Biomedical Engineering Reference
In-Depth Information
Input spectra
Output spectra
Stokes
Pump
Stokes
Pump
∆
I
S
∆
I
P
SRG
SRL
ω
vib
ω
vib
ω
vib
CARS
ω
ω
ω
2
ω
1
ω
2
ω
1
ω
AS
FIgurE 4.7
Input and output spectra of SRS and CARS: SRS leads to an intensity increase in the Stokes beam
(SRG) and an intensity decrease in the pump beam (SRL); CARS generates new signal at the anti-Stokes frequency.
(From Freudiger, C. W. et al. 2008. Label-free biomedical imaging with high sensitivity by stimulated Raman scat-
tering microscopy.
Science
322:1857-1861. Reproduced with permission of AAAS.)
is used to monitor the intensity of one of the input laser beams after it has gone through the sample.
Lastly, CARS and SRS microscopes mostly employ picosecond lasers instead of the femtosecond sources
mostly commonly used for SHG, THG, and TPEF. This is because the shorter pulses have bandwidths
(~10 nm FWHM) broader than that of vibrational bands (<1 nm). For CARS, this greatly increases the
ubiquitous nonresonant background, decreasing the attainable signal-to-noise ratio. This is not neces-
sarily problematic for SRS, but still decreases selectivity in the crowded fingerprint region. Thus, while
the use of longer pulses decreases the peak power and the resulting CARS and SRS signals, the trade-off
is the increased sensitivity to specific bonds and better background rejection.
For all the NLO imaging techniques described earlier, the polarization of the excitation laser beam
and/or the emitted light can be manipulated for special considerations. For example, the information
content in SHG imaging can be enhanced by measuring the signal anisotropy measurement, where a
linearly polarized laser is used to excite the sample, and the resulting SHG emission signals parallel and
perpendicular to the excitation polarization are collected separately and can be further analyzed [36-
38]. Other measurements show the dependence of the SHG signal on the laser polarization. Collectively,
these measurements relate to the underlying matrix elements of χ
(2)
[39]. For CARS, polarizers were used
for both excitation laser beams (pump and Stokes) and emission light (CARS signal) to suppress the
nonresonant background as a means to enhance SNR [26].
4.4 Representative Biological Applications of SHG imaging
and its comparison with other nLo techniques
4.4.1 Representative SHG examples and Unique Aspects
Based on Equation 4.3, SHG requires that the target sample contain noncentrosymmetric molecules
packed in a well-ordered manner. This attribute implies that SHG microscopy is sensitive to the bulk
property (susceptibility) of the material χ
(2)
. In this context, let us consider the structure of collagen.
Collagen is the most abundant protein in mammals and makes up 25-35% of the whole-body protein
content. Collagen has a molecular weight about 300 kD and has dimensions of 300 nm in length and
1 nm in width. The triple helical collagen molecules are aligned with one another and crosslinked to
form fibrils of 10-300 nm, which then aggregate into collagen fibers with a diameter on the order of
