Page 347 - Vibrational Spectroscopic Imaging for Biomedical Applications

T issue Imaging with CARS Micr oscopy 321

composed of parts that depend on the presence of a vibrational
mode and parts that are purely electronic in nature, which are
known as the resonant and nonresonant contributions, respectively.
3
()
For spectroscopic measurements, the resonant part χ is of interest,
r
which was the subject of extensive study in early experiments on
the nonlinear properties of solids and liquids. 8–10 In 1974, Begley et
al. summarized the most important advantages of vibrational spec-
11
troscopy based on nonlinear anti-Stokes generation. First, the
coherent anti-Stokes mechanism offers signals that are over five
orders of magnitude stronger relative to spontaneous Raman scat-
tering. Second, this nonlinear technique avoids interference with a
one-photon excited fluorescence background that often plagues
conventional Raman measurements. By baptizing the technique
with the name coherent anti-Stokes Raman spectroscopy (CARS),
Begley advertised the method as an attractive tool for rapid vibra-
tional spectroscopy.
The much stronger signals compared to spontaneous Raman
scattering has made CARS the method of choice for the rapid
identification of chemicals present in flames and combustion
processes. 12,13 An added advantage of CARS is that the signal
strength is also temperature dependent, which enables an accurate
temperature analysis of hot gases and flames. 14,15 Thermometry
and chemical analysis of hot gases continues to be one of the major
applications of the CARS technique.
The technique received a next boost when the advent of ultrafast
picosecond lasers opened up the possibility of directly time-resolving
16
the vibrational relaxation of selective molecular modes. The broad
bandwidth pulses that became available when femtosecond lasers
entered the laboratories of spectroscopists enabled furthermore the
coherent excitation of multiple Raman modes. 17–19 In femtosecond
CARS, the time-resolved signal typically displays oscillatory features,
which is a direct manifestation of mutual destructive and construc-
tive interferences, often called quantum beats, between the different
modes. Using Fourier transform methods, the time-resolved CARS
trace can be related to the Raman spectrum. Femtosecond CARS on
nonabsorbing substances can thus be seen as a form of Fourier trans-
form Raman spectroscopy.
Although CARS is a third order nonlinear process, the technique is
unable to resolve information beyond what is contained in the Raman
spectrum, such as the mutual coupling between vibrational modes. To
observe such couplings, higher order coherent Raman experiments are
20
required. When applied to systems with electronic resonances, how-
ever, femtosecond CARS may reveal information on time-dependent
vibronic relaxation, which cannot be probed with spontaneous Raman
21
scattering techniques. These advantages have kept the popularity of
time-resolved CARS spectroscopy as a probing tool for the ultrafast
molecular dynamics in the condensed phase at a high level.