The invention relates to a droplet dividing and clustering method, device, medium and program, the method is applied to a droplet-based digital
nucleic acid amplification quantitative analysis
system, and the method comprises the following steps: reading scatter data of each droplet in a droplet
fluorescence image, including position and brightness; the microdroplet
fluorescence image is divided into a plurality of grids, all the grids are sequenced and numbered, all microdroplets in each grid form a sub-array, and the sub-arrays are connected to form a microdroplet array; mapping the microdroplet array into a target image with a set size by taking the index of the microdroplet in the microdroplet array as a horizontal coordinate and the brightness of the microdroplet as a
vertical coordinate to perform
image segmentation so as to obtain a reference microdroplet; and carrying out microdroplet division and clustering based on the reference microdroplet. According to the method, the robustness and reliability during clustering can be obviously improved, and particularly the problem that the robustness and reliability of threshold division and clustering results are poor under the conditions of different sample concentrations, different negative and
positive contrast ratios, uneven light fields and the like is solved.