Heuristically optimal path scanning for high-speed multiphoton circuit imaging

Abstract

Population dynamics of patterned neuronal firing are fundamental to information processing in the brain. Multiphoton microscopy in combination with calcium indicator dyes allows circuit dynamics to be imaged with single-neuron resolution. However, the temporal resolution of fluorescent measures is constrained by the imaging frequency imposed by standard raster scanning techniques. As a result, traditional raster scans limit the ability to detect the relative timing of action potentials in the imaged neuronal population. To maximize the speed of fluorescence measures from large populations of neurons using a standard multiphoton laser scanning microscope (MPLSM) setup, we have developed heuristically optimal path scanning (HOPS). HOPS optimizes the laser travel path length, and thus the temporal resolution of neuronal fluorescent measures, using standard galvanometer scan mirrors. Minimizing the scan path alone is insufficient for prolonged high-speed imaging of neuronal populations. Path stability and the signal-to-noise ratio become increasingly important factors as scan rates increase. HOPS addresses this by characterizing the scan mirror galvanometers to achieve prolonged path stability. In addition, the neuronal dwell time is optimized to sharpen the detection of action potentials while maximizing scan rate. The combination of shortest path calculation and minimization of mirror positioning time allows us to optically monitor a population of neurons in a field of view at high rates with single-spike resolution, ∼ 125 Hz for 50 neurons and ∼ 8.5 Hz for 1,000 neurons. Our approach introduces an accessible method for rapid imaging of large neuronal populations using traditional MPLSMs, facilitating new insights into neuronal circuit dynamics.

Multineuronal activity patterns identify selective synaptic connections under realistic experimental constraints

“…This study complements related work by emphasizing performance under the conditions of realistic optical experiments (constrained recording durations and restricted sampling) (Sadovsky et al 2011). We purposefully designed our simulations to pose substantial challenges to inference, including weak conductance-based synapses, random connectivity, occlusion of 60% of the neurons in the network, and limited recording times.…”

“…We found it better to use a 10-ms lag window than a 5-ms lag window, measuring functional relationships that closely mirrored the time course of excitatory synaptic conductances in our model. With the use of HOPS scanning (Sadovsky et al 2011), a 10-ms frame duration corresponds to ϳ75 neurons imaged. Note that equal or better precision could be obtained by considering lags spanning multiple 5-ms frames rather than a single 10-ms frame.…”

“…Even with dense uniform sampling within a large field of view (Sadovsky et al 2011), connected neurons reside above, below, and outside the imaged field. Unobserved activity can lead to ambiguity in the reconstruction of synaptic connectivity from spiking because of misleading statistical dependencies (Chicharro and Panzeri 2014), particularly in resolving true synaptic connections vs. unconnected neurons with shared inputs (Nykamp 2007(Nykamp , 2008.…”

“…8A). With the use of HOPS scanning ex vivo with single-frame recruitment windows, these frame durations correspond to ϳ300, 75, and 30 neurons scanned, respectively (Sadovsky et al 2011). To investigate how choice of single-frame lag window impacts inference without the confound of changing sample size, we obscured 60% of the 1,000 excitatory model cells in each trial.…”

Circuit reactivation dynamically regulates synaptic plasticity in neocortex

“…A power increase in d-frequency as measured in the membrane voltage recordings of patch-clamped neurons accompanied the transition to the UP state (16.1±12.7-fold; n ¼ 100 UP states; n ¼ 8 neurons). We tracked spiking activity within the surrounding neuronal population using multiphoton laser-scanning microscopy of neurons loaded with the calcium indicator dye Fura-2AM 23,24 . Spike trains were inferred from somatic fluorescence measures 23,24 .…”