Biomedical Engineering Reference
In-Depth Information
two laser beams, and thus concurrently generates two SHG signals. Each SHG signal provides redundant
data, which may increase the signal-to-noise ratio. The CARS setup also allows sum frequency genera-
tion (SFG) between the two laser lines. SFG refers to the process of scattering of two photons at different
frequencies ω
1
and ω
2
into a photon at the frequency ω
1
+ ω
2
. Though similar to SHG, SFG can provide
higher signals from noncentrosymmetric molecules like collagen [18].
13.2.1.3 coherent Anti-Stokes Raman Scattering
CARS is a nonlinear optical imaging method that exhibits chemical specificity by probing the vibra-
tional mode of a molecule [17,19]. Excellent reviews of the various techniques and processes involved in
CARS microscopy have been published; see, for example, [20-22]. We summarize here the fundamen-
tal aspects and advantages of this imaging modality, which was described in more detail in Chapter
4. In addition to the benefits associated with nonlinear optical microscopy mentioned above, CARS
presents several other advantages for biological studies [19,22]: (1) Vibrational contrast allows imaging
with chemical specificity and suppresses the need for staining. Hence, the risk of perturbing the bio-
logical function of the molecule under study with a fluorescent probe is removed. Note that SFG also
has vibrational contrast but only with surface sensitivity rather than volume and surface sensitivity
for forward-CARS (F-CARS) and epi-CARS (E-CARS), respectively [20,21]; (2) Similar to harmonic
processes, there is no photobleaching or photodamage at reasonable laser powers [22] since there are no
transitions to an electronically excited state; (3) There is reduced saturation compared with MEF imag-
ing, which can saturate at moderate power levels. This is due to the quasi-instantaneous nature of the
nonresonant scattering process, which has a decay time of <1 ps, as opposed to the relatively long fluo-
rescence lifetimes of the order of several nanoseconds; (4) Coherent summation of the CARS fields from
the sample volume results in a quadratic signal increase with the number of oscillators, in contrast to
the linear increase obtained with incoherent processes such as MEF or spontaneous Raman scattering.
This can be problematic at low concentrations since the sensitivity equally reduces quadratically with
the number of oscillators; (5) Resonant CARS is highly directional, a feature that improves collection
efficiency and allows real-time imaging. Real-time measurements of water diffusion rate in living cells
using CARS imaging have been reported [23]; and (6) Because the CARS signal is blue shifted, it can be
easily separated from the fluorescence.
CARS is a third-order process in which the interaction of a pump field at the frequency ω
p
and a red-
shifted Stokes field at the frequency ω
s
results in the emission of an anti-Stokes field at the frequency
ω
as
= 2ω
p
− ω
s
. When the beat frequency ω
p
− ω
s
is tuned to a Raman-active vibrational band Ω of the
probed molecule, the CARS signal is enhanced by at least five orders of magnitude compared to sponta-
neous Raman scattering [21]. Thus, CARS microscopy provides vibrational contrast and allows imaging
with chemical specificity. Unfortunately, CARS images are not background free and are degraded by the
presence of a nonresonant signal from the solvent that effectively limits the sensitivity (see the energy
diagram in Figure 13.2). The CARS signal intensity
I
CARS
is proportional to the third-order susceptibility
χ
(3)
of the molecule,
I
χ δ
3 2 2
where δ = Ω − (ω
p
− ω
s
) is the detuning from the vibrational
band Ω,
I
p
,
and
I
s
refer to the intensity of the pump and Stokes beams, respectively. χ
(3)
(ω) is a complex
quantity, which is the sum of a resonant component
χ
( 3
(
δ
and a constant nonresonant component
χ
N
( 3
in the absence of two-photon electronic resonance (see the energy diagram in Figure 13.2). The CARS
intensity is therefore proportional to [24-26]
∝
|
( )
( )
|
I I
,
CARS
p s
+
(
)
+
2
2
( )
3
( )
3
( )
3
( )
3
(13.1)
I
CARS
∝
χ δ
( )
χ
2
Re
χ δ χ
(
)
R
NR
R
NR
The first and second terms represent the Lorentzian line signal and an offset, respectively. The third
term is due to the interference between the resonant and nonresonant components and causes the CARS
spectrum to disperse [21,24]. These components are shown in Figure 13.3 and explain the well-known
peak and dip of the CARS spectrum.
