CARS Microscopy

CARS stands for Coherent Anti-Stokes Raman Scattering. It is a non-linear Raman spectroscopy method which not only uses an excitation laser, but also irradiates the sample with a second, red-shifted so-called Stokes-laser.

By Dr. Jakob Bierwagen, AHF analysentechnik AG

The frequency shift of the second laser is chosen to correspond to the energy difference between the ground state and the (first) excited vibrational state of a dedicated Raman line of the molecule to be looked at. This leads to the coherent population of the, almost empty, excited vibrational state. From there, the molecule is again promoted to a higher virtual state. Subsequently, the molecule can return to the ground state. Thereby giving off a photon with more energy – so-called Anti-Stokes line.

The process is coherent because the population of the vibrational excited state takes place in a coherent process; it is a kind of ‘stimulated emission’ of the virtual state into the vibrational excited state. This process can amplify the Anti-Stokes Raman signal up to a factor of 106. The advantage of the method is that the Anti-Stokes line has a shorter wavelength than the laser wavelengths, so one does not have to compete with autofluorescence or other emission sources in the signal, which leads to a good noise ratio and has very little background. The disadvantage is that not all of the signal comes from the coherent Anti-Stokes line. Due to this the line width is quite high and thus the specificity of the method is reduced.

The method usually operates in transmitted light mode, since the Anti-Stokes line is also emitted coherently, so in the same direction as the two excitation lasers. Despite the strong enhancement of the signal by the coherent excitation, it is still very weak and the quantum yield is low.

In order to be able to separate the signal from the strong pulsed lasers, one needs filters with a very high optical density. Special beam splitters are also required for coupling the two laser beams. AHF can assist you in your project with a set of optical filters specially adapted for CARS microscopy and will gladly assist you in choosing the right filters.