Raman based spectroscopy techniques are highly sensitive to the biochemical nature of materials and have the high spatial resolution associated with optical microscopy. Fast identification of biochemical components in turbid materials has generated strong interest in live tissue spectroscopy since it makes disease diagnosis possibly achievable in situ
. For example, the diagnostic potential of Raman spectroscopy has generated a large amount of literature in oncology alone [1
]. However, it has become clear that a clinically useful technique should also provide context for diagnosis through imaging, something impossible with low sensitivity techniques such as spontaneous Raman spectroscopy.
Coherent anti-Stokes Raman scattering [3
] is a nonlinear technique with the necessary sensitivity for fast chemically-specific imaging. It requires two or more laser pulses of different wavelengths to coherently excite a Raman active vibrational mode in a molecule. On the one hand, the nonlinear nature of the process demands short pulses (ps or fs) with high peak power, and on the other hand a system with good tunability or a large bandwidth is necessary for spectroscopic measurements. Typical CARS systems based on optical parametric oscillators [5
] or synchronized Ti:Sapphire lasers [7
] have been successful for imaging lipids, myelin and water [4
] (for a review see [10
]) but their relatively slow tunability has hampered their use in spectroscopy. Broadband methods based on transform-limited ultrashort pulses [11
] or continuum generation [14
] have been successful for spectral imaging of optically thin systems. However, their need for a spectrometer to resolve the vibrational spectra results in very poor collection efficiency in turbid materials such as tissue where diffusion is important. Methods using spectral focussing of broadband excitation pulses avoid the need for a spectrometer but rely on a mechanical delay line for vibrational tuning [15
We propose a strategy where the Raman lines are excited sequentially at very high speed by narrowband picosecond pulses. The wavelength-swept coherent anti-Stokes Raman scattering system presented here is based on a master oscillator power amplifier (MOPA) pump laser synchronized with a rapidly tunable programmable laser (PL) as the Stokes beam. The spectral bandwidth of this instrument covers most of the high wavenumber region (2700–2950 cm−1). This strategy has many advantages compared to other existing methods. With the spectroscopic information encoded in time (), the detection can be done using fast and sensitive photomultiplier tubes. This is especially important for CARS spectroscopy in thick tissue where the largeétendue of the scattered signal is not compatible with the small entrance slit of spectrometers, resulting in poor collection efficiencies. Furthermore, this system allows random access to any Raman line within its bandwidth. Since the acquisition time scales linearly with the number of spectral points, this should prove to be an essential feature for applications where speed is critical. Finally, the high flexibility of the wavelength sweep rate can easily accommodate rapid single point spectroscopy or hyperspectral imaging where a whole image is acquired for every Raman line (). In this manuscript, we use the system for WS-CARS spectroscopy as well as single-line and hyperspectral imaging in thick tissue both in forward and epi-detection.
Fig. 1 (a) In wavelength-swept CARS spectroscopy, the Raman vibrations (Ω) are excited sequentially and the spectroscopic information is encoded in time. (b) Hyper spectral images are constructed by raster scanning of the sample for every Raman line. (more ...)