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Since it first revealed the existence of cells, the microscope has remained one of our most important tools for learning about the biological world. Early microscopes exploited density differences to produce images, but these images contained no chemical information. Today our knowledge of biology has advanced to the point where we seek a molecular understanding of the mechanisms governing basic cellular processes. The microscopes currently used by biologists require artificial fluorescent labels to identify and follow particular chemical species. These labels are not always easy to apply, and they only identify a fraction of the species present. In addition, they are subject to photobleaching and phototoxicity, and their use may perturb the system under study. This is where CARS microscopy comes in...

Although there are a number of naturally fluorescent species, an endogenous form of contrast that exists for every molecule is its Raman spectrum. Raman spectra, with well-defined transitions in the "fingerprint" region can readily identify different chemical species. A multiphoton version of spontaneous Raman scattering is CARS, which provides orders of magnitude signal enhancement over Raman scattering, allowing it to be used at low excitation levels appropriate to the study of living systems. The fact that CARS is a multiphoton process means that the excitation is localized at the laser focus, providing high axial resolution without the need for confocal detection, and making 3D imaging straightforward. The near-infrared wavelengths that are suitable for CARS also provide substantial depth penetration, a distinct advantage for microscopy of living systems.

We recently demonstrated a simple time-domain CARS microscopy method called Fourier transform CARS (FTCARS) (get the paper here). FTCARS has a number of advantages: it uses a single laser source, and provides essentially unlimited spectral resolution for CARS imaging over a broad spectral range. Because FTCARS is a time-domain technique it effectively eliminates the nonresonant background problem faced by all CARS microscopies. Most CARS methods image a single CARS mode, prohibiting them from following multiple chemical species simultaneously.

We have been working to improve the sensitivity of FTCARS microscopy to permit imaging of biological molecules that are found in low concentrations. We have implemented an interferometric detection scheme that provides an order of magnitude amplification of the CARS signal. Read about it here.