Heuristically Optimal Path Scanning (HOPS) for High Speed Multiphoton Circuit
چکیده
10 11 Population dynamics of patterned neuronal firing is fundamental to information 12 processing in the brain. Multiphoton microscopy in combination with calcium indicator 13 dyes allows circuit dynamics to be imaged with single neuron resolution. However, the 14 temporal resolution of fluorescent measures is constrained by the imaging frequency 15 imposed by standard raster scanning techniques. As a result, traditional raster scans 16 limit the ability to detect the relative timing of action potentials in the imaged neuronal 17 population. To maximize the speed of fluorescence measures from large populations of 18 neurons using a standard MPLSM setup we have developed Heuristically Optimal Path 19 Scanning (HOPS). HOPS optimizes the laser travel path length and thus the temporal 20 resolution of neuronal fluorescent measures using standard galvanometer scan mirrors. 21 Minimizing the scan path alone is insufficient for prolonged high speed imaging of 22 neuronal populations. Path stability and the SNR become increasingly important factors 23 as scan rates increase. HOPS addresses this by characterizing the scan mirror 24 galvanometers to achieve prolonged path stability. In addition, the neuronal dwell time is 25 optimized to sharpen the detection of action potentials while maximizing scan rate. The 26 combination of shortest path calculation and minimization of mirror positioning time 27 allows us to optically monitor a population of neurons in a field of view at high rates with 28 single spike resolution, ~125 Hz for 50 neurons and ~8.5 Hz for 1000 neurons. Our 29 approach introduces an accessible method for rapid imaging of large neuronal 30 populations using traditional multiphoton laser scanning microscopes – facilitating new 31 insights into neuronal circuit dynamics. 32 33 34