Spatial Encoding with Amplitude Modulation in Serial Flow Cytometry

Serial flow cytometry was recently introduced as a method that can estimate measurement uncertainty (i.e., imprecision, the coefficient of variation of repeated measurements of individual particles) independent from population characteristics. Replication of light sources and detectors at multiple sites along a flow cytometer's microchannel requires more equipment and can complicate detector synchronization. Here, we introduce amplitude modulation to encode each region of a serial cytometer with a unique carrier frequency, which enables demultiplexing of the combined signal incident on a single photodetector by fast Fourier transform (FFT) peak magnitude. To facilitate validation of detection, matching, and uncertainty quantification of fluorescence signals, we designed a microfluidic amplitude modulation (AM) serial flow cytometer that has ground truth detectors on individual regions (serial cytometry) in parallel with the combined channel detection for AM demultiplexing. With this report, we present metrics for event detection and dynamic range, prevalence and processing of overlapping detections, region-decoding accuracy, process yield, and uncertainty quantification on a brightness ladder of calibration microspheres. Despite being operated with reduced light intensities, the AM cytometer was capable of high-fidelity performance in comparison to conventional serial cytometry. For events above the detection limit, over 97% were analyzed. Both conventional and AM serial cytometers achieved median imprecisions in the range of 0.53% to 2.1% after outlier removal, which was well below the inherent intensity distribution of any of the microsphere subpopulations. Overall, AM cytometry supports uncertainty quantification and temporal analyses of serial cytometry data with a reduced number of photodetectors, which offers simplification of chip design with multiple measurement regions and wide-field detectors.