Fix or freeze? Spectral differences arising from tissue preparation in chemical imaging

Abstract Spectrochemical imaging has emerged as a powerful, label-free modality for visualizing the bio-chemical composition of tissues based on intrinsic vibrational signatures. Specifically, mid-infrared spectrochemical imaging (MIRSI) is becoming essential for fundamental biomedical research studying disease mechanisms, identifying biomarkers, and guiding drug development. However, the sensitivity of MIRSI to sample preparation protocols and its impact on spectral data interpretation remain poorly characterized. Here, we systematically compared spectral data collected from rat kidney and liver tissues prepared using standard fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissue processing methods using quantum cascade laser (QCL)–based MIRSI. We applied frequently used spectral data processing techniques, including uniform manifold approximation and projection (UMAP), correlation matrices, and second-derivative spectral analysis, to characterize preparation-induced differences. FF samples preserved a broader range of biochemical signals, retaining the innate chemical composition of tissues, while FFPE tissues showed reduced spectral diversity and absorption signal intensity. Moreover, a unique spectral band at 1026 cm −1 , likely arising from formalin-induced collagen crosslinking, was identified specifically in FFPE samples. Our findings demonstrate that tissue preparation substantially alters chemical and morphological information captured by MIRSI, necessitating careful consideration of processing protocols in workflows involving chemical imaging and Artificial Intelligence (AI) - based spectral analysis.