Biomedical Engineering Reference
In-Depth Information
2
sin
∆
∆
(
kz
kz
/
2
)
(4.8)
I
∝
(
χ
3
( )
)
2 2
I I
AS
p S
/
2
where
I
P
and
I
S
are intensities of the pump and Stokes waves, respectively,
z
is the sample thickness, and
Δ
k
=
k
AS
− (2
k
1
−
k
2
) is the wavevector mismatch. Equation 4.8 indicates that CARS signal is strongest
when Δ
k
= 0, a phase-matching condition similar to the one introduced for SHG, where the signal wave
copropagates with the excitation laser.
When two laser beams of strong intensity are concurrently present as the sample with the frequency
difference matching a Raman-active vibration for CARS generation, another χ
(3)
process, SRS [9] also
occurs simultaneously, which enhances the spontaneous Stokes Raman signal (ω
S
) induced by the
Stokes wave ω
1
(Figure 4.3). This has been successfully used in Xie's group as an alternative to CARS for
vibrational imaging [27]. SRS can be viewed as a four-wave mixing process, where the Stokes Raman
scattering, as described by the solid arrow and the dashed arrow on the right in Figure 4.3, is enhanced
by the coherent interaction between the optical waves (the two solid arrows on the left in Figure 4.3)
and the molecule (denoted by the energy levels). Figure 4.3 indicates that the photon energies of the two
applied optical waves, ω
1
and ω
2
, satisfy the energetics for both CARS and SRS (Δω = ω
1
− ω
2
= ω
vib
). A
major difference is that CARS leads to a new wavelength generation at ω
AS
= ω
1
− ω
vib
, while for SRS the
emission frequency (ω
S
) is the same as the stimulating frequency ω
2
resulting an intensity increase for
the stimulating laser beam (ω
2
; Raman gain) and a decrease of the Stokes beam (ω
1
; Raman loss). This
difference has a significant consequence for signal retrieval in the experimental design (see Section 4.3),
as the CARS emission is a signal over zero background while the SRS signal has a strong background.
However, CARS can also occur by means of a nonresonant χ
(3)
process in any material, where a real
upper vibrational state is absent, and thus this nonresonant contribution to ω
AS
is manifested as an
ever-present nonspecific background and unfavorably interferes with the resonant CARS signal. This
has the result that the CARS spectrum is not equivalent to the Raman spectrum and phase retrieval
efforts must be employed to extract the latter. On the other hand, the nonresonant background is not
a problem for SRS because of the detection scheme, that only is sensitive to the upper vibrational state
when Δω matches ω
vib
[27,30]. As an additional benefit over CARS, SRS detection results in the true
Raman spectrum.
4.3 instrumentation of nLo Microscopes
For all of the NLO effects introduced in the previous section, the intensity of the generated optical signal
scales nonlinear with the input optical waves, that is,
I
ω1
2
2
for SHG,
TPF, THG, CARS, and SRS, respectively. Pulsed lasers with high instantaneous peak power, typically
high-repetition-rate, mode-locked Ti:sapphire lasers, are used to drive the nonlinear effects. For tightly
focused laser beams in the material, such relationships dictate that the nonlinear effects can be confined
to the focal point only, where the peak power is most intense. Owing to the similar nature of nonlin-
ear signal generation, the NLO microscopes share a common instrumental design with some subtle
differences among themselves. Experimentally, all NLO microscopes are based on the idea of point
illumination and detection of point scanning microscopy, which is also the approach used for confocal
microscopy [31]. At the early phase of development, point scanning was implemented via sample scan-
ning with an XYZ transitional stage, and this has switched to laser beam scanning in later years, which
greatly increases the image acquisition speed, and microscopy based on this method is generally known
as laser scanning microscopy (LSM).
Conceptually, laser scanning microscopy contrasts with the traditional wide field microscopy, where
a region of interest is uniformly illuminated and the whole image is projected on a light-sensitive surface
detector such as a CCD camera. Thus, laser scanning does not employ Kohler illumination, although
the laser must be aligned with the Kohler image planes. While the imaging acquisition speed of LSM
and
I
NLO
∝
ω
I
2
,
I
2
,
I
3
,
I
2
I
I
ω1
ω
ω1
ω
2
