Coherent Raman Scattering Microscopy - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 110

allowing the visualization of fast dynamical processes in living cells and tissue
at video rates [26].
Beyond imaging, the combination of CRS microscopy with spectroscopic
techniques has been used to obtain the full wealth of the chemical and the
physical structure information of submicron-sized samples. In the frequency
domain, multiplex CRS microspectroscopy allows the chemical identification
of molecules on the basis of their characteristic Raman spectra and the extrac-
tion of their physical properties, e.g., their thermodynamic state. In the time
domain, time-resolved CRS microscopy allows the recording of the localized
Raman free induction decay occurring on the femtosecond and picosecond
time scales. CRS correlation spectroscopy can probe three-dimensional diffu-
sion dynamics with chemical selectivity.
This review is dedicated to the common underlying physical principles
behind CARS and SRS microscopy, the comparison of their image contrast
mechanisms and microspectroscopy implementations. We will discuss exem-
plary applications in biomedical areas with chosen illustrations taken from re-
cent literature. This chapter is organized as follows: Section 6.2 starts out with
a brief review of the fundamentals of CRS in general and the excitation pulse
schemes providing high vibrational selectivity in particular. This identifies the
analogies between CARS and SRS microscopy, while concurrently indicating
their distinct image contrast mechanisms. Section 6.3 gives an overview of
the general characteristics, the efforts for increasing the detection sensitiv-
ity, and biomedical applications of CARS imaging and microspectroscopies.
Section 6.4 is dedicated to the principles behind and the applications of SRS
imaging and microspectroscopy. A summary and a brief perspective conclude
this review.
6.2 Fundamentals of Coherent Raman Scattering
6.2.1 Basic Theory
Coherent Raman scattering (CRS) is classically described as a parametric
four-wave mixing process. For ease of experimental implementation, the ar-
rangement most commonly used in microscopy is frequency-degenerate CRS
requiring two synchronized laser fields at different frequencies, with at least
one of them being tunable to adjust the desired Raman shift. Here, the pump
and Stokes laser pulses with electromagnetic field amplitudes E p and E S at
frequencies ω p and ω S ( ω p >ω S ), respectively, interact with the sample and
induce a third-order nonlinear polarization not only at the frequencies of the
input fields but also at their combination frequencies. As a result, a variety
of distinct CRS processes concurrently occur. Among the different CRS de-
tection schemes, coherent anti-Stokes Raman scattering (CARS), stimulated
Raman gain (SRG), and stimulated Raman loss (SRL) have been success-
fully exploited to generate contrast in microscopy. The induced third-order
polarizations are given by [27-29]