Raman microscopy is a powerful tool for analytical imaging. The wavelength shift of Raman scattering corresponds to molecular vibrational energy. Therefore, we can access rich chemical information, such as distribution, concentration, and chemical environment of sample molecules. Despite these strengths of Raman microscopy, the spatial resolution has been a limiting factor for many practical applications. In this study, we developed a large-area, high-resolution Raman microscope by utilizing structured illumination microscopy (SIM) to overcome the spatial resolution limit.
A structured line-illumination (SLI) Raman microscope was constructed. The structured illumination is introduced along the line direction by the interference of two line-shaped beams. In SIM, the spatial frequency mixing between structured illumination and Raman scattering from the sample allows access to the high spatial frequency information beyond the conventional cut-off. As a result, the FWHM of 40-nm fluorescence particle images showed a clear resolution enhancement in the line direction: 366 nm in LI and 199 nm in SLI microscope. Using the developed microscope, we successfully demonstrated high-resolution Raman imaging of various kinds of specimens, such as few-layer graphene, graphite, mouse brain tissue, and polymer nanoparticles.
The high resolution Raman images showed the capability to extract original spectral features from the mixed Raman spectra of a multi-component sample because of the enhanced spatial resolution, which is advantageous in observing complex spectral features. The Raman microscopy technique reported here enables us to see the detailed chemical structures of chemical, biological, and medical samples with a spatial resolution smaller than 200 nm.
Raman spectral imaging has become a more and more popular technique in biological studies because it can extract
chemical information from living cells in a label-free manner. One of the most challenging issues in the label-free
Raman imaging of biological samples is to increase the molecular specificity in the spectra for better chemical contrast.
Usually, the Raman spectrum from a cell is dominated by a few strong Raman bands such as the amide I band around
1650 cm<sup>-1</sup>, CH<sub>2</sub> bend around 1445 cm<sup>-1</sup> or the amide III band around 1300 cm<sup>-1</sup> and it is not easy to get chemical contrast from other Raman bands that overlap with them. In this study, we aim to manipulate the chemical contrast in a living cell by exploiting the polarisation effects in Raman spectroscopy. By simply putting an analyser before the spectrometer, we can take the Raman image at the parallel and perpendicular polarisation against the incident light at the sample. The Raman spectra at the two orthogonal polarisations represent the Raman signals with different molecular orientation and symmetry of vibrations. Our experimental results demonstrate that at certain Raman shifts the two orthogonal polarisations indeed present different chemical contrasts. This indicates that polarized Raman imaging can help us visualise the different chemical contrasts that overlap at the same Raman shift and therefore increase the amount of chemical information we can get from cells.