CARS Microscopy of Spinal Cord

The spontaneous Raman effect is the process by which a photon sees its frequency changed after scattering off a molecule. This change in frequency is due to the fact that the photon gives energy to the molecule to initiate a vibration (called a Stokes process) or takes energy away from the molecule to stop a vibration (called an anti-Stokes process).

The change in frequency is simply equal to the vibration frequency of the molecule. Since all molecules have a set of well-defined vibration frequencies that uniquely characterizes them, the measurement of the complete spectrum of photons that have scattered from a molecule can permit the identification of the molecule simply with light. This is very powerful, but somewhat inefficient.

Recent advances in laser physics have enabled the development of a new kind of microscopy based on stimulated Raman scattering. This new technique known as Coherent anti-Stokes Raman scattering (CARS) is sensitive to the same vibrational signatures of molecules seen in Raman spectroscopy. Unlike spontaneous Raman spectroscopy (where only one beam of light is used to excite the sample), CARS employs multiple simultaneous photons (that is, multiple simultaneous beams) to excite the molecular vibrations, and produces a signal where the emitted waves are coherent with one another. As a result, the CARS signal is much stronger than the spontaneous Raman emission for similar excitation powers.

 

CARS is a third-order nonlinear optical process involving three laser beams (two of which have the same frequency in our setup): a pump beam of frequency ωp, a Stokes beam of frequency ωs and a probe beam at frequency ωpr (in our case ωp=ωpr). These beams interact with the sample and generate a coherent optical signal at the anti-Stokes frequency (ωp – ωs + ωpr). The latter is resonantly enhanced when the frequency difference between the pump and the Stokes beams (ωp – ωs) coincides with the frequency of a Raman resonance, which is the basis of the technique’s intrinsic vibrational contrast mechanism.

CARS microscopy allows vibrational imaging with high sensitivity, high spectral resolution and three-dimensional sectioning capabilities. It allows noninvasive characterization and imaging of chemical species and biological systems without preparation or labeling with natural or artificial fluorophores that are prone to photobleaching.