DeepSpot: A deep neural network for RNA spot enhancement in single-molecule fluorescence in-situ hybridization microscopy images.

Detection of RNA spots in single-molecule fluorescence in-situ hybridization microscopy images remains a difficult task, especially when applied to large volumes of data. The variable intensity of RNA spots combined with the high noise level of the images often requires manual adjustment of the spot detection thresholds for each image. In this work, we introduce DeepSpot, a Deep Learning-based tool specifically designed for RNA spot enhancement that enables spot detection without the need to resort to image per image parameter tuning. We show how our method can enable downstream accurate spot detection. DeepSpot's architecture is inspired by small object detection approaches. It incorporates dilated convolutions into a module specifically designed for context aggregation for small object and uses Residual Convolutions to propagate this information along the network. This enables DeepSpot to enhance all RNA spots to the same intensity, and thus circumvents the need for parameter tuning. We evaluated how easily spots can be detected in images enhanced with our method by testing DeepSpot on 20 simulated and 3 experimental datasets, and showed that accuracy of more than 97% is achieved. Moreover, comparison with alternative deep learning approaches for mRNA spot detection (deepBlink) indicated that DeepSpot provides more precise mRNA detection. In addition, we generated single-molecule fluorescence in-situ hybridization images of mouse fibroblasts in a wound healing assay to evaluate whether DeepSpot enhancement can enable seamless mRNA spot detection and thus streamline studies of localized mRNA expression in cells.