Locating molecules using optical microscopy: 3-D imaging by coherent antistokes Raman scattering

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 but generally does not reveal bulk species. All chemical species have a characteristic Raman spectrum. 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.