Coherent Raman Scattering Microscopy - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 138

A B

as (CH 2 -aliph.)
s (CH 2 -aliph.)

m

5

2800 2850 2900 2950

Raman shift / cm -1

Fig. 6.13. A Stimulated Raman loss (SRL) imaging of self-assembled 2 . 4-μm
polystyrene beads in water recorded at a Raman shift of 2904 cm − 1 . B Measured
SRL spectrum (filled circles) when focused on a single bead. Both the aliphatic
symmetric ν s (CH 2 ) and antisymmetric ν as (CH 2 ) Raman modes of polystyrene at
2853 cm − 1 and 2912 cm − 1 , respectively, are resolved, directly reproducing the spon-
taneous Raman spectrum (solid line) of bulk polystyrene (Adapted from Nandaku-
mar et al. [13].)

nonresonant background, no signal from water is detected. When focused on
a single bead, linear proportionality to both the pump and Stokes average
laser powers is observed. Figure 6.13B displays the spectral dependence of the
SRL signal on the Raman shift from a single bead within the range where
CH- stretching vibrations reside. The observed SRL spectrum reproduces the
corresponding spontaneous Raman spectrum measured for bulk polystyrene.
All observations combine to verify that the information content obtained by
SRS and by spontaneous Raman scattering is identical. Consequently, the
recorded image pixel intensity in Fig. 6.13A can now be readily interpreted
as the number density of ν as (CH 2 ) Raman modes of polystyrene inside the
probe volume.

Biomedical Applications

The potential of SRS imaging as a label-free and noninvasive tool in biological
and biomedical research has been demonstrated in visualizing distributions of
lipids in living cells [12, 13, 22]. As such, Freudiger et al. [12] followed the
uptake of polyunsaturated omega-3 fatty acids by living human lung cancer
cells through SRL imaging in the high-wavenumber Raman shift region. Nan-
dakumar et al. [13] applied SRL imaging at a Raman shift C C stretching
vibrations to map the subcellular distribution of lipid droplet organelles in un-
stained differentiated human leukemia cells that are rich in unsaturated lipids.
Ozeki et al. [22] demonstrated the three-dimensional visualization of the cell
wall and the nucleus in an unstained tobacco BY-2 cell based on the SRS
contrast of the CH- stretching modes. Examples for SRS tissue imaging with-
out staining include the visualization of lipid-rich myelin sheaths surrounding