Fluorescence imaging underpins digital PCR (dPCR), microarrays, and microfluidic biosensors, yet precise image integration remains a technical bottleneck when the sample area exceeds the microscope field of view. Current stitching methods often rely on fiducial markers or manual tuning, limiting automation and robustness, particularly in portable or point-of-care devices. We present a marker-free image stitching algorithm that combines partition-detection-based registration with mask-based illumination correction. The algorithm aligns frames using intrinsic structural features and compensates for brightness inconsistencies in an adaptive manner, without requiring platform-specific parameter tuning. Application to three dPCR systems, including droplet- and chip-based formats, showed an increased number of matched feature points within overlapping regions, improving the reliability of image stitching. In addition, it enhanced intensity uniformity by ≈ 29.6% compared with conventional methods. The proposed algorithm was further validated on microarrays and bead-based chips, demonstrating consistent stitching accuracy and signal integrity across different modalities. This generalized and automation-compatible solution supports high-throughput microfluidic imaging, quantitative bioanalysis, and integration with artificial intelligence-enabled diagnostic workflows.

Bioinspired Engineering and Biomechanics Center Xi'an Jiaotong University Xi'an 710049 P R China

Department of Microelectronics Faculty of Electrical Engineering and Communication Brno University of Technology Technická 3058 10 Brno 616 00 Czech Republic

Ministry of Education Key Laboratory of Micro and Nano Systems for Aerospace School of Mechanical Engineering Northwestern Polytechnical University 127 West Youyi Road Xi'an Shaanxi 710072 P R China

School of Marine Science and Technology Northwestern Polytechnical University Xi'an Shaanxi 710072 P R China

State Industry Education Integration Center for Medical Innovations at Xi'an Jiaotong University P R China

The Key Laboratory of Biomedical Information Engineering of the Ministry of Education School of Life Science and Technology Xi'an Jiaotong University Xi'an Shaanxi 710049 P R China

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