Automatic detection of fluorescent droplets for droplet digital PCR: a device capable of processing multiple microscope images†

Abstract

Droplet digital PCR (ddPCR) is recognized as a high-precision method for nucleic acid quantification, extensively utilized in biomedical research and clinical diagnostics. This technique employs microfluidic technology to partition the nucleic acid-containing reaction mixture into discrete droplets for amplification, achieving absolute quantification by identifying and enumerating the number of fluorescent droplets. The accuracy of droplet quantification is pivotal to the success of the assay. However, current image-processing tools are operationally complex, and commercial instruments are costly. Moreover, the designed algorithms exhibit a need for enhanced accuracy and are often restricted to use by trained personnel with specific microscopy equipment. In response to these challenges, we introduce an automated device (A-MMD), capable of detecting fluorescent droplets in ddPCR images captured by multiple microscopes. The device integrates three distinct algorithms tailored for the image processing of Laser Scanning Confocal Microscopy (LSCM), inverted microscopy, and self-assembled microscopy. Experimental validation using λ DNA demonstrated a 100.00% identification rate for positive droplets across all three image types, and the average identification rates for total droplets being 99.27% for LSCM, 98.96% for inverted microscopy, and 99.08% for self-assembled microscopy. Furthermore, the A-MMD is equipped with a user-friendly interface (UI) that streamlines the operational process, enabling non-specialists to efficiently perform droplet detection tasks. Our device not only has good environmental adaptability and identification accuracy, but also significantly reduces costs and operational complexity. It offers an economical, efficient, and user-friendly solution for ddPCR image analysis, thereby further propelling the advancement and application of nucleic acid detection technology.