Metrology for Raman Spectroscopy

Identifying cancer proteins from biopsies, or pollutant chemicals in air samples are just two uses of Raman spectroscopy. This technique measures the light scattered by samples to identify their constituent molecules. Improved calibration methods are needed to enable concentrations of molecules to be accurately determined in healthcare, nanotechnology and forensic science.

The EMRP project Metrology for Raman Spectroscopy (Raman) developed measurement techniques and reference materials to provide a reliable calibration chain for Raman measurements, and developed modelling methods and real-time 3D imaging that now enable evaluation of the dynamic processes occurring in living cells.

The project:

• Developed a standardised Raman spectroscopy depth profiling procedure based on reference materials and used it to achieve the required sensitivity to detect very low target bio-molecule concentrations in samples.
• Developed a stimulated 3D Raman scattering microscope and associated software capable of rapidly determining the concentration of molecules such as in DNA, collagen, fat, and cytoplasm in label-free bio-samples from video images.
• Established a method using single-walled nanotubes for measuring sub-nanometre dimensions of Raman tips. This is important for precisely determining 2D distance measurements made in tip-enhanced Raman spectroscopy.

The methods developed in this project have made Raman spectroscopy a very attractive technique for label-free quantitative imaging of molecules in their native environments, and work has already begun to adapt Raman spectroscopy to new applications.

A video-rate Raman imaging microscope developed during this project, allows real-time images to be produced of chemical and biological processes in molecules within living cells. For instance, the facility is being used to cost effectively investigate UV damage caused to surrogate skin samples in assessing the stability of a sun-cream formulation and in studies of drug uptake into living cells.

The greatest barrier to the more widespread adoption of precise tip-enhanced Raman spectroscopy is the production of reliable gold-coated tips for use. The project evaluated tips and successfully implemented an improved production method that now produces quality tips in greater numbers than previously possible. Using tip-enhanced Raman spectroscopy, surface structure resolution is now possible below 100nm enabling generation of 3D information from sub-surface measurements.

Project developments will pave the way for Raman spectroscopy to be used in new research areas such as the identification of microbes in soil samples and exploring the application of nanotechnology in biological systems.