Raman spectroscopy is based on the effect of inelastic light scattering due to changes in the polarizability of the electron cloud in molecule and associated with measuring of molecular vibrations. This method provides “fingerprint” spectra that are unique to each specific compound and contain information about chemical composition and structure. Moreover, the coupling of Raman spectroscopy with microscopy (Raman Microspectroscopy) enables high spatial resolution (below ~ 1 µm) and sensitivity.
Advantages of Raman Microspectroscopy
- RM provides fingerprint spectra
- No or little sample preparation is needed
- No staining is necessary
- Spectra are obtained non-invasively without interference from water
- RM Allows analysis with spatial resolution in µm range
Due to the low quantum efficiency of the Raman effect (typically 10-6–10-8), RM suffers from limited sensitivity. However, Raman scattering can be significantly enhanced if a molecule is attached, or in immediate proximity, to nanometer-roughened metal (Ag, Au, or Cu) surfaces. This effect, known as surface-enhanced Raman scattering (SERS), leads to Raman signal enhancements in the range of 103–106. Under certain conditions (at “hot spots” – closely spaced particles or rough nanostructures), enhancement factors up to 1011 (sufficient for single-molecule sensitivity) can be achieved. At least two effects contribute to the observed total enhancement. The electromagnetic enhancement effect is based on “localized surface plasmon resonance”, that takes place on the nanometer scale of SERS substrates, while chemical enhancement or “charge transfer”, is assumed to involve an electronic coupling between the adsorbed analyte and metallic substrate.