NeuroART: Real-time analysis and targeting of neuronal population activity during calcium imaging for informed closed loop experiments.

Two-photon calcium imaging allows for the activity readout of large populations of neurons at single cell resolution in living organisms, yielding new insights into how the brain processes information. Holographic optogenetics allows us to trigger activity of this population directly, raising the possibility of injecting information into a living brain. Optogenetic triggering of activity that mimics "natural" information, however, requires identification of stimulation targets based on real-time analysis of the functional network. We have developed NeuroART (Neuronal Analysis in Real Time), software that provides real-time readout of neuronal activity integrated with downstream analysis of correlations and synchrony, and of sensory metadata. On the example of auditory stimuli, we demonstrate real-time inference of the contribution of each neuron in the field of view to sensory information processing. To avoid the limitations of microscope hardware and enable collaboration of multiple research groups, NeuroART taps into microscope data streams without the need for modification of microscope control software and is compatible with a wide range of microscope platforms. NeuroART also integrates the capability to drive a Spatial Light Modulator (SLM) for holographic photostimulation of optimal stimulation targets, enabling real-time modification of functional networks. Neurons used for photostimulation experiments were extracted from Sprague Dawley rat embryos of both sexes.Significance Statement We have developed a software platform, Neuronal Analysis in Real Time (NeuroART), which addresses the growing need in neuronal imaging studies for real-time analysis capabilities and has unique capabilities when compared to other recently developed software (Giovannucci, Friedrich et al. 2017, Mitani and Komiyama 2018, Zhang, Russell et al. 2018, Giovannucci, Friedrich et al. 2019, Sheng, Zhao et al. 2022). NeuroART stands out in its real-time inclusion of functional network analysis, correlation analysis, synchrony analysis, holographic optogenetic photostimulation, and integration of sensory information metadata. Furthermore, this tool enables experimenters to assess data quality in real-time. With these unique features and its demonstrated ability to work with several widely used microscope platforms, NeuroART is poised to enable novel closed-loop model guided experiments.