Measuring Single-Cell Aging with an Imaging-based Biomarker of Chromatin and Epigenetic Aging.

To advance personalized healthcare and assess potential rejuvenation strategies, biomarkers reflecting biological age are crucial. We have introduced imaging-based chromatin and epigenetic age (ImAge), an imaging-based biomarker derived from the spatial organization of chromatin and epigenetic marks within single nuclei, capturing intrinsic age-related changes. ImAge effectively captures and quantifies the effects of interventions modulating biological age, showing sensitivity through significant decreases following rejuvenation strategies (such as caloric restriction and partial reprogramming through transient Oct4, Sox2, Klf4, and Myc (OSKM) expression) and increases after treatments like chemotherapy. Furthermore, lower ImAge correlated with higher locomotor activity, suggesting it captures functional improvements alongside biological age reversal, offering a promising single-cell tool to evaluate longevity interventions. As ImAge is an integrative method requiring good-quality imaging and meticulous computational analysis, we aim to provide thorough guidance to extract and validate it. This comprehensive protocol will detail the workflow for both wet laboratory procedures (sample preparation and imaging) and the proper utilization of our open-sourced computational pipeline (environment setup and processing of the imaging data) to extract and validate ImAge.