Fluorescence crosstalk reduction by modulated excitation-synchronous acquisition for multispectral analysis in high-throughput droplet microfluidics

Crosstalk between fluorescent biomarkers significantly limits the resolution of multispectral fluorescence analysis in real-time droplet-microfluidics applications. The crosstalk is a result of overlapping emission and excitation spectra of different fluorophores in multiplexed analyses. To mitigate this crosstalk, we present a method that modulates multiple laser beams to selectively and sequentially excite the fluorophores by a single beam of a particular wavelength using acousto-optic modulators at a frequency of 0.1 MHz. An FPGA based data acquisition algorithm synchronized with the modulation signal then acquires the emission signals only from the fluorescence channel that corresponds to the excitation wavelength provided in that particular time window. We applied our method for fluorescence-based droplet analysis in microfluidics and demonstrate that the method is able to reduce crosstalk contribution between channels by >97% and can resolve fluorescence populations that are indistinguishable with conventional droplet analysis methods.