Fabrication and characterization 8-nm-sized conjugated polymer nanoparticles (CPNs) and two-photon (2P) imaging of CPN labeled endothelial cells in a collagen-gel-based microfluidic device is described. CPNs exhibit super brightness and photostability comparable to quantum dots. The hydrophilicity and non-toxicity of CPNs enable long-term monitoring of cells in a tissue model, supporting CPNs' potential in biological and biomedical applications.