In voltage imaging, where the membrane potentials of individual neurons are recorded at from hundreds to thousand frames per second using fluorescence microscopy, data processing presents a challenge. Even a fraction of a minute of recording with a limited image size yields gigabytes of video data consisting of tens of thousands of frames, which can be time-consuming to process. Moreover, millisecond-level short exposures lead to noisy video frames, obscuring neuron footprints especially in deep-brain samples where noisy signals are buried in background fluorescence. To address this challenge, we propose a fast neuron segmentation method able to detect multiple, potentially overlapping, spiking neurons from noisy video frames, and implement a data processing pipeline incorporating the proposed segmentation method along with GPU-accelerated motion correction. By testing on existing datasets as well as on new datasets we introduce, we show that our pipeline extracts neuron footprints that agree well with human annotation even from cluttered datasets, and demonstrate real-time processing of voltage imaging data on a single desktop computer for the first time.
