The limited per-pixel bandwidth of most microscopy methods requires compromises between field of view, sampling density and imaging speed. This limitation constrains studies involving complex motion or fast cellular signaling, and presents a major bottleneck for high-throughput structural imaging. Here, we combine high-speed intensified camera technology with a versatile, reconfigurable and dramatically improved Swept, Confocally Aligned Planar Excitation (SCAPE) microscope design that can achieve high-resolution volumetric imaging at over 300 volumes-persecond and over 1.2 GHz pixel rates. We demonstrate near-isotropic sampling in freely moving C. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and ‘straightened’ freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90–100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze.
The brain is a complex system that operates based on coordinated neuronal activities. Brainwide cellular calcium imaging techniques have quickly advanced in recent years and become powerful tools for understanding the neuronal activities of small animal models. The whole brain imaging generally requires to extract the neuronal activities from three-dimensional (3D) image series. Unfortunately, the 3D image series are obtained under imaging conditions different among laboratories and extracting neuronal activities from the data requires multiple processes. Therefore researchers need to develop their own software, which has prevented the application of wholebrain imaging experiments in more laboratories. Here, we combined traditional image processing techniques with the powerful deep-learning method which can be flexibly modified to fit 3D image data in the nematode Caenorhabditis elegans obtained under different conditions. We first trained the 3D U-net deep network to classify each pixel into cell and non-cell categories. Cells merged as a whole region were further separated into individual cells by watershed segmentation. The cells were then tracked in 3D space over time with the combination of a feedforward network and a point set registration method to use local and global relative positions of the cells, respectively. Remarkably, one manually annotated 3D image combined with data augmentation was sufficient for training the deep networks to obtain satisfactory tracking results. Our method correctly tracked more than 98% of neurons in three different image datasets and successfully extracted brain-wide neuronal activities. Our method worked well even when the sampling rate was reduced: 86% correct in case 4/5 frames were removed, and when artificial noise was added into the raw images: 91% correct in case 35 times of background-level noise was added. Our results proved that deep learning is widely applicable to different datasets and can help us in establishing a flexible pipeline for extracting whole brain activities.
To ensure survival, animals must update the internal representations of their environment in a trial-and-error fashion. Psychological studies of associative learning and neurophysiological analyses of dopaminergic neurons have suggested that this updating process involves the temporal-difference (TD) method in the basal ganglia network. However, the way in which the component variables of the TD method are implemented at the neuronal level is unclear. To investigate the underlying neural mechanisms, we trained domestic chicks to associate color cues with food rewards. We recorded neuronal activities from the medial striatum or tegmentum in a freely behaving condition and examined how reward omission changed neuronal firing. To compare neuronal activities with the signals assumed in the TD method, we simulated the behavioral task in the form of a finite sequence composed of discrete steps of time. The three signals assumed in the simulated task were the prediction signal, the target signal for updating, and the TD-error signal. In both the medial striatum and tegmentum, the majority of recorded neurons were categorized into three types according to their fitness for three models, though these neurons tended to form a continuum spectrum without distinct differences in the firing rate. Specifically, two types of striatal neurons successfully mimicked the target signal and the prediction signal. A linear summation of these two types of striatum neurons was a good fit for the activity of one type of tegmental neurons mimicking the TD-error signal. The present study thus demonstrates that the striatum and tegmentum can convey the signals critically required for the TD method. Based on the theoretical and neurophysiological studies, together with tract-tracing data, we propose a novel model to explain how the convergence of signals represented in the striatum could lead to the computation of TD error in tegmental dopaminergic neurons.
The brain performs various functions by coordinating and integrating activities of its units, called nerve cells or "neurons". Therefore, the mechanisms underlying brain functions should be explored by analyzing neuronal activities in the entire brain [i.e. whole brain activities (WBAs)]. Because the brain consists of a very large number of neurons, extraction of useful information from dynamic WBAs requires powerful mathematical methods. In this review for non-specialists of brain science, we introduce the methods for measuring WBAs, the methods for analyzing these data using classic mathematical and recent machine learning techniques, the results of such analyses in various research contexts, and the challenges in those studies. We also propose an approach for extracting dynamic activity flow in the brain with a mathematical model and validating the model with cutting-edge biological experiments.
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