Abstract: A segmentation system processes images of a sample that includes fluorescent entities. The segmentation system applies a threshold image that represents background illumination distinct from fluorescence of target entities to pre-process the images. The segmentation system determines numbers of target entities in pre-processed images and determines whether an estimated number of target entities in the sample meets a threshold certainty. The segmentation system continues to analyze one or more images until the threshold certainty is determined. When the threshold certainty is met, the estimated number of target entities may be used to generate a user interface output (e.g., displaying the pre-processed images and visual indicators of the locations of target entities.