Raman microscopy is a technique which interacts laser light of a particular wavelength with a target sample resulting in this light being scattered by the sample, the changes in energy of the scattered light is then measured. These changes in energy relate to vibrations from different molecules and produce a vibrational fingerprint of the sample relating to the molecular composition. When conducted using a microscope and a stage which moves, multiple Raman spectra in 2 and 3 dimensions can be acquired to produce an image of the sample based on the intensity and the location of particular vibrations within the sample. This is referred to as a Raman map and is very often a false colour map laid on top of a standard magnified microscope image of the sample, a white light image, e.g. a heat map of intensity of say a protein vibration overlaid on the image of a cell. Conventional Raman microscopy is normally in a confocal mode which means that the highest resolution in spatial terms is half the wavelength of the excitation light so typically around 250 nm. Biological structures and processes are on a much smaller scale and this is a limitation of Raman spectroscopy. An advantage of Raman spectroscopy is that it is label free and reliant on the specific molecular vibrations from the molecules in the interrogation volume, unlike fluorescence microscopy, which is the most commonly used form of optical microscopy in life sciences. However fluorescence microscopy requires addition of a label to the sample which changes the sample composition and can affect the intrinsic biological processes of a biological system. This proposal will produce a new tool to acquire Raman maps and then process the data to enhance the spatial resolution possible from a Raman confocal microscope. We propose to generate sub 100 nm spatial resolution using this tool which will greatly transform the use of Raman spectroscopy and microscopy in the life sciences. This tool will require no addition of labels or hardware modifications to existing Raman microscope instruments.
A new way of producing super resolution Raman images has been investigated and a possible way to achieve this discovered. How to best extend this to the community is currently under review.
|Effective start/end date||20/11/17 → 19/02/19|
- BBSRC (Biotech & Biological Sciences Research Council): £145,439.00