Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows.

The landscape of tissue-based imaging modalities is constantly and rapidly evolving. While formalin-fixed, paraffin-embedded material is still useful for histological imaging, the fixation process irreversibly changes the molecular composition of the sample. Therefore, many imaging approaches require fresh-frozen material to get meaningful results. This is particularly true for molecular imaging techniques such as mass spectrometry imaging, which are widely used to probe the spatial arrangement of the tissue metabolome. As high-quality fresh-frozen tissues are limited in their availability, any sample preparation workflow they are subjected to needs to ensure morphological and molecular preservation of the tissues and be compatible with as many of the established and emerging imaging techniques as possible to obtain the maximum possible insights from the tissues. Here we describe a universal sample preparation workflow, from the initial step of freezing the tissues to the cold embedding in a new hydroxypropyl methylcellulose/polyvinylpyrrolidone-enriched hydrogel and the generation of thin tissue sections for analysis. Moreover, we highlight the optimized storage conditions that limit molecular and morphological degradation of the sections. The protocol is compatible with human and plant tissues and can be easily adapted for the preparation of alternative sample formats (e.g., three-dimensional cell cultures). The integrated workflow is universally compatible with histological tissue analysis, mass spectrometry imaging and imaging mass cytometry, as well as spatial proteomic, genomic and transcriptomic tissue analysis. The protocol can be completed within 4 h and requires minimal prior experience in the preparation of tissue samples for multimodal imaging experiments.