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

Abstract: Droplet microfluidics has been widely used to analyze chemicals and biological reactions at the single-molecule level. Current digital approaches use commercial microscope imaging systems, such as optical, fluorescence, and confocal microscopes, to obtain high-resolution images of droplets and software to extract quantitative data. Although microscopic imaging systems provide high-resolution images, they are expensive, only allow endpoint analysis, and have a narrow field of view when using high-magnification objective lenses, which limits the analysis of wide-field images or large quantities of samples, leading to longer experimental times and an increased demand for human resources. 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 represents a simple and cost-effective tool for obtaining wide-field images using a 50.6-megapixel CMOS image sensor-based DSLR camera and a macro lens. This platform uses dFinder software—a homemade droplet analysis tool for multiplexed and absolute quantification of numerous droplets. 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 μm, and the maximum detection area was 201.84 cm^2, suggesting that the system can theoretically analyze 3,103,377 droplets in real-time. We validated the capabilities of the platform in terms of multiplexing, high throughput, and detection accuracy through experimental comparisons with traditional technologies, including the microbroth dilution method and colony-forming unit assay. Then, the DropVIST was employed as a diagnostic approach for the real-time monitoring and quantification of living bacteria. Conventional diagnostic methods include microbial culture, disk diffusion, and identification using a microscopy system. Although these methods serve as standard techniques for detecting bacteria, those methods are time-consuming, labor-intensive, and require complex experimental procedures. To improve the mortality rate, rapid and accurate diagnostic approaches are demanded for precise treatment for the patient. Conventional methods face challenges in analyzing bacteria at concentrations of ≤10^5 cells/mL within 6 h, whereas DropVIST identifies bacteria at concentrations of ~10^2 cells/mL within 6 h when DropVIST was validated with five types of clinically isolated bacteria. This platform provides a rapid, simple, and accurate tool for the real-time monitoring and quantification of living bacteria in a single-cell manner.