Dr. Lorenzo Scipioni - ESPRESSO: Spatiotemporal Omics Based on Organelle Phenotyping Reveals Phenotypic Transitions

Dr. Lorenzo Scipioni - ESPRESSO: Spatiotemporal Omics Based on Organelle Phenotyping Reveals Phenotypic Transitions

Lorenzo Scipioni
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Centre de Recherche en Cancérologie de Toulouse

ESPRESSO: Spatiotemporal Omics Based on Organelle Phenotyping Reveals Phenotypic Transitions

Omics technologies, while fundamentally revolutionizing our molecular understanding of biological systems, are inherently limited in their capacity to fully capture the complexity of life. These bulk or even single-cell snapshot methodologies often fail to account for the crucial interplay of time and space within the living cell, primarily due to the impossibility of following the dynamic state transition of individual cells as a function of time. This limitation means that critical, transient cellular events—such as the immediate response to a drug or the subtle shifts during differentiation—are often averaged out or missed entirely, preventing a complete mechanistic understanding of cellular decisions. To bridge this critical gap between the static molecular profile and the dynamic functional reality, we introduce ESPRESSO (Environmental Sensor Phenotyping RElayed by Subcellular Structures and Organelles), a novel and powerful technique designed to achieve high-dimensional phenotyping resolved in both space and time at the single-cell level.
By quantifying the dynamic reorganization of the subcellular landscape, ESPRESSO allows us to unravel cellular heterogeneity with unprecedented resolution, moving beyond simple molecular categories to identify distinct functional subpopulations. This capability is paramount for unveiling specific stress and drug response pathways, as organelle reorganization often precedes major transcriptional changes, providing an earlier and more nuanced understanding of cellular fate. Furthermore, the temporal resolution allows us to precisely trace differentiation trajectories in complex cultures, observing the continuous path rather than jumping between discrete endpoints. Ultimately, ESPRESSO provides a crucial, missing bridge between molecular omics and the active physical state of the cell, providing the context necessary to link genomic potential to realized function. Here, we will demonstrate the broad applicability of this methodology by presenting ESPRESSO applied to several critical biological and translational contexts, including the study of phenotypic transitions in stress response, keratinocyte differentiation, macrophage polarization and dissecting drug response in heterogeneous breast cancer tumor spheroids. These examples highlight the method's unparalleled power in characterizing dynamic cellular behaviors relevant to disease and development.

Selected references

1\. Lorenzo Scipioni, Giulia Tedeschi, Mariana Navarro, Yunlong Jia, Scott Atwood, Jennifer A. Prescher, Michelle Digman (2025) ESPRESSO: Spatiotemporal omics based on organelle phenotyping. Nature Methods 22, 2349–2361 (2025)
2\. Giulia Tedeschi, Francesco Palomba, Lorenzo Scipioni\, Michelle A. Digman\ (2025) Multimodal Phasor Approach to Study Breast Cancer Cell Invasion in a 3D Spheroid Model. Chem. Biomed. Imaging 3, 7, 433–442 (2025)
3\. Scipioni, Lorenzo, Rossetta, A., Tedeschi, G., & Gratton, E. (2021). Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging. Nature Methods 18(5), 542–550 (2021)
4\. Yao\, Z., Brennan\, C. K., Scipioni\*, L., Chen, H., Ng, K. K., Tedeschi, G., … Others. Multiplexed bioluminescence microscopy via phasor analysis. Nature Methods, 19(7), 893–898 (2022)