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Coherent Anti-Stokes Raman Scattering (CARS) Microscopy The low natural optical contrast of biological cells
and tissues necessitates staining of such samples with various chromophores
and fluorescent markers. However, exogenous staining often affects cellular
metabolism and thus is of limited utility for imaging of living cells. CARS
microscopy allows for imaging living cells with intrinsic chemical contrast.
Similarly to spontaneous Raman scattering, CARS is sensitive to the molecular
structure of the material and its efficiency is greatly enhanced in the vicinity
of the molecular vibrational resonances. The coherent nature of this process
can make the CARS signal much greater than that of spontaneous Raman
scattering. Since the
first demonstration of CARS microscopy in 1982, and through its extensive
development during the last decade, practically all methods have been
variations of scanning techniques, based on co-propagating focused pump and
Stokes laser beams. We explore
a wide-field illumination scheme for picosecond CARS microscopy, in which the
whole image is registered simultaneously - similar to conventional microscopy
with a focal plane array detector. Such microscopes could have faster image
acquisition rates, significantly relaxed requirements on laser stability, and
simpler mechanical design with none of the moving parts inherent to scanning
systems. To prevent
CARS generation in a homogeneous bulk material, we use a non-phase-matching
sample illumination, and rely on light refraction or scattering within the
sample that brings some portion of the incident radiation into the phase
matching condition. Thus, the CARS signal is generated only in the presence
of a scattering or refracting object. If such an object, or its immediate
surrounding, contains molecules with Raman-active vibrational modes, the CARS
signal will be greatly enhanced. In this respect, such illumination geometry
resembles dark field illumination in conventional microscopy. However,
nonlinear optical transformation confers to this technique a new quality
– the CARS signal, which depends on both the optical properties of the
object and its chemical composition.
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