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Virtual fluorescent labeling of engineered vascular networks with embedded tracer particles
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DOI：10.1016/j.actbio.2026.04.059.png)
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Abstract
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Functional microvascular networks in engineered tissues depend on coordinated endothelial–stromal interactions and evolving extracellular matrix (ECM) mechanics, yet fluorescent staining precludes longitudinal studies and is incompatible with repeated particle-based microrheology and traction force measurements. We develop a deep-learning virtual labeling approach that recovers nuclear, cytoskeletal, and endothelial fluorescence from label-free images acquired in fibrin scaffolds. Human umbilical vein endothelial cells and lung fibroblasts were co-cultured in three-dimensional (3D) fibrin hydrogels containing 2μm silica microbeads that can be used to probe local matrix mechanics. Paired transmission and confocal fluorescence z-stacks (DAPI, phalloidin, UEA I) were used to train a 3D U-Net to generate virtual nuclei, actin, and vascular channels directly from bead-containing label-free volumes. To match channel-specific morphology, edge- and structure-preserving losses were assigned to phalloidin and UEA I, while a sparsity-aware loss was applied to DAPI, improving reconstruction quality across mean squared error, structural similarity, peak signal-to-noise ratio, and correlation. Virtual phalloidin preserved fibrillar density and orientation, virtual UEA I reproduced vessel continuity and density without false matrix labeling, and virtual DAPI enabled nuclei segmentation with Dice scores and total cell counts indistinguishable from ground truth (GT). Microbeads did not generate spurious signal in any channel, demonstrating robustness to scattering particles. Together, these results show that virtual fluorescent labeling can replace destructive staining for quantifying fibrillar architecture, vessel density, and cell number in bead-laden fibrin constructs, and establish a practical route toward longitudinal studies of coupled microvascular morphogenesis and ECM mechanics.
Keywords:
Deep learning
Virtual staining
Label-free microscopy
Vascular morphogenesis
Engineered tissues
3D U-net
Active microrheology
Extracellular matrix mechanics
Fibrin hydrogel
Endothelial cells
Confocal microscopy
Longitudinal imaging
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