Holographic functional calcium imaging of neuronal circuit activity

Functional imaging provides efficient optical recording of Ca²⁺ activity from single neurons as well as networks of neurons forming a circuit. Sufficient spatial resolution needs to be achieved in order to resolve cell bodies as well as fine neuronal structures such as dendrites and spines. On the other hand, the temporal resolution should be sufficient to capture Ca²⁺ events from multiple sites across the sample. Here, we briefly describe various imaging modalities and focus on two-photon holographic multi-foci excitation to perform multi-site Ca²⁺ imaging. Holographically projected multi-foci can be used to excite two-photon fluorescence from multiple sites along the dendritic tree of a single neuron or multiple cell bodies in a neuronal network. By simultaneously projecting multiple foci onto the sample, the fluorescence emanating from the different foci can be collected simultaneously using a camera, thereby setting the temporal resolution to the frame-rate of the camera. We describe the generation of appropriate holograms as well as discuss the limitations of the approach. We also discuss a solution to improve the signal-to-noise ratio via the application of temporal gating. The multi-foci holographic technique is a promising approach to perform high-speed optical recording of neuronal activity.