Optical microscopy is unique among current imaging modalities in its ability to probe living specimens with subcellular resolution, enabling the visualization of morphological details in tissue and cells on the scale of a few hundred nanometers. To date, however, optical microscopy has not been completely successful in providing high-resolution morphological information with chemical specificity. The contrast in confocal reflectance microscopy and optical coherence tomography (OCT), for instance, is based on refractive index differences, and cannot directly probe the chemical composition of tissue structures. Fluorescence microscopy, while extremely sensitive and widely used, is limited in chemical selectivity by the small number of intrinsic fluorophores, such as NAD(P)H, riboflavins and elastin. The introduction of extrinsic fluorophores provides specific probes, but often causes unwanted perturbations. Second harmonic generation (SHG) microscopy is useful for visualizing well-ordered protein assemblies such as collagen fibers but has inadequate sensitivity and specificity for other tissue components.

Vibrational spectra of biological specimens contain a multitude of molecular signatures that can be used for identifying biochemical constituents in tissue. Conventional vibrational microscopy methods, however, lack the sensitivity required for rapid tissue examination. Infrared microscopy is limited by low spatial resolution due to the long wavelength of infrared light, as well as strong water absorption in biological specimens. Raman microspectroscopy, while capable of discriminating healthy from diseased tissue in vivo , is hampered by long integration times and/or high laser powers that are not desirable in biomedical applications.

Much stronger vibrational signals can be attained with coherent anti-Stokes Raman scattering (CARS), a nonlinear Raman technique. 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 = 2 ω p - ω S . When the beat frequency between the pump and Stokes ( ω p - ω S ) matches the frequency of a particular Raman active molecular vibration ( O ), the resonant oscillators are coherently driven. This results in a significantly enhanced anti-Stokes signal, providing the vibrational contrast of CARS microscopy. The CARS energy diagram is shown in Figure 1.

Typical CARS signals from sub-micrometer objects are orders of magnitude stronger than the corresponding spontaneous Raman response. Since CARS is a nonlinear effect, the signal is generated only at the laser focus, which allows for point-by-point three-dimensional imaging of thick specimens, similar to two-photon fluorescence microscopy.

The first systematic study of this nonlinear Raman process was carried out by Maker and Terhune (Phys. Rev. 137, A801-818, 1965) at Ford Motor Company. However, the name of CARS spectroscopy appeared 9 years later (Appl. Phys. Lett. 25, 387-390, 1974). The first demonstration of CARS microscopy dates back to 1982 (Duncan et al, Opt. Lett. ), but it was not until 1999, through a series of technical advances, that high-sensitivity 3D CARS imaging of live cells was demonstrated by our group (Zumbusch et al, PRL). Presently, our research program focuses on developing CARS-related techniques for nonlinear optical imaging and microspectroscopy, and seeks to use these methods to unravel key processes in living systems.

[Left] 3D-rendering of a mouse sebaceous gland imaged with coherent anti-Stokes Raman scattering (CARS) microscopy. The contrast is derUived from the CH 2 symmetric stretching vibration, which is abundant in lipids. A crescent-shaped sebaceous gland surrounding a hair shaft is revealed to be composed of multiple cells, each filled with numerous micrometer-sized CH 2 -rich sebum reservoirs. A nonlinear vibrational imaging technique, CARS microscopy enables video-rate examination of tissue in vivo without the use of exogenous labels.

Why CARS Microscopy?

• Intrinsic vibrational contrast, circumventing the need for extrinsic labels.

• The coherent signal accumulation in the CARS process produces a strong, directional signal, making CARS microscopy much more sensitive than conventional vibrational microscopy. Consequently, CARS microscopy requires only moderate average powers that are easily tolerable by biological samples.

• The nonlinear CARS signal is generated only at the focus where the excitation intensities are the highest. This leads to a 3D sectioning capability, which is essential for imaging tissues or cell structures.

• The CARS signal is higher in frequency than one-photon fluorescence, allowing it to easily detected in presence of a strong fluorescent background.

• There is little scattering of the near-infrared excitation beams, allowing deep penetration in tissues.

• There is little absorption of the near-infrared excitation beams, significantly reducing the photodamage to biological samples.