Vibrational Spectroscopy of Fluorescent Proteins: A Tool to Investigate the Structure of the Chromophore and Its Environment - Fluorescent Proteins: From Understanding to Design - page 131

2 Experimental Techniques

A molecular vibration occurs when atoms in a molecule are in periodic motion
while the molecule as a whole has constant translational and rotational
motion. A molecule can vibrate in many ways, or better, a complicated vibrational
motion can be considered, in a first approximation, as a superposition of simple
harmonic motions; each of these is called a vibrational mode. In a mode, all the
atoms move harmonically in (or out-of) phase with a single frequency, in a
concerted motion with directions and amplitudes typical of the given mode; one
example of these directions and amplitudes for a delocalized mode of the GFP
chromophore is shown in Fig. 1b . Molecules with N atoms have 3N-6 vibrational
modes (3N-5 if the molecule is linear), each characterized by its vibrational
frequency, . A molecular vibration is excited when the molecule absorbs a

quantum of energy E

n

, where h is the Planck constant. The energies of
vibrations are usually measured in inverse-wavenumbers k , with units cm 1 , linked
to the usual units of energy by the relation E

¼ h n

hck , where c is the velocity of light.
The fingerprint-region energies for organic molecules are mostly within the infrared
(IR) portion of the electromagnetic spectrum, in particular in the mid IR, ranging
from ~300 cm 1 to ~4,000 cm 1 (corresponding to wavelength

¼

m).
The vibrational states of a molecule can be probed in a variety of ways. Within
biophysics and biology, the most often used techniques are infrared absorption (IR)
and inelastic light scattering (Raman), which will be described in Sect. 2.1 and 2.2 ;
a scheme of the quantum processes at the basis of these techniques is shown in the
top panels of Fig. 1c , while examples of the results on HBDI are shown in its bottom
panels. In Sect. 2.3 multiphoton and time-resolved variations of these techniques
will be presented.

l ~ 30-2.5 m

2.1 IR Absorption

The infrared absorption spectrum of a sample is recorded by transmitting a beam of
infrared light through the sample, analogously to the UV/Vis absorption technique.
Examination of the transmitted light reveals how much energy is absorbed at each
wavelength. This can be done with a monochromatic beam, which changes in
wavelength over time (dispersive configuration), or by using a multichannel dis-
persive spectrograph or a Fourier transform instrument (FTIR configuration) to
measure all wavelengths at once. From this, a transmittance or absorbance spectrum
can be produced, as schematized in Fig. 2a .
In the dispersive configuration, the setup is usually very similar to the ones used
for UV/Vis spectrometers. Most often, a beam of infrared light is split into two
separate beams passing through the sample and through a reference (often the same
substance where the sample is dissolved). In the lock-in configuration, the splitter
actually alternates the light path between sample and reference (usually 10-100
times a second); the beams are both directed toward a detector, and the resulting