CARS is a four-wave mixing process in which a pump beam at frequency ω_p and a Stokes beam at frequency ω_s interact with a sample to generate an anti-Stokes signal at frequency ω_as = 2ω_p - ω_S . When the beat frequency between the pump and Stokes Δω matches the frequency of a particular Raman active molecular vibration Ω, the resonant oscillators are coherently driven. This generates a nonlinear polarization of the sample that radiates the anti-Stokes signal. Because the radiation is emitted coherently, the CARS signal is strongly enhanced compared to the spontaneous Raman scattering signal, for which Stokes and anti-Stokes photons are emitted incoherently.

In contrast to SRS, CARS is a parametric process, i.e., no energy is transferred into the sample but the difference energy between the pump- and Stokes-photon is carried away by the anti-Stokes field. As a result, CARS can also occur when there are no resonant molecules in the focal volumes. This gives rise to a nonresonant background that can limit the sensitivity and alter the CARS spectra from spontaneous Raman spectra. We developed several detection schemes for CARS microscopy to overcome this background.

Detection Schemes for CARS:

• Forward CARS (F-CARS)

The signal is detected in the phase-matched direction and the signal is selected by a set of spectral filters. The F-CARS signals are generally very strong and can sometimes be observed by the naked eye.

• Epi CARS (E-CARS)

The forward CARS signal is accompanied by a strong nonresonant background which may overshadow weak signals that are of interest. When detected in the backward direction, the nonresonant signal from the solvent (water) is completely eliminated. E-CARS is particularly sensitive to objects in focus that are smaller than the optical wavelength. When the sample is highly scattering, the forward propagating CARS signal can be backscattered, giving rise to a strong epi-signal .

[Left] CARS tissue image of small adipocytes of the subcutaneous layer from mouse ear tissue. The image was taken at a Raman shift of 2845 cm^-1 to address the CH₂ stretching frequency.

• Polarization sensitive CARS (P-CARS)

By taking advantage of the Raman depolarization ratio of certain modes, the resonant signal can be separated from the nonresonant background when polarization sensitive detection is employed. P-CARS significantly enhances the image quality by eliminating the nonresonant background completely.

• Time-resolved CARS

By employing a femtosecond time-scale delay between the excitation and probe pulses, time-resolved CARS allows for the complete suppression of nonresonant signals.

• Interferometric CARS (iCARS)

By interfering with the CARS signal from a sample with a local oscillator CARS signal it is possible to selectively image the real and imaginary part of the third-order nonlinear susceptibility. iCARS enables rapid, amplified Raman imaging that is free of the nonresonant background.

CARS Microspectroscopy Techniques:

In addition to imaging, CARS spectroscopy can be carried out with the microscope. We have developed the following tools:

• Multiplex CARS (M-CARS)

A picosecond laser is combined with a femtosecond laser to cover a broad range of vibrational frequencies. The CARS signal is collected and analyzed on a spectrometer. Chemical details in microscopic volumes can be quickly characterized with this method.

• CARS correlation spectroscopy (CARS-CS)

Analogous to fluorescence correlation spectroscopy, the autocorrelation of time-resolved CARS signals reflects rapid diffusion dynamics of small Raman scatterers in focus without the need for labeling.

• Interferometric CARS microspectroscopy

Essentially a combination of interferometric CARS microscopy and multiplex CARS, interferometric CARS microspectroscopy enables detection of the real and imaginary components of the CARS signal.