Integration of wide-field imaging system with droplet microfluidics for monitoring living bacteria

Droplet microfluidics has been widely used to analyze chemicals and biological reactions at the single-molecule level. Microscopic systems are commonly used for imaging and analyzing droplets using high-magnification objective lenses. However, these systems are expensive, complex, and large, limiting the high-throughput characterization of droplets in reactions targeting disease-related biomarkers, including nucleic acids, proteins, and pathogens. Another concern is the time gap between droplet analysis within a microfluidic channel and imaging chamber, which can cause discrepancies in reaction time between droplets. To address this issue, we introduce the droplet analysis system for vast imaging and statistical tool (DropVIST)-a wide-field imaging system integrated with droplet microfluidics for rapid and accurate droplet analysis. DropVIST can detect eight differently colored droplets simultaneously using a wide-field imaging system and dFinder software, which can quantify the reacted droplets. The smallest droplet diameter for detection was 30 mu m, and the maximum detection area was 201.84 cm2, suggesting that the system can theoretically analyze 3103,377 droplets in realtime. We validated the monitoring performance of DropVIST using clinical isolates of five types of living pathogenic bacteria and compared this with conventional approaches, including the microbroth dilution method and colony-forming unit assay. This platform provides a rapid, simple, and accurate tool for monitoring living bacteria at the single-cell level.