Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering

Abstract

Confocal [1] and multiphoton [2] fluorescence microscopy have become powerful techniques for threedimensional imaging of chemical and biological samples, especially for live cells. This coincides with developments of various natural and artificial fluorescent probes for cellular constituents [3]. For chemical species or cellular components that either do not fluoresce or cannot tolerate labeling, infrared microscopy and spontaneous Raman microscopy can be used as contrast mechanisms based on vibrational properties. Conventional infrared microscopy is limited to low spatial resolution because of the long wavelength of light used. High resolution Raman microscopy of biological samples has been demonstrated with a confocal microscope [4]. However, the intrinsically weak Raman signal necessitates high laser powers (typically .10 mW) and is often overwhelmed by the fluorescence background of the sample. A multiphoton microscopy based on coherent anti-Stokes Raman scattering (CARS) [5] was put forward as an alternative way of providing vibrational contrast [6]. However, the sensitivity of CARS microscopy was limited by the nonresonant background signal, and high resolution three-dimensional sectioning was not achieved. In the study reported here, we demonstrate CARS microscopy in the chemically interesting vibrational spectral region around 3000 cm21 with high spatial resolution and three-dimensional sectioning capability. Most importantly, the use of near-infrared laser pulses generated by a Ti:sapphire laser s,855 nmd and an optical parametric oscillator/amplifier s,1.2 mmd allows a significant improvement in signal to background ratio in CARS detection. Unlike spontaneous Raman microscopy, the highly sensitive CARS microscopy requires only a moderate average power for excitation s,0.1 mWd, tolerable by most biological samples. CARS spectroscopy has been extensively used as a spectroscopic tool for chemical analyses in the condensed and gas phases [7]. In doing CARS spectroscopy, a pump laser and a Stokes laser beam, with center frequencies of np and nS , respectively, are spatially overlapped. The CARS signal at 2np 2 nS is generated in a direction determined by the phase-matching conditions. When the frequency difference np 2 nS coincides with the frequency of a molecular vibration of the sample, the CARS