What is Raman Spectroscopy?

Raman spectroscopy is an optical spectroscopic technique that can be used to produce a fingerprint spectrum of a sample on the basis of the chemical bonds present. While the underlying mechanisms are different, much of the equipment required for Raman spectroscopic imaging is similar to that for conventional imaging techniques such as confocal fluorescence microscopy.

1. Raman Scattering
2. Raman Spectra
3. Biological and Clinical Applications of Raman Spectroscopy
4. Raman Spectroscopy Equipment

1. Raman Scattering

When incident light interacts with a sample, one of four possible outcomes can occur:

The light is transmitted through the sample
The light is absorbed by the sample and later re-emitted as fluorescence
The light is elastically scattered (Rayleigh scattering, photon energy is conserved)
The light is inelastically scattered (Raman scattering, photon energy is not conserved)

Figure 1 - Absoprtion and scattering processes. On the left hand side is a schematic of the different scattering processes that can occur, while on the right hand side is a Jablonski diagram detailing the energy transitions that occur for both elastic and inelastic scattering events.

The Raman scattering signal is due to the interaction of the incident light with a vibrating molecule, which results in a change in the polarizability of the molecule. The resulting change in energy of the incident photon is thus linked to the vibrational modes of polarizable molecules, which acts to provide a fingerprint spectrum detailing the chemical composition of the sample.
Raman scattering is a relatively rare scattering event, occurring for only 1 in ~10^8 scattering events and thus requires both sensitive detectors and long acquisition times to generate sufficient signal. This also means that for fluorescent samples, fluorescent signal will quickly swamp any Raman signal generated, which is a key consideration when applying Raman spectroscopy to auto-fluorescent samples such as biological tissues.

2. Raman Spectra

Upon illumination of a sample by incident laser light, Raman (inelastically) scattered light is collected and passed to a spectrometer. The spectrometer disperses the light according to its wavelength onto a camera of a charged coupled device, to produce a spectrum. Thus the Raman spectrum provides information about the different bonds that are present in the sample, which appear as peaks at different points along the Raman spectrum (Figure 2). By comparing the positions of the different peaks to the Raman spectra of pure compounds, we can start to build information as to the chemical content of complex samples such as cells and tissues.

Figure 2 - An example Raman spectrum of a cell, acquired using a 532 nm wavelength laser at ~35 mW with a 1 second acquisition time. On the right hand side is a magnified view of the fingerprint region (500 - 1800 cm^-1), with a few key Raman spectral peaks highlighted.

In addition to providing information about what molecules are present in a sample, Raman spectroscopy can be used to quantify how much of a given molecule is present in a sample. This is because the height of a Raman peak is linearly proportional to the concentration of specific molecules.

Perhaps most importantly in the context of biological samples is the spectral separation of the water peak (3000 - 3600 cm^-1) from the fingerprint region (600 - 1800 cm^-1), which enables Raman spectroscopy to be performed on hydrated samples such as live cells and tissues.

For a more comprehensive discussion on Raman spectra, please see: Understanding a Raman Spectrum

3. Biological and Clinical Applications of Raman Spectroscopy

Raman spectroscopy is well suited to the study of biological samples such as cells and tissues for several reasons:

It provides detailed biochemical information
It does not require sample processing
It can be performed label-free
It can be performed on hydrated samples
It is capable of live imaging

As such, Raman spectroscopy has been extensively applied for in vitro studies of fixed and living cells, ex vivo studies of tissues, as well as for in vivo cancer diagnostic applications.

4. Raman Spectroscopy Equipment

While Raman spectroscopy may at first appear complex, the good news is that much of the equipment required to perform Raman spectroscopy is similar to that for a confocal fluorescence microscope. Indeed, there are several commercial Raman spectroscopy systems available, and here we have provided detailed instructions for the assembly of your own, custom Raman microscope along with open source software (implemented in MATLAB) for control of this system: see Hardware and Software.

Briefly, the basic equipment required for Raman spectroscopic imaging is as follows:

Computer
Laser source
Spectrometer
Modular Microscope
Objective
Optical fibres