Multimodal Nanoscale Imaging Reveals Cellular and Metabolic Complexity of Human Breast Milk

Abstract Human milk contains diverse live cells that contribute to infant nutrition, immune protection, and maternal-infant health, yet their cellular biology remains difficult to resolve within this lipid-rich and heterogeneous biofluid. Here, we adapted correlative light, electron, and ion microscopy (CLEIM) for nanoscale imaging and metabolic profiling of human milk cells (HMCs) and integrated these spatial measurements with single-cell transcriptomic analysis. This workflow combines confocal microscopy for cell-type identification, electron microscopy (EM) for ultrastructural mapping, nanoscale secondary ion mass spectrometry (NanoSIMS) for spatial elemental and isotope analysis, and single-cell RNA sequencing (scRNA-seq) for transcriptional interpretation. Using this platform, we generated an ultrastructural atlas of HMCs, including lactocytes, neutrophils, macrophages, dendritic cells, and bacteria-like structures. NanoSIMS revealed sulfur- and iron-enriched neutrophil granules, consistent with antimicrobial functions. Stable isotope tracing further uncovered distinct metabolic specializations among HMCs: 13C-glucose-derived carbon was preferentially incorporated into macrophage lipid droplets, supported by lipid-handling transcriptional features, whereas 15N-amino acid tracing identified metabolically active lactocyte subsets associated with translation and secretory programs. These findings reveal cellular and metabolic heterogeneity in human milk that is not captured by conventional profiling methods. More broadly, this work establishes a spatial multimodal framework for linking cell identity, ultrastructure, metabolic activity, and transcriptional state in complex biological fluids, providing a foundation for future studies of human milk biology and maternal-infant health.