Bioorthogonal Raman and IR probes for live cell metabolomics: A library

Vibrational techniques using bioorthogonal Raman and infrared (IR) probes that emit unique spectral signals in the silent region (2800–1800 cm⁻¹) have gained considerable attention across various fields of biomedicine. Labeled Raman and infrared microscopies are proving to be highly beneficial in studying the dynamics of metabolic processes within single living cells. Disruption or deregulation of these systems leads to a range of diseases and makes understanding the biochemistry of metabolism one of the greatest challenges in biology. In this study, we built a library of Raman and IR probes to assess metabolic changes, categorizing them into four groups related to glucose and lipid metabolism, as well as the activity of the cell nucleus and mitochondria. We highlight the effectiveness of vibrational labeling in three key areas: tracking specific metabolic pathways, multiplex imaging, and multimodal imaging. Single-pathway analysis was achieved using selected molecular probes (e.g., azido-palmitic acid, EdU, d₇-glucose, MitoBADY), each targeting different metabolic pathways. Multiplex imaging allowed for the simultaneous tracking of lipid and mitochondrial metabolism, providing a more comprehensive view of the cell's biomolecular state. Furthermore, a multimodal approach, which combined Raman and optical photothermal infrared (O-PTIR) techniques, enabled the complementary characterization of endothelial cells labeled with azide- and deuterium-palmitic acid. Both high-resolution imaging techniques and microscopy facilitated subcellular analysis in living and fixed cells, also in aqueous environments. These findings underscore the value of integrating vibrational labeling with advanced cell imaging to expand the horizons of metabolomics research.