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.

中文翻译：

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用于液滴数字 PCR 的荧光液滴自动检测：一种能够处理多个显微镜图像的设备


微滴式数字 PCR （ddPCR） 是公认的高精度核酸定量方法，广泛用于生物医学研究和临床诊断。该技术采用微流控技术将含核酸的反应混合物分成离散的液滴进行扩增，通过识别和计数荧光液滴的数量来实现绝对定量。液滴定量的准确性对于检测成功至关重要。然而，目前的图像处理工具操作复杂，商用仪器成本高昂。此外，所设计的算法表现出对提高准确性的需求，并且通常仅限于由具有特定显微镜设备的受过培训的人员使用。为了应对这些挑战，我们推出了一种自动化设备 （A-MMD），能够检测多台显微镜捕获的 ddPCR 图像中的荧光液滴。该设备集成了三种不同的算法，专为激光扫描共聚焦显微镜 （LSCM）、倒置显微镜和自组装显微镜的图像处理而量身定制。使用 λ DNA 的实验验证表明，在所有三种图像类型中，阳性液滴的鉴定率为 100.00%，LSCM 总液滴的平均鉴定率为 99.27%，倒置显微镜为 98.96%，自组装显微镜为 99.08%。此外，A-MMD 配备了用户友好的界面 （UI），可简化操作过程，使非专业人员能够有效地执行液滴检测任务。我们的设备不仅具有良好的环境适应性和识别准确性，而且还显著降低了成本和操作复杂性。 它为 ddPCR 图像分析提供了一种经济、高效和用户友好的解决方案，从而进一步推动了核酸检测技术的进步和应用。