Most microscopy methods do not directly reveal the chemical identity responsible for image formation. Fluorescence imaging, resulting from either intrinsic species or added dyes, is molecule specific hut generally does not reveal bulk species. All chemical species have a characteristic Raman spectnun. We demonstrate a nonlinear microscopy method that provides a new solution to the problems of signal level and background fluorescence present in Raman microscopy. Signal production is due to vibrational, not electronic, resonances. Unlike fluorescence microscopy, Coherent Anti-Stokes Raman Scattering, or CARS, is resistant to photobleaching.Live cell imaging is performed routinely. Like other multiphoton microscopy methods, 3-D images are created by scanning the focal region of the laser beams through the sample.
SIGNAL PRODUCTION IN CARSThe CARS process is a special case of four-wave mixing [l], in which three optical fields are incident upon a sample, and the resulting polarizability is described hy the third-order nonlinear susceptibility XFL (equation 1). The sum of all contributions due to materials interacting with the light fields generate the overall response which produces the generated signal S , , is described by equation 2, which contains the laser intensities I, and I s , and the square modulus of xpk. The Raman vibrational characteristics are described by the first term inxy& , including the containing the concentration C, the amplimde &b. and the frequency and linewidths w ,~ and rGb. The incident light fields iu1: brought into resonance with the sample by adjusting the frequency difference between the pump and Stokes' shifted beams w, -ws. The energy levels of the molecule and the light waves are schematically indicated in Figure 1. t CARS x Y e W Figure 1. Energy level diagram showing contributionsto CARS signal production. Arrows are pump P and Stokes S incident laser beams, and the detected laser-like CARS beam AS. The left diagram represents vibrational resonances, and the right diagram shows electronic terms that contribute to a non-resonant background signal.
