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The brain atlas, which provides information about the distribution of genes, proteins, neurons, or anatomical regions, plays a crucial role in contemporary neuroscience research. To analyze the spatial distribution of those substances based on images from different brain samples, we often need to warp and register individual brain images to a standard brain template. However, the process of warping and registration may lead to spatial errors, thereby severely reducing the accuracy of the analysis. To address this issue, we develop an automated method for segmenting neuropils in the Drosophila brain for fluorescence images from the FlyCircuit database. This technique allows future brain atlas studies to be conducted accurately at the individual level without warping and aligning to a standard brain template. Our method, LYNSU (Locating by YOLO and Segmenting by U-Net), consists of two stages. In the first stage, we use the YOLOv7 model to quickly locate neuropils and rapidly extract small-scale 3D images as input for the second stage model. This stage achieves a 99.4% accuracy rate in neuropil localization. In the second stage, we employ the 3D U-Net model to segment neuropils. LYNSU can achieve high accuracy in segmentation using a small training set consisting of images from merely 16 brains. We demonstrate LYNSU on six distinct neuropils or structures, achieving a high segmentation accuracy comparable to professional manual annotations with a 3D Intersection-over-Union (IoU) reaching up to 0.869. Our method takes only about 7 s to segment a neuropil while achieving a similar level of performance as the human annotators. To demonstrate a use case of LYNSU, we applied it to all female Drosophila brains from the FlyCircuit database to investigate the asymmetry of the mushroom bodies (MBs), the learning center of fruit flies. We used LYNSU to segment bilateral MBs and compare the volumes between left and right for each individual. Notably, of 8,703 valid brain samples, 10.14% showed bilateral volume differences that exceeded 10%. The study demonstrated the potential of the proposed method in high-throughput anatomical analysis and connectomics construction of the Drosophila brain.

Segmenting individual neurons from a large number of noisy raw images is the first step in building a comprehensive map of neuron-to-neuron connections for predicting information flow in the brain. Thousands of fluorescence-labeled brain neurons have been imaged. However, mapping a complete connectome remains challenging because imaged neurons are often entangled and manual segmentation of a large population of single neurons is laborious and prone to bias. In this study, we report an automatic algorithm, NeuroRetriever, for unbiased large-scale segmentation of confocal fluorescence images of single neurons in the adult Drosophila brain. NeuroRetriever uses a high-dynamic-range thresholding method to segment three-dimensional morphology of single neurons based on branch-specific structural features. Applying NeuroRetriever to automatically segment single neurons in 22,037 raw brain images, we successfully retrieved 28,125 individual neurons validated by human segmentation. Thus, automated NeuroRetriever will greatly accelerate 3D reconstruction of the single neurons for constructing the complete connectomes.