Correlative multimodal imaging of tissue structure, metabolome and elemental distribution using stimulated Raman scattering, desorption electrospray ionisation and MeV ion beam analysis.

BACKGROUND: Combining imaging modalities at high spatial resolution is needed to gain a comprehensive understanding of disease pathogenesis and therapeutics. Specifically, it is desirable to perform label free imaging of lipids, proteins and elements at sub-micron spatial resolution and to detect small molecules with high chemical specificity. This work addresses this challenge by developing a workflow for carrying out stimulated Raman scattering (SRS) microscopy, desorption electrospray ionisation (DESI) and MeV ion beam analysis (IBA) on the same tissue section.

RESULTS: The first challenge was to find a substrate compatible with all three imaging modalities. It was found that PET membranes can be used to image tissues using all three techniques. Next, a strategy for performing the techniques in sequence was developed. It was found that prior SRS analysis has no detectable effect on subsequent DESI or PIXE imaging, and that DESI does not delocalise elements in skin tissue; therefore the techniques can be performed on the same section of tissue in the order SRS < DESI < PIXE. This work also shows that a methanol:ethanol DESI spray solvent can be used to detect biologically relevant lipids in negative ion mode.

SIGNIFICANCE: The compatibility of SRS with PET-mounted tissues opens the possibility of sequential analysis using laser capture microdissection or other X-ray spectrometry techniques. Fresh frozen porcine skin was used to highlight the ability to correlate structural, chemical and elemental information, highlighting the co-localisation of lipid hotspots (SRS), with chemical characterisation from DESI and calcium deposits (PIXE) in follicular structures.