A Quantitative Metric of Confidence for Segmentation of Nuclei in Large Spatially Variable Image Volumes

Nuclei segmentation is an important step for quantitative analysis of fluorescence microscopy images. A large volume generally has many different regions containing nuclei with varying spatial characteristics. Automatically identifying nuclei that are challenging to segment can speed up the analysis of biological tissues. Here we show a segmentation technique that provides a metric of segmentation “confidence” for each segmented object in an image volume. This confidence metric can be used either to generate a “confidence map” for visual distinction of reliable from unreliable regions, or in the data space to identify questionable measurements that can be analyzed separately or eliminated from analysis. In an analysis of nuclei in a 3-dimensional image volume, we show that the confidence map correlates well with visual evaluations of segmentation quality, and that the confidence metric correlates well with F1 scores within subregions of the image volume. In addition, we also describe three visualization methods that can visualize the segmentation differences between a segmented volume and a reference volume.