Coherent Anti-Stokes Raman Scattering (CARS)

CARS is the most recent MPM imaging contrast. CARS microscopy derives contrast directly from Raman- active vibrational modes within molecules and requires two synchronised laser pulses of different wavelengths.

CARS is a third order nonlinear optical process in which a pump field E_p and a Stokes field E_s interact with a sample to generate a signal field E_as at the anti-Stokes frequency of ω_as = 2ω_p - ω_s. When ω_p - ω_s is tuned to be resonant with a molecular vibration (&Omega), the CARS signal can be significantly enhanced, producing a vibrational contrast.

Unlike spontaneous Raman scattering, CARS produces a highly directional field. The two excitation beams (ω_p and ω_s) form a beating field with frequency ω_p - ω_s. When ω_p - ω_s matches &Omega all the molecules within the interaction volume vibrate in-phase.

Advantages

CARS microscopy permits biological imaging with several advantages:

• Raman resonance enhancement provides chemical selectivity without the need for labelling.
• There is little scattering of the near-infrared excitation beams, allowing deep penetration in tissues.
• Due to the anti-Stokes shift, the CARS signal is of shorter wavelength than one-photon fluorescence. This allows detection in the presence of a strong fluorescent background.
• Coherent addition of CARS fields generates a large signal;
• Nonlinear dependence on excitation intensities produces inherent 3D resolution.
• Low absorption of the near-infrared excitation beams, significantly reduces the photodamage in biological samples.