Position-Independent Particle Sizing in High-Precision Impedance Cytometry via Full-Waveform Feature Calibration.

Accurate particle characterization is critical for biomedical research, yet electrical impedance flow cytometry suffers from measurement inaccuracies due to the positional heterogeneity of particles within microfluidic channels. In large-scale microchannels, off-axis particle trajectories lead to significant waveform distortion and the attenuation of key characteristic features, undermining measurement precision. To address this issue, this study presents a novel signal-processing framework centered on a full-waveform calibration factor (CF). The CF is theoretically grounded in the exponential decay of the electric field along the vertical axis of coplanar electrodes and is derived from comprehensive waveform features rather than localized extrema, ensuring robustness against the distortions prevalent in high-flow conditions. Coupled with a dedicated real-time algorithm, it corrects for vertical position-induced errors in particle sizing. The effectiveness of this method was validated through a combination of finite element simulation and experimental impedance measurement. The system's performance is demonstrated by accurately discriminating polystyrene microsphere populations (10  μ m , 15  μ m , and their mixture) and by resolving the size distributions of viable versus non-viable HEK-293T cells. Statistical analysis ( N ≥ 500 events per group, p < 0.001 ) confirms significant improvement in size-distribution accuracy after calibration. Furthermore, this study designs an inverted microfluidic chip enabling real-time, optical sensing of particle stream height within the impedance detection zone and supporting position-independent impedance cytometry applicable to continuous-flow particle streams. Experimental calibration achieves a strong linear correlation ( R 2 = 0.970 ) between the sensor response and measured height, confirming the method's utility for spatially resolved impedance cytometry.