Raman scattering is a valuable spectroscopic technique that can be applied to solid, liquid or gas samples. The Raman effect occurs when a sample is irradiated with light of any wavelength and a very small fraction (10-6-10-8) of the incident radiation is scattered and shifted at lower frequency (longer wavelength) that corresponds to a vibrational transition specific of the sample. Because the defining quantity is the difference in energy (frequency) between the incident and scattered light, Raman transitions are commonly defined in wavenumber (cm-1).
Complex molecules display very congested Raman spectra especially in the so-called fingerprint region (500 - 1,500 cm-1). To achieve sufficient resolution, it is therefore necessary that the laser source be spectrally narrow (<1 cm-1), better if single frequency. In a typical Raman spectrum acquisition experiment, the Raman light is collected, filtered and scanned in wavelength before it reaches the detector to produce a high-resolution spectrum. With many thousands of commercial Raman Spectrum analyzers deployed in scientific and industrial research laboratories, Raman spectroscopy has become a fundamental analytical tool in chemistry, pharmaceutical industry, and life and material sciences.
In this section only CW-excited Raman spectroscopy is described, while pico- or femtosecond Raman techniques are discussed elsewhere. Although the excitation light can be at any wavelength, it is convenient to select a wavelength that maximizes the signal-to-noise ratio of the weak Raman signal. Short visible wavelengths (blue and green lasers) produce a slightly stronger Raman signal but also a stronger competing fluorescence while red or near-IR light is less efficiently Raman-scattered but the fluorescence background will also be much smaller. For this reason, it is best to employ a set of different wavelength sources, the most common being 488 nm, 532 nm, 631 nm and 750 nm. Coherent produces lasers with wavelength and spectral characteristics that address all the Raman needs. These lasers include the Sapphire SF series, the Innova ion lasers that provide a wide variety of wavelengths in one single laser and the Surelock diodes.
UV resonance Raman is a variation of standard Raman spectroscopy where the proximity of electronic resonances greatly enhances the signal. To take advantage of this, the Raman source must be very short in wavelength (<270 nm) to minimize competing fluorescence. Coherent provides unique CW sources at 257 nm and 244 nm – the Innova Fred Ion lasers.
Raman scattering can be exploited also using picosecond and femtosecond laser pulses. However, while CW Raman is a spontaneous process, and therefore has very low probability, in picosecond or femtosecond Raman experiments, the process is a stimulated one, similarly to the way in which stimulated emission is used to coherently amplify light in a laser oscillator. To take advantage of this effect, the Raman transition of interest should be known in advance. CARS or Coherent Anti-Stokes Raman spectroscopy is a technique that requires two pico- or femtosecond synchronized laser pulses to hit the sample at the same time. Here the wavelength difference between the two pulses is tuned to match a strong and known Raman transition.
Conventionally the shorter wavelength beam is called the Raman pump and the longer wavelength beam is called the Raman Stokes. The combination of these photons sends the molecule under test to the vibrational level corresponding to the selected Raman vibration; a second pump photon brings the molecule to a higher energy vibrational level from which the molecule decays instantaneously releasing a photon at higher energy than both the pump and the Stokes (AntiStokes beam). This offers the advantage of easy wavelength separation by filtering.
CARS microscopy was first developed in 1992 by Sunney Xie and coworkers, and found immediate applications in microscopy biological samples. Most CARS microscopy experiments are performed using the C-H2 molecular “stretch” typical of lipids because it provides a particularly strong transition.
A more recent variation of CARS is SRS (Stimulated Raman scattering). Here the same two laser wavelengths are used but, rather than detecting the antistokes wavelength, the measurable quantity is the relative intensity loss in the Raman pump or gain in the Raman Stokes when the pump wavelength is switched on and off at a very rapid frequency (several MHz). Because of the very minute change in the pump or Stokes intensity (~ 10-5), lock-in techniques and low-noise lasers are required to detect these weak signals. While for many narrowly spaced Raman transitions picosecond pulses are required, signal from lipids is the strongest when a 100-200 fs source is used. CARS/SRS microscopy are popular when imaging biological structures and dynamics involving lipids. Furthermore, because CARS/SRS do not require an external chemical marker (unlike fluorescence) they are suitable for in-vivo applications even in human subjects; for this reason, CARS is sometime added to a multiphoton and harmonic generation imaging of tissue samples to develop potential tools for all-optical histopathology or intraoperative aid for cancer surgery.
Coherent lasers best suited for CARS/SRS are Chameleon with its MPX OPO and Chameleon Discovery.
While CARS and SRS are used prevalently for imaging, Femtosecond Stimulated Raman Scattering (FSRS) is a sophisticated technology used to measure the Raman spectra of excited states. There are two major differences between SRS microscopy and FSRS spectroscopy: first, FSRS studies the Raman spectra of excited species, while SRS uses the Raman spectrum associated with the fundamental electronic level of the sample molecule. Second, in SRS the laser beams are tuned to a well-known Raman transition as the purpose is to identify and image structures that contain molecules exhibiting this transition. On the contrary, in FSRS the purpose is to extract a complete and a priori unknown spectrum. For this reason, it is necessary to add a third beam (sometime called “actinic pump” for historical reasons) that excites the sample; to provide the broadest possible transient Raman spectrum, the Stokes beam has to be broadband. FSRS experiments require a short pulse amplifier like Coherent Legend Elite to provide the actinic pump and the Raman Stokes probe beam (via an OPA). In addition, part of the beam has to be filtered to provide a narrower (picosecond) Raman pump; alternatively, a Second Harmonic Bandwidth Compressor (SHBC) can be added to pump a picosecond TOPAS OPA.
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