Biomedical Engineering Reference
In-Depth Information
happens when the beat frequency between the pump and Stokes beams, .! P ! S /,
matches the frequency of a particular Raman active molecular vibration, ! vib .
This results in enhanced anti-Stokes signal, providing the vibrational contrast for
CARS microscopy [ 42 ]. CARS can selectively image molecules by exciting their
specific vibrational mode with high sensitivity. Since its signal frequency is blue-
shifted from the excitation frequencies, CARS can be used in the presence of
high fluorescence background. Normally, pump and Stokes field are near infrared
laser which can penetrate deeply into biological system with little absorption. This
permits CARS to be noninvasive in imaging living cells. The CARS signal is
generated by the induced third-order nonlinear polarization
.! AS / D " o X
jkl
P .3/
i
.3/
ijkl .! AS /E j .! P /E k .! P /E l .! S /;
(7.4)
where E.! P / is the pump beam, E.! S / is the Stokes beam, the 3rd-order sus-
ceptibility .3/
ijkl is the fourth rank tensor, and indices ijkl are summed over linear
polarization directions of x, y,and z .
Raman scattering-based spectroscopy and imaging offers unique capabilities for
live cell chemical contrast without using fluorescent markers. Inherent Raman sig-
nals do not photobleach, enabling long-term cellular studies. There have been new
developments in CARS microscopy such as (a) F-CARS, forward-detection with
parallel-polarized pump and Stokes beams; (b) E-CARS, epidetection with parallel-
polarized pump and Stokes beam; (c) P-CARS, forward-detection with polarization;
and (d) C-CARS, detection with counter propagating parallel-polarized pump
and Stokes beams. The four kinds of the CARS microscopy can be applied
under different circumstances. F-CARS microscopy produces a large signal at low
excitation power. It is suitable for the vibration with a large resonant CARS signal.
However, the resonant F-CARS signal is often overwhelmed by the nonresonant
background from the scatterers and solvent. The E-CARS and C-CARS beam
geometries introduce a phase mismatch, which acts as a size filter that effectively
rejects the signal from the bulk solvent. E-CARS permits high-sensitivity imaging
of small features embedded in a nonlinear medium.
CARS microscopy has advantages in imaging small molecules such as lipids and
hormones. The function of these small molecules is prone to be affected when they
are labeled with fluorescence tags. It has a potential to be used in fields such as
membrane biology, neurobiology, and pharmacology.
7.3.5
Laser Sources in Nonlinear Microscopy
Collagen was the first biological material studied using a Q-switched ruby laser
at a wavelength of 694 nm but resulted in strong absorption of the SHG signal
at 347 nm [ 60 ]. Another Q-switched Nd-YAG laser at 1064 nm with nanosecond
pulses was used to observe connective tissues [ 32 ]. More recently, femtosecond
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