Code-multiplexed multi-frequency impedance cytometry with a unified deep-unfolding network

Impedance flow cytometry (IFC) is a label-free, single-cell measurement technique that captures biophysical properties beyond traditional biochemical markers. Code-multiplexing allows parallelization of IFC with simple hardware but requires advanced signal processing algorithms to resolve overlaps in signals originating from different channels. Existing methods, however, rely on multiple task-specific networks with template-based linear fitting, which loses accuracy under nonlinear or unstable conditions common in microfluidic experiments. Prior studies have also been restricted to demultiplexing single-frequency impedance measurements. To this end, we develop a unified deep-unfolding network for analyzing code-multiplexed, multi-frequency IFC data. We unfold the successive-interference cancellation (SIC) algorithm into a deep-learning network, where repeated stages of a single multitask network implement iterative signal estimation and interference cancellation that reflect the structural prior of SIC. To mitigate nonlinear signal stretching and amplification, our network recognizes events by predicting bit-level intensity and duration. For multi-frequency impedance profiling, we apply least-squares fitting to the predicted single-frequency real-impedance trace to map the real and imaginary impedance traces at other frequencies. On the cell-bead mixture evaluation dataset, our pipeline resolves overlaps from singlets to triplets reliably, reconstructs impedance-intensity distributions accurately, and enables multi-frequency impedance profiling. As a demonstration of principle, we use our pipeline to perform label-free quantification of basophil activation from code-multiplexed, multi-frequency IFC measurements. Consistent with our previous study, impedance opacity correlates well with activation levels measured by fluorescence flow cytometry. In summary, our study demonstrates the feasibility and utility of a deep-unfolding network that extends code-multiplexed, multi-frequency IFC to label-free single-cell functional assays.