Staining processes have constraints: Dyes bleach with time, can be phototoxic, and can influence subsequent research methods and camouflage subsequent research information. Processes with stained specimens are not suitable for long term analysis: Atmospheric conditions destroy dyes, and dyes alter when in contact with air or moisture – any change to environmental condition will influence the experimental result. Also, the staining process is time consuming. Some samples cannot be stained at all, because the attachment of a fluorophore or the presence of the dye changes the functionality of the molecule or the organism.
Coherent Anti-Stokes Raman Spectroscopy – the technique
CARS is a third-order, nonlinear process that involves a pump beam at a frequency of wp and a Stokes beam at a frequency of ws. The specimen is stimulated through a wave-mixing process. The anti-Stokes signal at ϖas=2ϖp–ϖs is generated in the phase matching direction as vibrational contrast at the frequency difference ∆ν=vsp–vs between the pump beam and the Stokes beam. This equals the frequency of the vibrational energy of a particular chemical bond.
High resolution images
CARS at video rate
Making CARS microscopy accessible
Prof. Andreas Zumbusch, Department of Chemistry, University of Konstanz, Germany, heads a research group for Physical Chemistry. He focuses on single molecule fluorescence spectroscopy and microscopy, as well as on the development and application of non-linear optical microscopy: "Many research areas, whether from a life science or materials science background, require fast, non-invasive imaging with high spatial resolution, high molecular specificity, and high sensitivity. As an optical method by which contrast is generated on the basis of spectroscopic properties intrinsic to the sample, CARS microscopy can offer all of this. In neuroscience, for example, CARS microscopy can have an impact comparable to two-photon microscopy, except that it does not rely on the introduction of fluorescent labels.
The advent of commercial CARS microscopy systems is certainly a major step toward making the technique accessible to researchers interested in its application. Attractive system concepts offer hands-off operation while allowing the full potential of CARS microscopy to be used."