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Raman micro-spectroscopy (RMS) is a powerful technique for the identification, classification, and diagnosis of cancer cells and tissues.1 The requirement for long acquisition times of 1-30 s have impeded clinical application. The slow acquisition time can be overcome by the use of coherent Raman scattering (CRS), a class of thirdorder nonlinear optical spectroscopies that employ a sequence of light pulses to set-up a vibrational coherence within the ensemble of molecules inside the laser focus. The two most widely employed CRS techniques are coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) both of which achieve extremely high acquisition speeds up to the video rate, but traditional architectures are limited in terms of “single frequency” detection resulting from the use of picosecond pump and Stokes pulses with an optical bandwidth of a few wavenumbers. An important breakthrough has been recently achieved by the Cicerone group2 using a femtosecond Er:fiber laser oscillator followed by two erbium doped fiber amplifier arms; one arm is frequency doubled to generate narrow-band pulses at 770 nm with a flat-top 3.8 ps temporal profile, while the other is spectrally broadened in a highly non-linear fiber to generate a broad supercontinuum spanning the 900–1350 nm wavelength region. This pulse combination enables extraction of the CARS response by both the two-color and the three-color mechanism. While all previous broadband CARS systems failed to provide a low-noise spectrum in the fingerprint region, this approach has enabled Raman spectra in the whole biologically relevant frequency region (500–3500 cm-1) to be captured with 10 cm-1 resolution and 3.5 ms acquisition. Here, we provide guidance on the initial setup and optimization of this bCARS micro-spectroscopy system, with specific examples of the common pitfalls encountered during the setup. This is particularly useful for those coming from a background of designing spontaneous Raman spectroscopy systems for biomedical applications.
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