Multimodal Molecular Imaging combining Raman Spectroscopy and Mass Spectrometry for the study of biological samples

Biological samples such as tissues, cells or microbial communities including biofilms are intricate molecular systems. Their complexity is mainly expressed by the heterogeneity of their microstructure, both in terms of spatial molecular distribution and molecular composition. Therefore, the analysis of biological samples requires analytical techniques able to visualise the distribution of the molecular components of the sample with a high spatial resolution (down to the single cell level) and with high specificity needed to resolve the complex molecular mixture. However, to date, no single analytical technique used in isolation can fulfil all the required criteria for an optimal imaging analysis of biological samples. Thus, in order to maximise the molecular information and to overcome the limitations inherent to an individual technique, several complementary imaging techniques can be combined in a multimodal molecular imaging approach. In this context, the combination of Raman Spectroscopy and Laser Desorption/Ionisation Mass Spectrometry imaging modalities represents a promising avenue for the study of various biological samples as these techniques respectively offer high spatial resolution and high specificity. Here we show the application of Surface-Enhanced Raman Spectroscopy (SERS) Imaging and Surface-Assisted Laser Desorption/Ionisation Mass Spectrometry (SALDI-MS) Imaging on selected biological samples, namely bacterial biofilms. Both techniques employ nanomaterials to improve the performances of the respective “classical” counterpart technique. In SERS, the deposition of metallic nanoparticles on the sample surface greatly increases the sensitivity by amplifying the Raman signal and leads to the quenching of the sample autofluorescence, which are major drawbacks of Raman Spectroscopy. On the other hand, contrasting with the traditional well-known MALDI-MS, SALDI-MS is a novel technique employing nanostructured substrates instead of organic matrices to promote the desorption and ionisation of the analytes. SALDI-MS represents an interesting alternative to MALDI-MS as the use of nanosubstrates allows to get around the matrix-related issues encountered in MALDI-MS and which can be really problematic during the analysis of metabolites. We showed that SALDI-MS is particularly effective for the analysis of small molecules in both ion modes (negative and positive) with the same nanosubstrates and that the deposition of nanosubstrates instead of an organic matrix provides visually better images with increasing spatial resolution.