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ADVANCES IN RAMAN TECHNIQUES21eueuMicroscopyandAnalysis | July/August 2017Typically, laser setups using two laser beams of different colour and in pulsed mode enable a more elaborate, but coherent process, that generates a signal which is generally orders of magnitude higher. In practise, the advantage of coherent Raman scattering over linear Raman is this overall higher signal.In Coherent Antistokes Raman Scattering (CARS), the difference between the two wavelengths is used to coherently drive a Raman transition. Typically, two narrow bandwidth picosecond electronically synchronized laser pulse trains have been used, one of which is scanned in the wavelength to do CARS spectroscopy. The shape of the CARS signal is different from that of linear Raman, also the overall sensitivity is limited by the distinction between the resonant and non-resonant part of the signal. An overview of the method is given in a book by Xie and colleagues10.A variant of Coherent Raman is Stimulated Raman Scattering (SRS), which again uses lasers of two different colours with synchronized output pulses to coherently drive a nonlinear process. The advantage is greatly reduced non-resonant background, and that the output trace is the same as conventional Raman allowing existing databases of compound results to be used, as well as quantitative interpretation of the measured data11.SRS makes use of modulation of one of the laser beams, a double modulation technique Stimulated Raman Gain and Opposite Loss Detection (SRGOLD) proposes background free detection and is the latest variant using a three colour laser beam excitation scheme12.Both CARS and SRS can in principal be done in versions using femtosecond or picosecond lasers (and even nanosecond lasers), each with different advantages and drawbacks. Historically, two electronically synchronized tuneable laser sources have been used13, or more typically today, optical parametric oscillators, providing optically synchronised pulses.However, todays available commercial implementations are often hampered with limited tuning speed, and hence slow in acquisition of a CARS/SRS spectrum14. One elegant way for considerably increasing the acquisition speed or even enabling single beam capability is the use of multiplex CARS15, 16 or multiplex SRS17, 18. With this technique, a narrow band pump and a broadband Stokes pulse, or even one ultra-broadband spectrum with phase shaping supply all wavelengths needed to simultaneously record a large number of Raman shifts. However, parallel detection may require dedicated sophisticated detectors and electronics18.With extremely broadband laser oscillators offering typical bandwidth on the order of 400 nm in the NIR and also high repetition rate amplified systems like an OPCPA offering a bandwidth of greater 300 nm being readily available, broadband single beam Raman techniques have become possible. The available bandwidth allows a wavenumber range of up to 4000 cm-1. It also allows for an efficient combination of multiple modalities, or for very fast spectroscopic CARS modality using a quite simple detector in form of a photo diode.One implementation of CARS using broad bandwidth uses the principle of spectral focussing19. A simple implementation relying on glass stretchers to measure two vibrational frequencies with two (or more) pairs of pump/stokes pulses simultaneously is called differential CARS (or D-CARS). One advantage of this method is the fast acquisition speed of a few seconds for complete images and the effectively rejection of non-resonant background using the D-CARS signal. The simple analysis scheme allows for rapid distinguishing between saturated and unsaturated lipids20.The laser source providing a sufficiently large bandwidth not only enables single beam CARS, but also has been used in a multimodal, or hyperspectral scheme combining D-CARS covering a wavenumber range of 2600 cm-1 with a resolution of 10 cm-1 together with second harmonic generation (SHG) as well as two photon fluorescence (TPF) imaging – all from a single beam at the same time, using a pulse shaper21, 22. An example image is given in fig. 4 providing much richer information than from one imaging modality alone.An early implementation of spectral focusing using an octave spanning oscillator (Laser Quantum venteon) and a spatial light modulator to do single-pulse (single beam) CARS microscopy is described by Isobe et al23. In fig. 5, CARS images of unstained HeLa cells are shown from this experiment demonstrating the applicability to biological samples.The use of an octave spanning oscillator allowed the researchers to focus into narrow spectral regions ranging from approx. 1000 cm-1 to 4800 cm-1.Aimed at fast measurement of spectroscopic CARS, the method of Fourier Transform (FT) CARS shows great potential. The principle has been shown by Ogilve et al24 using a 100 MHz oscillator and is based on a pump probe type dual pulse setup. In this method, the CARS spectrum is obtained through the Fourier transform of the CARS interferogram – i.e. the CARS emission as a function of time delay between two identical collinear excitation pulse trains with a generated time delay between successive pulses. The non-resonant background can be removed by simply removing the contribution around zero time delay. The detection of the CARS signal can be accomplished with a simple Si-diode.Using a 5 fs oscillator with a spectral bandwidth of 4000 cm-1 the applicability of FT CARS to biological samples covering the bandwidth in a single measurement has been demonstrated23. An image of unstained HeLa cells has been obtained by FT CARS (fig. 6) shows the differentiating of mitochondria or endoplasmic reticulum, the nucleus and water.The speed of the acquisition of a complete CARS spectrum using FT CARS depends on how fast the two pulses can pass through the needed delay times that contribute to the CARS interferogram.The refresh time then depends on how fast this cycle can be repeated. The time delays which contribute to the interferogram depend on the dephasing time of the coherent Raman excitation, which is typically on the picosecond (ps) timescale. The interferometric, non-resonant background being present at short delay times only and hence can be completely suppressed by suitably positioning the numerical apodization window26.In the implementation of the work published by Isobe and colleagues a mechanical delay line is used23. The first step to speed up the measurement is to use an ASynchronous OPtical Sampling (ASOPS) dual comb type setup where two repetition rate Figure 4 TPF (green), CARS from subcutaneous lipid deposits (red) and SHG from collagen (blue) of tissue from a mouse tailReprinted (adapted) with permission from by Pope et al.9Copyright (2013) Optical Society of America