Voltage imaging of many neurons simultaneously at single-cell resolution is hampered by the difficulty of detecting small voltage signals from overlapping neuronal processes in neural tissue. Recent advances in genetically encoded voltage indicator (GEVI) imaging have shown single-cell resolution optical voltage recordings in intact tissue through imaging naturally sparse cell classes, sparse viral expression, soma restricted expression, advanced optical systems, or a combination of these. Widespread sparse and strong transgenic GEVI expression would enable straightforward optical access to a densely occurring cell type, such as cortical pyramidal cells. Here we demonstrate that a recently described sparse transgenic expression strategy can enable single-cell resolution voltage imaging of cortical pyramidal cells in intact brain tissue without restricting expression to the soma. We also quantify the functional crosstalk in brain tissue and discuss optimal imaging rates to inform future GEVI experimental design.
Light-field microscopy (LFM) is a type of all-optical imaging system that is able to capture 4D geometric information of light rays and can reconstruct a 3D model from a single snapshot. In this paper, we propose a new 3D localization approach to effectively detect 3D positions of neuronal cells from a single light-field image with high accuracy and outstanding robustness to light scattering. This is achieved by constructing a depth-aware dictionary and by combining it with convolutional sparse coding. Specifically, our approach includes 3 key parts: light-field calibration, depth-aware dictionary construction, and localization based on convolutional sparse coding (CSC). In the first part, an observed raw light-field image is calibrated and then decoded into a two-plane parameterized 4D format which leads to the epi-polar plane image (EPI). The second part involves simulating a set of light-fields using a wave-optics forward model for a ball-shaped volume that is located at different depths. Then, a depth-aware dictionary is constructed where each element is a synthetic EPI associated to a specific depth. Finally, by taking full advantage of the sparsity prior and shift-invariance property of EPI, 3D localization is achieved via convolutional sparse coding on an observed EPI with respect to the depthaware EPI dictionary. We evaluate our approach on both nonscattering specimen (fluorescent beads suspended in agarose gel) and scattering media (brain tissues of genetically encoded mice). Extensive experiments demonstrate that our approach can reliably detect the 3D positions of granular targets with small Root Mean Square Error (RMSE), high robustness to optical aberration and light scattering in mammalian brain tissues.
Recent years have seen substantial developments in technology for imaging neural circuits, raising the prospect of large scale imaging studies of neural populations involved in information processing, with the potential to lead to step changes in our understanding of brain function and dysfunction. In this article we will review some key recent advances: improved fluorophores for single cell resolution functional neuroimaging using a two photon microscope; improved approaches to the problem of scanning active circuits; and the prospect of scanless microscopes which overcome some of the bandwidth limitations of current imaging techniques. These advances in technology for experimental neuroscience have in themselves led to technical challenges, such as the need for the development of novel signal processing and data analysis tools in order to make the most of the new experimental tools. We review recent work in some active topics, such as region of interest segmentation algorithms capable of demixing overlapping signals, and new highly accurate algorithms for calcium transient detection. These advances motivate the development of new data analysis tools capable of dealing with spatial or spatiotemporal patterns of neural activity, that scale well with pattern size.
Cancer cells feature a resting membrane potential (Vm) that is depolarized compared to normal cells, and express active ionic conductances, which factor directly in their pathophysiological behavior. Despite similarities to ‘excitable’ tissues, relatively little is known about cancer cell Vm dynamics. Here high-throughput, cellular-resolution Vm imaging reveals that Vm fluctuates dynamically in several breast cancer cell lines compared to non-cancerous MCF-10A cells. We characterize Vm fluctuations of hundreds of human triple-negative breast cancer MDA-MB-231 cells. By quantifying their Dynamic Electrical Signatures (DESs) through an unsupervised machine-learning protocol, we identify four classes ranging from "noisy” to “blinking/waving“. The Vm of MDA-MB-231 cells exhibits spontaneous, transient hyperpolarizations inhibited by the voltage-gated sodium channel blocker tetrodotoxin, and by calcium-activated potassium channel inhibitors apamin and iberiotoxin. The Vm of MCF-10A cells is comparatively static, but fluctuations increase following treatment with transforming growth factor-β1, a canonical inducer of the epithelial-to-mesenchymal transition. These data suggest that the ability to generate Vm fluctuations may be a property of hybrid epithelial-mesenchymal cells or those originated from luminal progenitors.
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