Coherent Raman Scattering Microscopy - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 140

6.4.3 SRS Microspectroscopy

Like CARS microscopy, SRS microscopy also offers the possibility for spatially
resolved vibrational spectroscopy in the frequency and time domain and for
Raman correlation spectroscopy. In the frequency domain, an SRS spectrum
is recorded over a wide range of Raman shifts, whereas in the time domain
the SRS signal decay occurring on the femto- and picosecond time scales due
to vibrational dephasing processes is monitored. SRS correlation spectroscopy
can monitor the SRS signal fluctuation due to diffusion and transport of the
microscopic scatterer through the focal volume on time scales of micro- to
milliseconds. Among these SRS microspectroscopies, the feasibility of com-
bining frequency-multiplexed SRS spectroscopy and optical microscopy has
been experimentally demonstrated to date [21].
Analogous to the principal concept of multiplex CARS microspectroscopy
(cf. Sect. 6.3.5), in multiplex SRS detection a pair of a broad-bandwidth pulse,
eg., white-light femtosecond pulse, and a narrow-bandwidth picosecond pulse
that determine the spectral width of the SRS spectrum and its inherent spec-
tral resolution, respectively, is used to simultaneously excite multiple Raman
resonances in the sample. Due to SRS, modulations appear in the spectrum
of the transmitted broad-bandwidth pulse, which are read out using a pho-
todiode array detector. Unlike SRS imaging, it is dicult to integrate phase-
sensitive lock-in detection with a multiplex detector in order to directly re-
trieve the Raman spectrum from these modulations. Instead, two consecutive
spectra, i.e., one with the narrow-bandwidth picosecond beam present and one
with that beam blocked, are recorded. Their ratio allows the computation of
the linear Raman spectrum that can readily be interpreted in a quantitative
manner [49]. Unlike the spectral analysis of a multiplex CARS spectrum, no
retrieval of hidden phase information is required to obtain the spontaneous
Raman response in multiplex SRS microspectroscopy.
In a proof-of-principle experiment, Ploetz et al. [21] demonstrated mul-
tiplex SRL microspectroscopy of polystyrene spheres dispersed in water.
Figure 6.15A shows Raman spectra that are calculated from SRL spectra
recorded when focused on and next to an individual 20- m bead. Raster scan-

ning the sample in the focal plane while recording SRL spectra results in a
data set of Raman spectra, from which Raman images for a particular Raman
resonance can be reconstructed. This is shown in Fig. 6.15B, which displays
density maps of the ring breathing mode at 1003 cm − 1 , the aliphatic C-H
stretching mode at 2904 cm − 1 , and the aromatic C-H stretching modes of
the benzene ring at 3066 cm − 1 of polystyrene beads. In this demonstration,
however, the SRS detection sensitivity is more than three orders of mag-
nitude lower than that typically achieved in CARS/SRS imaging, and the
spectrum acquisition time per image pixel is more than two orders of magni-
tude longer than typically used in CARS microspectroscopy (cf. Sect. 6.3.5.).
To facilitate fast multiplex SRS microspectroscopy of biological samples at

μ