Understanding a Raman Spectrum

Interpreting a Raman spectrum can be a daunting task at first, so here we provide a close-up look at some of the key features of Raman spectra and highlight some of the key Raman peak assignments for Raman spectral imaging of cells and tissues.

1. Components of a Raman Spectrum
2. The Fingerprint Region
3. The High Wavenumber Region
4. The Silent Region
5. Raman Peak Assignments

1. Components of a Raman Spectrum

For biological samples such as cells and tissues, we think of the Raman spectrum as being composed of three distinct regions:

The Fingerprint region (600 - 1800 cm^-1)
The Silent region (1800 - 2800 cm^-1)
The High-wavenumber region (2800 - 3800 cm^-1)

Each of these regions of the Raman spectrum contains peaks with different biochemical information and together they can provide many insights into the underlying biology and chemistry of cells and tissues.

The position of a Raman peak gives information about the particular bond that is present. For bonds that are only found in a particular class of molecules or that are particularly abundant in a class of molecules, the presence of a Raman peak at a given Raman shift might indicate its presence in the sample under investigation.

The height of a Raman peak details the concentration of a particular bond (and hence potentially also the concentration of a particular class of molecules). By comparing normalised Raman spectra of two different samples acquired on the same system, the relative heights of Raman peaks can be used to perform a relative quantification of the amounts of different molecules present.

2. The Fingerprint Region

In most biological applications of Raman spectroscopy, the fingerprint regions provides the greatest wealth of biochemical information. Here we can find peaks that correspond to information about nucleic acids, proteins, and lipids, thus providing information about both the makeup and state of the cell or tissue under investigation.

3. The High Wavenumber Region

The high wavenumber region consists of two major peaks: the peak centred at ~2900 cm^-1 arises due to CH, CH2, and CH3 stretching and thus contains information about proteins and lipids, while the major peak at ~3400 cm^-1 occurs due to O-H stretching and thus gives information about the level of sample hydration and more.

4. The Silent Region

The silent region separates the fingerprint and high wavenumber regions of the Raman spectrum and gets its name due to the fact that no endogenous biomolecules display peaks in this region. However, far from having no use in biological Raman investigations, this region can be exploited using Raman active tags such as alkynes and deuterium that display signal in this region and can thus be used for highly specific Raman imaging.