Crosstalk characterization

To determine the degree of temporal and spatial crosstalk observed in scattering tissue, a mouse virally expressing SpikeyGi implanted with a cranial window was used. Tissue was scanned using one beam at a time (~30 mW), and fluorescence was collected across all eight subareas. Images consisting of 50-frame averages acquired at 389 Hz were taken at 30-μm steps from 0 to 300 μm below the pial surface. For the excited subarea in each image, pixels corresponding to SpikeyGi fluorescence were identified as those whose intensities were >95th percentile compared with other pixels in the same subarea. Per cent crosstalk was calculated as the mean signal measured in each of the nonexcited subareas divided by the mean signal in the excited subarea containing SpikeyGi fluorescence.