Improved tissue preparation for multimodal vibrational imaging of biological tissues

Abstract

The complementary nature of Infrared (IR) and Raman spectroscopies enables a thorough understanding of biological tissue – so called multimodal vibrational spectroscopic imaging. However, new approaches in terms of sample preparation and data analysis are required to release the full potential of multimodal spectroscopy. Herein, we propose an inexpensive and relatively simple sample preparation technique incorporating mirror-finished stainless-steel slides and polyethylene glycol as an embedding medium that is compatible for both infrared and Raman spectroscopy of tissue sections. K-Means Clustering and Principal Component Analysis (PCA) were used to evaluate the performance of multimodal vibrational spectroscopic imaging compared with IR and Raman spectroscopic imaging individually using a rat kidney as a model. The K-Means cluster maps generated with the multimodal dataset showed the best correlation between different tissue types identified by an adjacent section stained with Masson’s Trichrome compared to either Raman or IR spectroscopy analysed independently. PCA score maps of the multimodal dataset produced a clear separation of individual tissue types along the first three Principal Components. Additionally, PCA permitted the correlation of IR and Raman peaks arising mainly from collagen vibrational modes. Finally, polyethylene glycol embedding is shown as an attractive alternative to paraffin embedding for spectroscopic analyses, due to significantly less fluorescence in Raman measurements and retention of lipids in the tissue, without any retention of the medium within the tissue.