Complex molecular alterations underlying cancer pathophysiology are intensely studied with omics methods using bulk tissue extracts. For spatially resolved tissue diagnostics using needle biopsy cores, however, histopathological analysis using stained FFPE tissue and immuno-histochemistry (IHC) of few marker proteins is currently the main clinical focus. Today, spatial omics imaging using MSI or IRI are emerging diagnostic technologies for identification and classification of various cancer types. However, to conserve tissue-specific metabolomic states, fast, reliable and precise methods for preparation of fresh-frozen (FF) tissue sections are crucial. Such methods are often incompatible with clinical practice, since spatial metabolomics and routine histopathology of needle biopsies currently require two biopsies for FF and FFPE sampling, respectively. Therefore, we developed a device and corresponding laboratory and computational workflows for multimodal spatial omics analysis of fresh-frozen, longitudinally sectioned needle biopsies to accompany standard FFPE histopathology on the same biopsy core. As proof-of-concept, we analyzed surgical human liver cancer specimen by IRI and MSI with precise co-registration and, following FFPE processing, by sequential clinical pathology analysis on the same biopsy core. This workflow allowed spatial comparison between different spectral profiles and alterations in tissue histology, as well as direct comparison to histological diagnosis without the need of an extra biopsy.SIMPLE SUMMARYRoutine clinical approaches for cancer diagnosis demand fast, cost-efficient, and reliable methods, and their implementation within clinical settings. Currently, histopathology is the golden standard for tissue-based clinical diagnosis. Recently, spatially resolved molecular profiling techniques like mass spectrometry imaging (MSI) or infrared spectroscopy imaging (IRI) have increasingly contributed to clinical research, e.g., by differentiation of cancer subtypes using molecular fingerprints. However, adoption of the corresponding workflows in clinical routine remains challenging, especially for fresh-frozen tissue specimen. Here, we present a novel device based on 3D-printing technology, which facilitates sample preparation of needle biopsies for correlated clinical tissue analysis. It enables combination of MSI and IRI on fresh-frozen clinical samples with histopathological examination of the same needle core after formalin-fixation and paraffin-embedding (FFPE). This device and workflow can pave the way for a more profound understanding of biomolecular processes in cancer and, thus, aid more accurate diagnosis.Abstract Figure