Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 133

the wavelength scale is usually calibrated using a He-Ne laser, and is therefore very
accurate. Some advantages arise also from the facts that the impact of stray light is
negligible and that the resolution is the same at all wavelengths.
A disadvantage of IR absorption arises from the fact that many materials and
substances, and in particular water, have rich IR spectra that might obscure the ones
of the substance under investigation. This requires a careful preparation of the
sample, which can be contained in solvents with low IR absorption at the wave-
lengths of interest (e.g., Cl 4 , CHCl 3 ), can be dispersed in solid pellets or films dried
between polished salt (KBr) disks, could be in gaseous phase, or, if liquid, must be
sandwiched in a thin layer between two salt disks. A different configuration is used
in the attenuated total reflection (ATR) spectroscopy technique: in this case, an IR-
transparent, high refractive index internal reflection element (IRE) is “immersed”
into or pressed onto the sample, or the sample is deposited on it. A beam of infrared
light is passed through the IRE so that it totally reflects at least once off the internal
surface in contact with the sample; in practice, the light partially enters the solution
(evanescent wave) and can be absorbed. The reflected light is therefore attenuated,
causing a measurable reduction in the output signal dependent upon the absorbance
of the sample. ATR has been used, e.g., to study changes in the IR spectrum during
bacteriorhodopsin photoreaction on a thin film of the protein covering the IRE [ 6 ].
However, most of the preparations mentioned above are not the best options for
protein samples. Moreover, the application of infrared spectroscopy on fluorescent
proteins is challenging, because special means have to be used to resolve the
vibrational bands of the chromophore in the presence of the strong IR absorption
by the protein backbone or by other aminoacidic residues. To overcome these
difficulties, it is possible to analyze differential spectra, which highlight changes
between different configurations of a given fluorescent protein; starting from these
kinds of experiments, it is possible to relate the changes in the IR spectrum with
specific changes in the molecular structure of the sample. In the case of fast
reversible changes, it is even possible to collect only the differential spectrum,
e.g., with a lock-in technique. A variation of this technique, the time-resolved
infrared absorption (TRIR), will be described in Sects. 2.3 and 4.4 .
2.2 Raman and SERS
Raman spectroscopy is a technique used to study vibrational, rotational, and other
low-frequency modes in a system. It relies on inelastic scattering of monochromatic
light. The light interacts with excitations in the system, resulting in the energy of the
scattered photons being shifted down (up) by an amount corresponding to the
energy of the excited (depleted) mode, for Stokes (anti-Stokes) Raman scattering,
as schematized in the second panel of Fig. 1c . For molecular spectroscopy, the
derivative of the molecular polarization potential (i.e., the amount of deformation
of the electron cloud) with respect to the vibrational coordinate will determine the
Raman scattering intensity (see Sect. 3 ). The pattern of shifted frequencies is