Neuronal Imaging at 8‐Bit Depth to Combine High Spatial and High Temporal Resolution With Acquisition Rates Up To 40 <scp>kHz</scp>

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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.