Raman hyperspectral imaging is a powerful investigational tool in biophysics as it allows label-free imaging with chemical contrast. However, widespread diffusion of this technique is hindered by some of its intrinsic limitations, such as extremely small scattering cross-sections, that make it necessary using long acquisition times and high laser powers. These constraints imply that noninvasive cellular imaging is nearly impossible. Several approaches have been proposed to increase the intensity of the signal, including tip-enhanced Raman scattering, surface-enhanced Raman scattering and coherent spectroscopy.

The coherent anti-Stokes Raman spectroscopy (CARS) offers imaging possibilities with chemical contrast and is extremely promising in terms of applicability to a broad range of research areas, covering biophysics studies at sub-cellular level and material investigations at the nanoscale. Advantages offered by CARS microscopy with respect to conventional spontaneous-Raman investigations include: sub-diffraction-limit spatial resolution and intrinsic 3-D sectioning ability of thick samples, similar to two-photon fluorescence; access to phonon lifetimes and selective excitation of phononic states in nano-objects; video-rate imaging due to the large signals originating from the coherent buildup of the CARS process; penetration of up to approx. 0.3 mm into tissues for in-vivo studies; label-free imaging of live cells and tissues; insensitivity to fluorescence or luminescence backgrounds as a result of its blue-shifted anti-Stokes signal.

CARS is a four-wave mixing process: it requires illuminating the sample with three laser sources (pump, Stokes, and probe beams) and collecting the generated anti-Stokes photons. In practical implementations typically the pump and the probe beams are delivered from the same laser. Using a broadband Stokes field allows probing a large set of excited states yielding rich spectroscopic information on the specimen under investigation.

Fig.1: Schematics of the four-way mixing processes underlying coherent anti-Stokes emission.

Fig.2: Setup for CARS microscopy.

At CNI we are in the process of setting-up a CARS microscope to investigate a broad range of phenomena:

• In-vivo investigation of drug and/or nanoparticle penetration into tissues. The 3D sectioning ability of the CARS technique allows determining in real time the penetration of drugs and/or nanoparticles into a tissue sample, for example the skin of a living animal. Target research and application areas include nanotoxicology, pharmacology, and cosmetics.
• Measurement of phonon lifetime in nanomaterials, such as nanowires and graphene. This will allow, for example, exploring the interaction between graphene layers and the substrate material, which is of particular interest for graphene-based electro-optic devices and hydrogen-storage applications.
• In-vivo studies of cellular processes. Three-dimensional, video-rate, high-resolution images of living cells with chemical contrast can be readily acquired by CARS by taking advantage of the spectral fingerprints that different molecular bonds have in their Raman spectra. Additionally, since labeling or staining the target specie – e. g. with fluorophores or other molecules that could interfere with the cellular processes – is not required, CARS allows less invasive investigations with a simplified sample preparation.