Accurate neuron segmentation method for one-photon calcium imaging videos combining convolutional neural networks and clustering

One-photon fluorescent calcium imaging helps understand brain functions by recording large-scale neural activities in freely moving animals. Automatic, fast, and accurate active neuron segmentation algorithms are essential to extract and interpret information from these videos. One-photon imaging videos' low resolution, high noise, and high background fluctuation pose significant challenges. Here, we develop a software pipeline to address the challenges of processing one-photon calcium imaging videos. We extend our previous two-photon active neuron segmentation algorithm, Shallow U-Net Neuron Segmentation (SUNS), to better suppress background fluctuations in one-photon videos. We also develop additional neuron extraction (ANE) to locate small or dim neurons missed by SUNS. To train our segmentation method, we create ground truth neurons by developing a manual labeling pipeline assisted with semi-automatic refinement. Our method is more accurate and faster than state-of-the-art techniques when processing simulated videos and multiple experimental datasets acquired over various brain regions with different imaging conditions.