 Nonlinear Techniques: CARS and Time-Resolved
Pump-and-Probe-Based Techniques
While Raman is formally a second-order process, the intensity of the signal is only
proportional to the incoming light intensity, because the Raman process is usually
based on a “spontaneous emission.” The Raman signal can be increased using the
stimulated Raman scattering (SRS) technique [ 16 ]. SRS is analogous to the
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