BPS2026 – ESPRESSO: Spatiotemporal omics based on organelle phenotyping reveals dynamic cell state transitions

Omics technologies, while revolutionizing our molecular understanding of life, often fail to capture the crucial interplay of time and space within the living cell due to the impossibility to follow the state transition of individual cells as a function of time. We present environmental sensor phenotyping relayed by subcellular structures and organelles (ESPRESSO), a novel technique that achieves high-dimensional phenotyping resolved in both space and time at the single-cell level by leveraging morphological and functional information relative to the organelle network. ESPRESSO combines live-cell staining with organelle-specific, environment-sensitive probes, employs confocal hyperspectral imaging and spectral phasor unmixing to resolve multiple, overlapping signals simultaneously, enabling high-speed temporal acquisition. This allows us to quantitatively extract a large feature set of a dimensionality comparable to single-cell RNA sequencing, including the morphological and functional properties (such as mitochondrial membrane potential or lysosomal pH), generating an organelle-based omics signature that reflects the cell’s integrated biophysical state. This powerful framework allows us to unravel cellular heterogeneity, unveil distinct stress and drug response pathways based on dynamic organelle reorganization, and trace differentiation trajectories in complex cultures, thus providing a crucial bridge between molecular omics and the active physical state of the cell. Here, we will present ESPRESSO applied to the phenotypic transition in the contexts of stress response, keratinocyte differentiation, macrophage polarization and drug response in breast cancer tumor spheroids, highlighting the power and broad applicability of this methodology.