Coherent Anti-Stokes Raman Spectrum (CARS) microscopy is a label-free, chemical specific imaging modality based on the Raman spectrum,

or vibrational fingerprint of a molecule (Pegoraro et. al, 2009). It employs multiple photons to address the molecular vibrations and produces a signal in which the emitted waves are coherent with one another. CARS microscopy makes use of the principles of Raman Spectroscopy. Raman Spectroscopy detects information of molecular vibrations in a molecule. Molecular vibrations are very sensitive to the molecular species, structures, conditions and its environment (Hashimoto, et. al, 2009). CARS requires involvement of two very short laser pulses which are known as pump and Stokes, both of which having frequencies comparable to an infrared. These near-infrared frequencies avoid damage photochemically and allow the deep penetration into the tissue (Pegoraro & Stolow, 2009). The pump and the Stokes interact with the tissue to excite a vibrationally resonant Raman mode at frequency. An involvement of a third photon, the probe, is inelastically scattered off the coherent excitation, thus anti-Stokes light becomes emitted from the tissue (U.S. Department of Commerce, 2012). By proper choice of the pump and Stokes wavelengths it is possible to tune the CARS signal to a particular vibrational mode, and hence to obtain a chemical-specific image (Molecular Photonics Group, 2010).

(image from: http://www.photonics.com/Article.aspx?AID=40014) Traditionally, CARS is a nonlinear optical phenomenon. CARS make use of ultrafast pulsed lasers, and probe the vibrational response of molecules in a more efficient way. Physically, nonlinear Raman techniques make the molecule vibrate in unison, generating coherent signals that can be up to five orders of magnitude higher compared to conventional Raman spectroscopy (Potoma, 2012).However, the sensitivity of CARS microscopy is limited by the non-resonant background signal. The non-resonant component is entirely in phase with the

driving field of the laser and gives us no information about the chemical nature of the sample. The resonant component contains the chemical information and has frequency-dependent amplitude (U.S. Department of Commerce, 2012). Non-resonant signals reduce contrast on the image of the sample/tissue produced. The CARS spectroscopy process gives many advantages over traditional Raman microscopy. One example is that CARS, with its vastly stronger signal, enables real-time imaging (Pegoraro et.al, 2009).

REFERENCES: Hashimoto, M., Minamikawa, T., Niioka, H., & Araki, T. (2009). Multifocus cars microscopy for realtime vibrational imaging. 7507(1), doi: 10.1117/12.838084 Molecular Photonics Group. (2010). Microscopy of living cells. Retrieved from website: http://www.chem.queensu.ca/people/faculty/Stolow/Research/MLCCARS.html Pegoraro, A. F., Stolow, A., Risdale, A., Moffatt, D. J., Pezacki, J. P., & Jia, Y. (2009). Cars microscopy made simple. Biophotonics, Retrieved from http://www.photonics.com/Article.aspx?AID=40014 Potoma, E. O. (2012, July 17). Cars microscopy: Imaging characteristic vibrational contrast of molecules. Retrieved from http://www.leica-microsystems.com/science-lab/carsmicroscopy-imaging-characteristic-vibrational-contrast-of-molecules/ U.S. Department of Commerce, The National Institute of Standards and Technology. (2012). Broadband cars microscopy. Retrieved from website: http://www.nist.gov/mml/bbd/biomaterials/bcars.cfm