In-incubator continuous and endpoint monitoring of angiogenesis using a chip-sized holographic microscope.

Abstract

Continuous monitoring of cell-to-cell interactions and multicellular network dynamics is essential for in situ biosensing, drug screening and cell-based assays. However, conventional live-cell imaging relies on benchtop optical microscopes placed outside the incubator, which limits throughput and makes long-term, minimally perturbative monitoring difficult. Here, we present a chip-sized, lensless digital holographic microscope (CSM) designed for compact, scalable, low-cost and in-incubator imaging of live-cell assays. The CSM combines a CMOS sensor and a micro-LED array in an inline holographic configuration, enabling wide-field, label-free imaging directly inside a standard CO₂ incubator. The CSM was validated using a Vascular Endothelial Growth Factor (VEGF)-induced tube formation assay with primary human umbilical vein endothelial cells (HUVECs). Quantitative network metrics (total tube length, number of junctions and meshes, and mean mesh area) are extracted from numerically reconstructed images and are shown to capture dose-dependent effects of VEGF. At 24 h, the CSM resolved significant increases in tube length and junction count at 20-40 ng/mL VEGF-165, followed by a marked regression at 50 ng/mL, and achieved a morphological detection limit of 1383 μm² in mesh area with a corresponding morphological dynamic range of 1643. Time-lapse imaging every 30 min further resolves the full temporal evolution of tube formation and regression, revealing kinetic differences that are not accessible from endpoint measurements alone. These results establish the CSM as a versatile platform for quantitative, in-incubator monitoring of angiogenesis assays, bridging continuous imaging and endpoint read-outs.