Biomedical Engineering Reference
In-Depth Information
bation of the sample and where conventional Raman microscopy requires a
long averaging time and high laser powers will benefit from the use of CRS
imaging. For example, in vivo CRS microscopy opens exciting possibilities
to study chemical processes inside a living cell or organism in real time. It
thus has the potential to overcome many limitations of conventional cell bi-
ology techniques for the detection, identification, and quantification of the
chemical composition of intracellular components and organelles, which are
often destructive by nature, possess imperfect specificity, and can even per-
turb the cellular biology under investigation. Furthermore, multimodal CRS
microscopy that readily combines the chemical specificity of CRS with other
nonlinear optical image contrast mechanisms, such as TPF, SHG, and, THG,
allows tissue diagnostics based on a variety of simultaneously imaged endoge-
nous signals.
For a particular biomedical application of CRS microscopy, the best choice
whether to use CARS or SRS detection depends on the optimal balance be-
tween the pros and cons of each technique regarding its detection sensitivity,
image acquisition time, and interpretability of image contrast and spectrum.
In the following, we provide a critical discussion of the advantages and disad-
vantages of both complementary detection techniques:
Since CARS microscopy benefits from the fact that the signal can be selec-
tively detected through spectral filtering against the pump and Stokes beams,
it allows video-rate chemical mapping and fast hyperspectral CARS imaging
with acquisition times only limited by signal strength. Because the CARS
signal scales quadratically with the number density of vibrational modes in-
side the probe volume, samples with a high density of chemical bonds, e.g.,
lipids, are best studied by CARS contrast. CARS microscopy is then capable
of monitoring dynamic processes in real time. On the other hand, the modulus
square dependence of the CARS signal on the total third-order susceptibility
causes distorted Raman line shapes and limits the detection sensitivity due to
unavoidable nonresonant background contributions. Moreover, the CARS sig-
nal scales as the square of the spontaneous Raman scattering signal and as a
cube of laser power. Consequently, weak Raman resonances and low densities
of scatterers are not easily detected. In addition, the need to fulfil the phase
matching condition in CARS results in a signal that is dependent on both the
dimension of the microscopic scatterer and the microscope's geometry for the
propagation directions of the input and output fields. In consequence, unlike
in fluorescence and spontaneous Raman microscopy, both CARS images and
spectra cannot readily be interpreted in a quantitative manner. The ecient
suppression of nonresonant background signal or OHD-CARS schemes have to
be implemented to significantly simplify image interpretation. An alternative
approach to quantify the CARS image contrast is based on the separation of
individual susceptibility components by means of multiplex CARS microspec-
troscopy in combination with spectral phase retrieval using MEM.
The detection of stimulated Raman scattering (SRS) has the major advan-
tage that it inherently avoids these problems. A readily interpretable chemical
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