TOWARD ROBUST FULLY 3D FILOPODIUM SEGMENTATION AND TRACKING IN TIME-LAPSE FLUORESCENCE MICROSCOPY

Development, parameter tuning, and objective benchmarking of bioimage analysis workflows heavily rely on the availability of diverse bioimage datasets accompanied by reference annotations. In this paper, we present a new benchmark dataset, FiloData3D, designed for in-depth performance assessments of fully 3D filopodium segmentation and tracking algorithms that emerged recently in the field. It consists of 180 synthetic, fully annotated, 3D time-lapse sequences of single lung cancer cells, combining different cell shapes, signal-to-noise ratios, and anisotropy ratios, which are the well-known factors that influence the quality of segmentation and tracking results. Using FiloData3D, we show that the number of filopodia and their lengths extracted are significantly underestimated in the case of traditional 2D protocols that prevail in daily practice compared to fully 3D measurements, calling for a procedural change in filopodial analyses of 3D+t bioimage data.