Automating Event-detection of Brain Neuron Synaptic Activity and Action Potential Firing in vivo using a Random-access Multiphoton Laser Scanning Microscope for Real-time Analysis.

Determining how a neuron computes requires an understanding of the complex spatiotemporal relationship between its input (e.g. synaptic input as a result of external stimuli) and action potential output. Recent advances in in vivo, laser-scanning multiphoton technology, known as random-access microscopy (RAM), can capture this relationship by imaging fluorescent light, emitted from calcium-sensitive biosensors responding to synaptic and action potential firing in a neuron's full dendritic arbor and cell body. Ideally, a continuous output of fluorescent intensities from the neuron would be converted to a binary output (`event', 'or no-event'). These binary events can be used to correlate temporal and spatial associations between the input and output. However, neurons contain hundreds-to-thousands of synapses on the dendritic arbors generating an enormous quantity of data composed of physiological signals, which vary greatly in shape and size. Thus, automating data-processing tasks is essential to support high-throughput analysis for real-time/post-processing operations and to improve operators' comprehension of the data used to decipher neuron computations. Here, we describe an automated software algorithm to detect brain neuron events in real-time using an acousto-optic, multiphoton, laser scanning RAM developed in our laboratory. The fluorescent light intensities, from a genetically encoded, calcium biosensor (GCAMP 6m), are measured by our RAM system and are input to our 'event-detector', which converts them to a binary output meant for real-time applications. We evaluate three algorithms for this purpose: exponentially weighted moving average, cumulative sum, and template matching; present each algorithm's performance; and discuss user-feasibility of each. We validated our system in vivo, using the visual circuit of the Xenopus laevis.