Neuronal Imaging at 8-Bit Depth to Combine High Spatial and High Temporal Resolution With Acquisition Rates Up To 40 kHz.

A challenge in neuroimaging is acquiring frame sequences at high temporal resolution from the largest possible number of pixels. Measuring 1%-10% fluorescence changes normally requires 12-bit or higher bit depth, constraining the frame size allowing imaging in the kHz range. We resolved Ca or membrane potential signals from cell populations or single neurons in brain slices by acquiring fluorescence at 8-bit depth and by binning pixels offline, achieving unprecedented frame sizes at kHz rates. In hippocampal slices stained with the Ca indicator Fluo-4 AM, we resolved transients at 2 kHz from large frames. Along the apical dendrite of a layer-5 pyramidal neuron, we measured Ca signals associated with a back-propagating action potential at 10 kHz. Finally, in the axon initial segment of the same cell type, we recorded an action potential at 40 kHz by voltage-sensitive dye imaging. This approach unlocks the potential for a range of imaging measurements.