TrueSpot: a robust automated tool for quantifying signal puncta in fluorescent imaging

Characterizing the movement of biomolecules in single cells quantitatively is essential to understanding fundamental biological mechanisms. RNA fluorescent in situ hybridization (RNA-FISH) is a technique for visualizing RNA in fixed cells using fluorescent probes. Automated processing of the resulting images is essential for large datasets. Here we demonstrate that our RNA-FISH image processing tool, TrueSpot, is useful for automatically detecting the locations of RNA at single molecule resolution. TrueSpot also performs well on images with immunofluorescent and GFP-tagged clustered protein targets. Additionally, we show that our 3D spot detection approach substantially outperforms current 2D spot detection algorithms.