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

## TL;DR

This paper shows how to capture high-resolution neuronal activity using 8-bit imaging and pixel binning, enabling faster and more detailed brain imaging.

## Contribution

The novel approach combines 8-bit depth imaging with offline pixel binning to achieve high spatial and temporal resolution in neuronal imaging.

## Key findings

- Fluorescence transients from calcium and voltage-sensitive dyes were resolved using 8-bit depth and pixel binning.
- Action potentials were recorded at 40 kHz in the axon initial segment of layer-5 pyramidal neurons.
- High frame rates up to 2 kHz and 10 kHz were achieved in hippocampal slices and dendritic regions.

## Abstract

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.

We demonstrate that fluorescence transients from calcium or from voltage‐sensitive dyes, associated with neuronal signals, can be resolved by acquiring frames at 8‐bit digital depth and by binning pixels offline. With this approach, we could attain exceptional frame sizes, in terms of number of pixels, at very high temporal resolution.