Establishing a standard workflow for fluorescence-based nanoparticle tracking analysis for the reliable...

Fluorescence-based nanoparticle tracking analysis (Fl.NTA) enables selective detection and quantification of labelled nanoparticles. However, the lack of a standardized workflow has hindered the reproducibility and comparability of concentration measurements across different studies. Here, we established a comprehensive Fl.NTA protocol using fluorescent polystyrene bead standards (Fluoresbrite 50/100 nm; FluoSpheres 100/200 nm). We systematically optimized the standardized workflow by aligning excitation modules and long-pass emission filters with fluorophore spectra, and by rigorously defining key acquisition and processing parameters, such as camera level, camera shutter, and detection threshold. To ensure reliable quantification, the proposed protocol was validated through day-to-day repeatability tests and an intra-laboratory comparison involving five independent analysts. Our results demonstrate that while particle sizing remains robust (inter-analyst CVs 0.0-8.0%), concentration recovery is highly sensitive to optical configurations and signal detectability, particularly for sub-100 nm particles. The workflow was further applied to biological extracellular vesicles (EVs), including CD63-EGFP and CD9-QD625 labelled EVs. The significant variance in Fl:Sc concentration ratios (13.8% for EGFP vs. 124.5% for QD625) underscores the necessity of considering fluorophore photostability and labelling efficiency for reliable quantification. Overall, this study provides a practical, performance-driven standardized workflow for Fl.NTA that addresses existing operational gaps in ISO 19430:2024 and provides a practical foundation for the consistent and reproducible characterization of heterogeneous bio-nanoparticles in EV research and quality control.