Coherent Raman Scattering Microscopy - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 134

and photophysical processes of the fluorophore itself [159]. Correlation spec-
troscopy that measures the fluctuation of Raman scattering intensity, thus
providing vibrational selectivity, was reported by Schrof and coworkers using
confocal spontaneous Raman microscopy [160] and by Eggeling et al. [161] us-
ing surface-enhanced resonance Raman scattering microscopy. The limitations
inherent to the latter two methods can be circumvented by CARS correlation
spectroscopy (CARS-CS) [162, 163].
CARS-CS represents a novel type of CRS spectroscopy that is a direct
consequence of the spatial compartmentation provided by CARS microscopy.
With a probe volume that can be much smaller than the average volume
of particle occupancy given by the reciprocal particle concentration, CARS-
CS can monitor the number fluctuation of Raman-active particles caused by
Brownian diffusion. The CARS signal is typically investigated by means of
photon counting that registers distinct photon arrival times and is analyzed
in terms of its photon number trajectory. As an example, Fig. 6.12A shows
spontaneous stochastic CARS signal fluctuations about its equilibrium mean
value originating from freely diffusing 110-nm polystyrene spheres in water.
The CARS intensity changes as particles move in and out of the probe focal
volume. To achieve a high fluctuation contrast in CARS-CS, it is vital to
eciently suppress any nonresonant CARS from surrounding solvent and/or
auto-fluorescence background because these signals are stationary and show
no vibrational selectivity. The photon number trajectory is analyzed in the

A B

150

1.8

100

1.4

50

1.0

D

0

10 -1 10 0 10 1 10 2 10 3 10 4 10 5 0 1 2 3 4 5 6

correlation time / ms time/sec [bin width: 5 ms]

Fig. 6.12. A Typical CARS signal trajectory revealing the particle number fluctu-
ations of 110-nm polystyrene spheres undergoing free Brownian diffusion in water.
The epi-detected CARS contrast arises from the breathing vibration of the benzene
rings at ∼ 1003 cm − 1 . B Measured CARS intensity autocorrelation function for an
aqueous suspension of 200-nm polystyrene spheres at a Raman shift of 3050 cm − 1
where aromatic C-H stretch vibrations reside. The corresponding translational dif-
fusion time, τ D , of 20 ms is indicated. (Panel B courtesy of Andreas Zumbusch,
adapted from [162])