Simple SummaryRoutine clinical approaches for cancer diagnosis demand fast, cost-efficient, and reliable methods, and the implementation of these methods within clinical settings. Currently, histopathology is the gold standard for tissue-based clinical diagnosis. Recently, spatially resolved molecular profiling techniques such as mass spectrometry imaging (MSI) or infrared spectroscopy imaging (IRI) have increasingly contributed to clinical research, e.g., by differentiating cancer subtypes using molecular fingerprints. However, the adoption of the corresponding workflows in clinical routines remains challenging, especially for fresh-frozen tissue specimens. Here, we present a novel device based on 3D-printing technology, which facilitates the sample preparation of needle biopsies for correlated clinical tissue analysis. It enables the use of a combination of MSI and IRI on fresh-frozen clinical samples, with a 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, facilitate more accurate diagnosis.AbstractThe complex molecular alterations that underlie cancer pathophysiology are studied in depth with omics methods using bulk tissue extracts. For spatially resolved tissue diagnostics using needle biopsy cores, however, histopathological analysis using stained FFPE tissue and the immunohistochemistry (IHC) of a few marker proteins is currently the main clinical focus. Today, spatial omics imaging using MSI or IRI is an emerging diagnostic technology for the identification and classification of various cancer types. However, to conserve tissue-specific metabolomic states, fast, reliable, and precise methods for the preparation of fresh-frozen (FF) tissue sections are crucial. Such methods are often incompatible with clinical practice, since spatial metabolomics and the 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 the multimodal spatial omics analysis of fresh-frozen, longitudinally sectioned needle biopsies to accompany standard FFPE histopathology of the same biopsy core. As a proof-of-concept, we analyzed surgical human liver cancer specimens using IRI and MSI with precise co-registration and, following FFPE processing, by sequential clinical pathology analysis of the same biopsy core. This workflow allowed for a spatial comparison between different spectral profiles and alterations in tissue histology, as well as a direct comparison for histological diagnosis without the need for an extra biopsy.